Cell Culture Bioprocess Engineering

Cell Culture Bioprocess Engineering

Cell Culture Bioprocess Engineering Wei-Shou Hu Department of Chemical Engineering and Material Science University of Minnesota Minneapolis, MN

With Contributions From:

Weichang Zhou Gargi Seth Sadettin Ozturk Chun Zhang

Copyright © 2012 by Wei-Shou Hu ISBN: 978-0-9856626-0-8 http://www.cellprocessbook.com/

Preface For over two decades, we have assembled innovative guest lecturers to share their research and best-practices at our annual cell culture bioprocessing short course at the University of Minnesota. This course was created for industrial practitioners of the production of biologics. This book is the culmination of two decades of accumulated expertise, practical know-how and insight into future trends. There have been many books and courses on cell culture technology covering topics from a technical or business perspective. The goal of this course and this book is to bring new knowledge from cutting-edge research into the very practical setting of today’s industrial laboratories. A second goal of this course is to prepare industrial practitioners and students from different academic disciplines to collaborate in today’s cross-disciplinary teams. In the course of delivering a molecule from a gene sequence in the laboratory to a product in the manufacturing plant, scientists and engineers must quickly communicate, troubleshoot and innovate. The fundamental knowledge for practicing industrial cell culture spans from cell biology and physiology to process engineering principles in stoichiometry, reactor kinetics and scale up. Thus, we have designed this course for students of diverse backgrounds. The book is used in the classroom of our annual course. The layout of the book is thus designed to facilitate the delivery of information. The left panels are graphs, tables, diagrams, highlights of key points and space for note taking; while the right panels are descriptive text. This course has been given around the world: in Europe, East and South Asia, South America and as an internal course at many corporations. Over three thousand industrial biotechnologists have taken this course. With the technology of biologics production spreading to wider regions of the world, this book will meet a timely need of many who practice the technology but cannot attend the course in Minnesota. The book is published in an electronic form to allow for more frequent future updates, and for easy distribution to the parts of the world where the biologics manufacturing is quickly expanding. Wei-Shou Hu Department of Chemical Engineering and Material Science University of Minnesota

Acknowledgements The authoring of this book has been influenced by many who have lectured in the summer course at the University of Minnesota over the years. Foremost, thanks go to Anthony J. Sinskey, Michael C. Flickinger, Donald McClure and Fredrick Srienc who started the course with me originally. Konstantin Konstantinov, James Piret, James N. Thomas, Randall Kaufman, Florian Wurm, John Aunins, Michael Betenbaugh, Sadettin Ozturk, Matthew Croughan, Weichang Zhou, Chun Zhang and Gargi Seth all contributed to enrich the course. Many former and current members of my research laboratory at the University of Minnesota contributed to the preparation of course materials. These include Derek Adams, Marlene Castro, Bhanu Chandra Mulukutla, Anushree Chatterjee, Anna Europa, Patrick Fu, Chetan Gadgil, Mugdha Gadgil, Anshu Gambhir, Patrick Hossler, Claire Hypolite, Nitya M. Jacob, Kathryn Johnson, Anne Kantardjieff, Edmund Kao, Anurag Khetan, Rashmi Korke, Huong Le, Jongchan Lee, Marcela de Leon Gatti, Sarika Mehra, Jason D. Owens, Yonsil Park, Gargi Seth, Shikha Sharma, Kartik Subramanian, Siguang Sui, Katie Wlaschin, and Kathy Wong. Gargi Seth, Sadettin Ozturk, Weichang Zhou and Chun Zhang, whose participation in the course led to the development of new chapters, are noted as contributors. This book, which began as a set of lecture notes, has gone through many years of refinement in organization by many skillful hands. Kimberly Durand first took the notes to digital form in a CD ROM. Ruth Patton, Radha Dalal, Katherine Matthews, Heather Wooten, Kirsten Keefe, Jessica Raines-Jones, Kimberly Coffee and Kaitlyn Pladson continued to shape it. At the long last, Erin Fenton and Jenna Novotny took it to current form. Kimberly Durand also coordinated our final publication efforts. This book is dedicated to the students, fellows and staff formerly and currently in my laboratory at the University of Minnesota. It is through working with them that the materials used in the book were distilled. It was also through their educating me with new knowledge, new concepts, and new tools that this book took its shape. I must also thank my dear friend and close colleague, Miranda Yap of Bioprocess Technology Institute, Singapore, with whom I have had a wonderful and long collaboration. Finally, I wish for my lovely family, Jenny, Kenny and my wife, Sheau-Ping to share the joy of the book’s completion. Wei-Shou Hu Department of Chemical Engineering and Material Science University of Minnesota

Contents In Brief Overview of Cell Culture Technology. . . . . . . . . . . . . . . . . . . . . . . 1 Cell Biology for Bioprocessing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Cell Physiology for Process Engineering. . . . . . . . . . . . . . . . . . . . . 57 Medium Design for Cell Culture Processing. . . . . . . . . . . . . . . . . . 97 Cell Line Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Stoichiometry and Kinetics of Cell Cultivation. . . . . . . . . . . . . . . . 147 Cell Culture Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Metabolic Flux Analysis in Cell Culture Systems . . . . . . . . . . . . . . 175 Cell Culture Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Oxygen Transfer in Cell Culture Bioreactors . . . . . . . . . . . . . . . . . 213 Fedbatch Culture and Dynamic Nutrient Feeding. . . . . . . . . . . . . 233 Cell Retention and Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Scaling Up and Scaling Down for Cell Culture Bioreactors. . . . . . 263 Cell Culture Genomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

309

ACKNOWLEDGEMENTS | VII

Overview of Cell Culture Technology Cell Culture Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Cell Culture Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Virus Vaccines and Protein Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Protein Molecule as Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Industrial Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Biosimilars or Follow-on-Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Manufacturing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Alternative Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Product Quality and Process Robustness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Critical Feature of rDNA Proteins from Mammalian Cells . . . . . . . . . . . . . . . . . . 14 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Cell Culture Engineering In the past decade we have seen continuous growth in mammalian cell culture bioprocessing, driven primarily by the expansion of therapeutic antibody production in the pharmaceutical industry. The range and quantity of products have both significantly increased over the past ten years. Also fueling this growth are the increasing numbers of therapeutic protein candidates in the drug development pipeline that can potentially render many more untreatable diseases treatable. Recombinant therapeutic proteins have yielded major advances in healthcare. Their societal impacts may even rival those of antibiotics, whose discovery and clinical applications transformed much of modern medicine. Microbial fermentation technology enabled pharmaceutical industry to make penicillin widely available between 1950 and 1970. Today, we see cell culture processing technology enabling this new class of protein biologics to reach needy patients. As with any product, manufacturers are under continual pressure to produce more with less. In

OVERVIEW | 1

the case of cell culture bioprocess technology, we see increasing demand for therapeutic proteins, coupled with the strain of often prohibitively high investment costs for new manufacturing facilities. Thus, we must constantly re-evaluate, streamline and refine, to increase production without the luxury of totally new and improved facilities.

Fig. 1.1: Historical trend of penicillin titer and value

As we look to the future of cell culture processing, it is useful to look back at the history and development of penicillin. This specific case highlights lessons that are almost universally relevant for the manufacturing of other products. Today’s innovations will all travel through some variation of these phases, from the moment of discovery, expansion and distribution, maturation and even demise of the product. Pencillin is also representative of the many strides made in the broader field of microbial natural products that preceded today’s protein biologics.

Sir Alexander Fleming’s discovery of penicillin began a new chapter in biotechnology. In the twentyfive years following the first clinical applications (pioneered by Edward Penley Abraham) both the product titer and the production volume of penicillin increased almost exponentially. This rapid expansion in production quantities and titer was then followed by a period of slower but steady growth over the next fifty years. The roughly three orders of magnitude increase in production volume and product concentration was the result of relentless effort on the part of process scientists and engineers. These engineers looked for hidden opportunities for strain improvement, media development, and much more. As a result, we have seen steady productivity growth due to improvements in oxygen transfer, heat transfer, and mixing characteristics. Additional advances in on-line sensing, sterility control, equipment reliability and process control all contributed to technological success. It should be noted that the success of process technology also eventually drove down the price. Penicillin G is no longer produced in the United States; the cost of production is now

OVERVIEW OF CELL CULTURE TECHNOLOGY | 2

dramatically lower in other parts of the world.

Now, two decades after the first introduction of therapeutic biologics, we have seen titers in large manufacturing processes increase from tens of milligrams per liter to more than five grams per liter for many immunoglobulin products today. Although little published information is available, the production cost has also decreased by at least an order of magnitude since the beginning of cell culture products.

A graph of historical data for cell culture products plotted one or two decades from now will likely resemble that of penicillin. Cell culture production today is likely around the transition from the exponential growth stage to the steady and slower growth period. However, it is important to note that, in terms of both absolute quantity of product produced and economic value, the slower and steadier phase is as critical as the early rapid growth stage for the product life cycle.

Even for penicillin, there were tremendous process enhancements after the initial rapid growth phase. Due to these improvements, major medicines became affordable for the world’s population. The next question for bioprocess scientists and engineers is: How can cell culture processing accomplish what the antibiotics industry has achieved for our society? Bioprocess scientists and engineers possess genomics and genome engineering tools that were not previously available to antibiotic researchers or even to the early innovators of cell culture processes. These new genome-wide investigative and engineering tools will greatly facilitate the designing and engineering of cells with desired growth and production characteristics. Process technologists will need to harness the power of genomics and genome engineering to enhance productivity and process robustness. This will also facilitate the expansion of biosimilars (i.e., “Follow-on” biologics) and make many medicines available to needy patients around the world at an affordable cost. Much of the process technology employed in cell culture biologics was developed for antibiotic

OVERVIEW OF CELL CULTURE TECHNOLOGY | 3

Cell Culture Products

production. In transforming cell culture products from laboratory discovery to clinical reality, many innovations in the design and engineering of gene constructs, cells, products, and processes have been conceived and implemented. These technologies are also likely to help new technologies move forward. The next generation technology that will benefit most from cell culture innovations is stem cell based therapy. This technology is still in its infancy, but its significant potential impact on our society will compel cell culture technologists to push the evelope.

Virus Vaccines and Protein Therapeutics Cell culture processes have been used to produce viral vaccines for over half a century. Virus production in animals or in tissues has been in practice for over two centuries. The most notable example is the pox vaccine from cow. Most of the tissue-based production methods have since been replaced by cell culture processes. A tissue system that is still in use is the chick egg. This process is begun by seeding a virus into 10-day-old embryos in chicken eggs. A few days later, the replicated virus is then isolated from infected embryos. Early cell culture processes were an extension of tissue culture, using primary cells explanted from various tissues (such as chick embryos and monkey kidneys) for the virus to infect and replicate. The primary cells used in virus production have mostly been replaced by cell strains or even cell lines, which can be cultivated over many generations to build up stocks (or a cell bank) for routine use to ensure consistent quality.

Most viruses used as vaccines have been inactivated by formalin treatment to render the virus incapable of infection. However, the treated virus particles retain a small degree of immunogenicity to elicit the immune response in vaccine applications. There are cases in which live attenuated viruses are used. These attenuated viruses have been adapted,

OVERVIEW OF CELL CULTURE TECHNOLOGY | 4

Table 1. Principal Viral Vaccines Used in Prevention of Human Virus Diseases Disease

Source of vaccine

Condition of virus

Poliomyelitis

Tissue culture (human diploid cell line, monkey kidney)

Live attenuated, inactivated

Measles

Tissue culture (chicken embryo)

Live attenuated

Mumps

Tissue culture (chicken embryo)

Live attenuated

Rubella

Tissue culture (duck embryo, rabbit, or human Live attenuated diploid)

Smallpox (vaccinia)

Lymph from calf or sheep Live vaccinia (glycerolated, lyophilized)

Smallpox (vaccinia)

Chorioallantois, tissue cultures (lyophilized)

Vaccinia

Yellow fever

Tissue cultures and eggs (17D strain)

Live attenuated

Influenza

Highly purified subunit forms of chicken embryo allantoic fluid (formalinized UV irradiated)

Inactivated

Influenza

Cell culture (MDCK, Vero) Attenuated

Rabies

Duck embryo or human diploid cells

Inactivated

Adenovirus

Human diploid cell cultures

Live attenuated

Japanese B encephalitis

Mouse brain (formalinized), cell culture

Inactivated

Venezuelan equine cephalomyelitis

Guinea pig heart cell culture

Live attenuated

Eastern equine

Chicken embryo cell culture

Inactivated

Western equine

Chicken embryo cell culture

Inactivated

Russian spring summer encephalitis

Mouse brain (formalinized)

Inactivated

often by the prolonged cultivation in a non-human host species so that the adapted strain is no longer virulent to humans. These viruses are still capable of replication, which significantly reduces the dose required for immunization. However, they also carry a very low, but non-zero, risk of reverting to their wild type form and causing an infection in the patient.

Vaccine technology predated modern cell culture for recombinant protein production by over two decades. Although recombinant therapeutic proteins propelled the advances in cell culture technology, proteins derived from tissues, and even cell culture, were used for therapeutic purposes even before the arrival of recombinant DNA technology. These examples include insulin, urokinase, Factor VIII, and interferon.

The generation of recombinant DNA therapeutic proteins, such as human growth hormone and insulin, were first produced in microorganisms. The next wave were human proteins, which naturally circulate in human blood and require post-translational modifications, such as complex disulfide-bond formation and glycosylation. These proteins can not be replicated in microbial systems. For the production of those proteins, mammalian cells must be, and were, employed.

Initially, hybridoma cells were used. These are fusion products of the non-antibody-secreting, but continuously proliferating, myeloma cell and the antibody-secreting, but non-dividing, lymphocyte. This soon gave way to recombinant DNA technology. After the introduction of tissue plasminogen activation (tPA) by Genentech in 1987, erythropoietin (EPO) and Factor VIII also reached the market in following years. Antibody products and antibody-based fusion proteins have since blossomed. They make up the bulk of the protein drugs in clinical use.

OVERVIEW OF CELL CULTURE TECHNOLOGY | 5

Table 2. Therapeutic Protein Biologics Produced in Non-Mammalian Host Activity/Use Granulocyte colonystimulating factor (Neupogen)

White blood cell growth for Neutropenia

Insulin (Humulin)

Diabetes

α-Interferon (Intron-A)

Anticancer, viral infections

Somatropin [human growth hormone] (Humatrope)

Growth deficiencies

Somatropin [human growth hormone] (Protopin/ Nutropin)

Growth deficiencies

Interleukin-2 (Proleukin)

Kidney Cancer

Table 3. Non-Antibody Products Produced in Mammalian Cells Trade name

Type

Therapeutic Use

Manufacturer

U.S. approval year

Host

Aldurazyme

Laronidase

Mucopolysaccharid-eosis I

Genzyme

2006

CHO

Cerezyme

β-glucocerebrosidase Gaucher’s disease

Genzyme

1994

CHO

Myozyme Fabrazyme

-galactosidase

Pompe disease

Genzyme

2006

CHO

-galactosidase

Fabry disease

Genzyme

2003

CHO

Naglazyme

N-acetylgalactosamie Mucopolysaccharideosis VI 4-sulfatase

BioMarin Pharmaceutical 2005

CHO

Orencia

Ig-CTLA4 fusion

Rheumatoid arthritis

Bristol-Myers Squibb

2005

CHO

Luveris

Luteinizing hormone

Infertility

Serono

2004

CHO

Activase

Tissue plasminogen activator

Acute myocardial infraction

Genentech

1987

CHO

Epogen/ Procrit

EPO

Anemia

Amgen/Ortho Biotech

1989

CHO

Aranesp

EPO (engineered)

Anemia

Amgen

2001

CHO

Pulmozyme

Deoxyribonuclease I

Cystic fibrosis

Genentech

1993

CHO

Avonex

Interferon-β

Relapsing multiple sclerosis

Biogen Idec

1996

CHO

Rebif

Interferon-β

Relapsing multiple sclerosis

Serono

2002

CHO

Follistim/ Gonal-F

Follicle stimulating hormone

Infertility

Serono/NV Organon

1997

CHO

Benefix

Factor IX

Hemophillia A

Wyeth

2000

CHO

Enbrel

TNF receptor fusion

Rheumatoid arthritis

Amgen, Wyeth

1998

CHO

Tenecteplase

Tissue plasminogen activator (engineered)

Myocardial infraction

Genentech

2000

CHO

ReFacto

Factor VIII

Hemophilia A

Wyeth

2000

CHO

Advate

Factor VIII (engineered)

Hemophilia A

Baxter

2003

CHO

OVERVIEW OF CELL CULTURE TECHNOLOGY | 6

Table 4. Therapeutic Antibody Products Trade name

mAb type

Therapeutic Use

Manufacturer

U.S. approval year

Host

Orthoclone OKT3

Muromomab CD3

Reversal of acute kidney transplant rejection

Johnson & Johnson

1986 Hybridoma

ReoPro

Anti-Abciximab

Prevention of blood clots

Centocor

1994 SP2/0

Rituxan

Anti-CD20 mAb

Non-Hodgkin’s lymphoma

Genentech, Biogen IDEC

1997 CHO

Zenapax (Daclizumab)

Humanized, anti-αsubunit T cell IL-2 receptor

Prevention of acute kidney transplant rejection

Protein Design Labs

1997 NS0

Simulect (Basiliximab)

Chimeric, anti-αchain T cell IL-2 receptor

Prophylaxis of acute organ rejection in allogeneic renal transplantation

Novartis

1998

Synagis (Palivizumab)

Humanized, anti-A antigen of RSV

Prophylaxis of lowerrespiratory-tract disease

MedImmune

1998 CHO

Remicade

Anti-TNF- - mAb

Active Crohn’s disease

Centocor

1998 SP2/0

Herceptin

Anti-HER2 mAb

Metastatic breast cancer

Genentech

1998 CHO

Mylotarg

Anti-CD33

Acute myeloid leukemia

Wyeth

2000 CHO

Campath

Anti-CD52 mAb

Chronic lymphocytic leukemia

Millennium, Berlex, Genzyme

2001 CHO

Zevalin

Anti-CD20 murine mAb

Non-Hodgkins lymphoma

Biogen IDEC

2002 CHO

Humira

Anti-TNF- mAb

Rheumatoid arthritis

Abbott

2002 CHO

Xolair

Humanized, AntiIgE mAb

Moderate/severe asthema

Genentech

2003 CHO

BEXXAR

Anti- CD20 mAb

Follicular non-Hodgkins lymphoma

GSK

2003 CHO

Raptiva

Anti-CD11a mAb

Chronic psoriasis

Genentech

2003 CHO

Erbitux

Chimeric antibody raised against human EGF receptor

EGF receptor–expressing metastatic colorectal cancer

Imclone Systems, Bristol-Myers Squibb, Merck

2004 CHO

Avastin

Anti-VEGF

Metastatic colorectal cancer and lung cancer

Genetech

2004 CHO

Soliris

Antibody binding to C5

Paroxysmal nocturnal hemoglobinuria

Alexion

2007 NS0

Vectibix

Anti-EGFR mAb

Metastatic colorectal cancer

Amgen

2006 CHO

Protein Molecule as Therapeutics

The early generation of protein therapeutics consisted of all molecules native to humans. Many antibody molecules retained part of the sequence of the immunized species (e.g., mouse or rabbit), although later generations of antibody molecules were all humanized or were human antibodies. Some products are engineered molecules with altered amino acid sequences that enhance their drug characteristics.

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Table 5. Industrial Cell Lines Major Cell Strains and Lines for Human Biologics Production Human Vaccines Primary Cells

Green monkey kidney cells (no longer used) Chicken embryo cells

Cell strains

MRC5 (human lung fibroblast)

Cell line

Vero (monkey kidney epithelial cell), MDCK

Recombinant Proteins Species cell line derived from Human

HEK 293, Per C6

Mouse

C-127, NSO, hybridoma cells, SP2/0

Chinese Hamster

CHO

Syrian hamster

BHK

Subsequent products entail fusion proteins, in which domains (or fragments) of different human protein molecules are joined. A prominent example is the fusion molecule of the Fc fragment of IgG and the TNFα binding fragment of the TNFα receptor. This molecule was developed by then Immunex (now Amgen) for inhibition of TNFα to suppress its inflammatory effect. Another class of product entails completely foreign proteins, such as recombinant protein or designer proteins, which have enhanced potency for eliciting an immune response.

Table 6. Cell Lines Used in the Production of Veterinarian Vaccines* Vaccines

Cell line

Bovine viral diarrhea virus

MDBK

Bovine parainfluenza virus type 3

MDBK

Bovine rhinotracheitis virus

MDBK

Bovine respiratory syncytial virus

MDBK

Feline leukemia virus

FL72

Feline panleukopenia virus

CRFK

Feline chlamydia

CRFK

Canine parvovirus

CRFK

Canine distemper

Vero

Canine adenovirus type 2

Vero

Ehrlichia canis

DH82

Rabies

BHK-21

Eastern equine encephalitis virus

Vero

Western equine encephalitis virus

Vero

Equine rotavirus

MA104

Equine rhinopneumonitis virus type 1 and 4 Equine Dermal Equine influenza virus

MDCK

Foot and mouth disease virus

BHK-21

Swine parvovirus

ST, PK

Swine influenza virus

MDCK

*This table was provided by Terry Ng, 2001. Organisms in italics are intracellular parasitic bacteria.

OVERVIEW OF CELL CULTURE TECHNOLOGY | 8

Industrial Cell Lines

For the production of traditional viral vaccines, human diploid cell strains are the primary production vehicle. Viral products differ from protein products, in that the viral genome, along with the entire virus particle, is injected into the patient to elicit a response. Even though the virus particle is inactivated by formalin or other treatment, there is still a potential risk of recombination between the virus genome and the host cell genome that may result in the transmission of activated oncogenic or foreign genetic elements to the patient. Therefore, the vast majority of virus vaccines are still produced in normal diploid human cells. Vero and MDCK cells (along with chick embryos) are notable exceptions of non-human continuous cell lines used for human vaccine production. For veterinary vaccines, the selection of host cells is vastly wider. Both cell lines and tissue-derived cell strains with limited life spans are widely used. For the production of recombinant therapeutic proteins, the cell lines that are primarily used are of rodent origin and include mouse, chinese hamster, and syrian hamster cells. Human cells are only used for a handful of products. The vast majority is produced using chinese hamster ovary (CHO) cells.

Biosimilars or Follow-on-Biologics

Two decades after the introduction of mammalian cell-based therapeutic proteins, many of those medicines’ patents have expired. A number of commercially successful therapeutic proteins will go off patent between 2013 and 2017, including the blockbuster drugs Remicade and Humira. These prospects certainly have helped to draw in investments to follow-on biologics. Generic versions of those protein therapeutics have begun to reach patients throughout the world. The terms “biosimilar” or “follow-on biologic” refer to products that are marketed after the expiration of patents. They are expected to have similar properties to existing biologic products. Sandoz was the first company to launch a biosimilar-human growth hormone,

OVERVIEW OF CELL CULTURE TECHNOLOGY | 9

Omnitrope, in both Europe and the United States.

Follow-on biologics differ from traditional generic drugs, in that their biological activity, or the efficacy of their active ingredient, is not as easily defined as the traditional chemical and natural product drugs. Traditional drugs, like penicillin and statins, have very clearly defined chemical structures that also confer their biochemical activities. Protein therapeutics, on the other hand, cannot be entirely characterized by their chemical composition, or primary sequences. Therefore, their biological equivalency to their patented and branded counterparts cannot be established simply by structural similarity or identity.

Table 7. Approved Biosimilars in the EU Generic Name

Product

Launch

Recombinant human EPO-α

Medice Arzneimittel Putter (Germany)

2007

Binocrit

Recombinant human EPO-α

Sandoz (Austria)

2007

Epoetin alfa Hexal

Recombinant human EPO-α

Hexal Biotech (Germany)

2007

Retacrit

Hospira Enterprises

2007

Silapo

STADA Arzneimittel (Germany)

2007

Somatropin growth hormone

Sandoz (Austria)

2006

Somatropin growth hormone

Biopartners (Germany)

2006

Valtropin

Table 8. Marketed Biosimilars in India Company

Brand Name

Biosimilar

Launch

Ranbaxy

Ceriton

Epoetin

2003

Dr Reddy’s

Grastim

G-CSF

2001

Reditux

MabThera

2007

Wosulin

Insulin

2003

Wepox

Epoetin

2001

Biovac-B

Hepatitis B

2000

Insurgen

Insulin

2004

BioMab-EGFR

MabThera

2006

Recosulin

Epoetin

2004

Epofit, Erykine

Epoetin

2005

Neukine

G-CSF

2004

Shanpoietin

Epoetin

2005

Shanferon

IFN α 2b

2002

Shankinase

Strptokinase

2004

Shanvac B

Hepatitis B

1997

Wockhardt

Biocon

Intas Pharmaceuticals Shantha Biotechnics

The status of molecular folding, glycan composition, etc. may affect their activity profoundly. The particular host cell line that is used, as well as the production process, may affect subtle aspects of the protein’s properties, thus posing a greater uncertainty about the “quality” of the product produced by manufacturers of those follow-ons. While a biosimilar’s approval pathway has been established in Europe, the U.S. has yet to lay down any guidelines.

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Table 9. Marketed Biosimilars in China Company

Biosimilars

Dragon Pharmaceuticals

Epoetin, filgrastim

Dongbao

Insulin, G-CSF

Anhui Anke Biotechnology

HGH, interferon alpha

Amyotop

G-CSF, IL-11

GeneLeuk Biotech

G-CSF, PEG filgrastim, interferon

HangzhouJiuyan Gene

G-CSF, IL-11

Manufacturing Table 10. Dose of Some Antibody Product Approximate Formulation Configuration

Product

Disease Indication

Company

Amevive

Psoriasis

Biogen

7.5mg / 0.5ml; 15mg / 0.5ml

Enbrel

RA

Amgen

25mg

Heceptin

Breast Cancer

Genentech

440mg / 30cc

Humira

Rheumatoid arthritis

Abbott

40mg (1ml prefilled syringe)

Remicade

Crohn’s disease, RA

Johnson & Johnson, Centocor

100 mg / 20cc

Rituxan

NHL

Genetech/Idec

100mg / 10cc; 500mg / 50cc

Synagis

Respiratory syncytial virus

MedImmune

100mg

Xolair

Allergic Asthma

Genetech/ Tanox/Novatis

150mg / 5cc

Viral vaccines are administered to patients in relatively minute quantities because a small amount of antigen proteins is sufficient to elicit immune response. Cytokine, growth factors, or enzymetypes of proteins (such as EPO, human growth hormone or tPA) are also given in small doses, in terms of protein quantity. Depending on the market size, the production facility of these products may be relatively small. The biological effect of antibody products is largely based on their binding to antigen; this event requires antibody and antigen molecules to be in some stoichiometric ratio to elicit downstream target killing or neutralization. Antibodies are large molecules, as are many antibody-based fusion proteins. Thus, many antibody products are administered in relatively high doses. Thus, the product vessels, and the size of the manufacturing plant for antibody products, tend to be larger.

The manufacturing process of protein therapeutics is rather similar to that for traditional biochemical, such as antibiotics and E. coli-based recombinant proteins. A typical process entails a couple of seed expansion reactor cultures before reaching the production reactor. The process cycle tends to be longer. Many cell culture manufacturing processes are operated in fed-batch modes that last ten to fifteen days. Some are operated as continuous perfusion processes and last from two to six months. The recovery process of cell culture products is simpler than that for bacterial-based recombinant

OVERVIEW OF CELL CULTURE TECHNOLOGY | 11

Manufacturing Plants •

Genentech’s Vacaville Facility, California • Started construction in 2004, started operation in 2009. Currently inoperative due to capacity reasons • Investment: $800 million • Eight 25,000-liter bioreactor • Production of Herceptin, Avastin and Rituxan • Bristol Myer Squibb, Devens, Masschusetts • Started construction in 2007, validation in 2011 • Investment: $750 million • Six 20,000- liter bioreactors, one purification strain • Production of Orencia and other biologics • Biogen IDEC LSM Facility • 245,000 ft2 production • Multi-product facility • Six 15,000L production reactor capacity

proteins. The vast majority of processes now employ a medium with a relatively low concentration of proteins, to ease the purification operation. With the high product concentrations in the range of 5 – 10 g/L, the product molecule should be the predominant protein in the medium at the end of cell culture process. The product isolation and purification process is substantially simpler than separating intracellular protein products.

Fig. 2.2: Flow chart of a typical recombinant antibody production process

Alternative Technologies Other host cells used for biopharmaceutical production include E.coli and Sacchromyces cerevisiae. Alternative production systems include: • Insect cell culture • Yeast ( Pichia ) • Transgenic animals • Transgenic plants

Mammalian cells, especially CHO and myeloma cells such as NS0 and SP1/0, have been the workhorse for the production of protein therapeutics that require post-translational modifications (e.g., glycosylation, γ-carboxylation, etc). Although those post-translational modifications cannot be carried out in bacterial systems (primarily E. coli), there are a number of host systems that are capable of performing N- and O-glycosylation and other posttranslational modifications. They have been explored as the production vehicles of therapeutic proteins.

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Insect Cell Culture Table 11. Insect Cell Culture Application

Comments

Basic research

Hundreds of genes have been expressed using baculovirus.

Bioproduction

Using baculovirus expression systems.

Gene therapy

BV may be used as the gene-delivery vehicle.

Bioreagent production

A number of bioreagent suppliers use BV to make target proteins, viral components and other compounds for the research market.

Yeast

Table 12. Product

Company

Use

Status

Blood expander

On the market in Japan

Medway (recombinant human serum albumin)

Mitsubishi Tanabe Pharma Corporation, Osaka, Japan

Hepatitis B vaccine

Shantha Hepatitis B Biotechnics Ltd., India

On the market in India

Interferonalpha

Shantha Hepatitis C/ Biotechnics Ltd., Cancer India

On the market in India

DX-88

Dyax Corporation, Cambridge, Mass.

Hereditary angioedema (HAE), a debilitating condition characterized by acute attacks of inflammation.

BLA submitted

Recombinant Human Insulin

Biocon, India

Diabetes, all types

On the market in India

Recombinant collagen

Fibrogen Inc., South San Francisco

Medical research reagents and dermal filler

On the market

Botulism vaccine

USAMRIID/ DynPort

Botulism vaccine product

Phase I (U.S.)

Antithrombolytic

ThromboGenics Ltd.

Thrombosis Tx

Phase II

Insect cells were explored as a production vehicle for therapeutic proteins. The glycoforms of the proteins produced in insect systems are rather different from those produced in mammals. Overall, such efforts have largely subsided. However, for other applications, such as protein production for toxicity studies and for veterinary vaccine production, the insect cell culture remains attractive because the cultivation is relatively straightforward and the process development time can be relatively short. The yeast in the genus Pichia is capable of synthesizing N-glycans that are not the mannoserich types produced in Saccharomyces. They have been used in the production of recombinant proteins, including serum albumin. Advances have been made in ‘humanizing’ the glycosylation characteristics in Pichia systems for the production of therapeutic proteins. Glycofi (Merck) has worked towards a multistep genetic engineering process where non-human glycosylation enzymes were first eliminated and human glycosylation reactions were then introduced. A titer of ~ 1.4 g/L of recombinant proteins has been reported. With further improved secretion capacities and glycosylation patterns, these engineered yeast strains may be capable of producing proteins with consistent glycosylation patterns, or even with uniform glycans.

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Transgenic organisms for the production of biotherapeutics have been in development for two decades. These production systems require a low initial capital investment and have a relatively easy purification process for glycosylated products. However, so far, the FDA has approved only one product, ATryn, which is produced in transgenic goat’s milk by GTC Biotherapeutics.

Transgenic Animals

An advantage of transgenic animal production is its high titer in milk, on the order of 2 – 10 g/L. However, over the years, the titer in cell culture processes has also increased to 5 – 10 g/L range, thereby diminishing this particular advantage of transgenic animal production.

Table 13. Transgenic Animal Products Approved or Under Development Species

Company

Product

Status

Comments

Goat

GTC Biotherapeutics, MA

ATryn- recombinant human antithrombin-alpha

Approved

Glycosylation patterns differ slightly ( involves N-glycolylneuramic acid- not seen in humans), but was not a regulatory hurdle; Predicted sales of $6-$10 million in 2009.

Goat

PharmAthene, MD

Protexia- recombinant human butyrylcholinesterase (BChE)

Development

Rabbit

Pharming, Netherlands

Rhucin-Recombinant human C1 esterase inhibitor

Phase 3 trials For the treatment of hereditary angiodema.

mAb

Pilot Studies

Chickens Origen Therapeutics, (eggs) Medarex Inc., CA

Functional Mabs produced at 3 mg/egg; some differences in glycosylation; Half life in mouse serum half that of natural antibodies (reduced from 200-100h)

Product Quality and Process Robustness Critical Feature of rDNA Proteins from Mammalian Cells • Folding and disulfide bond • Glycosylation • N or O - glycosylation • Sulfation or phosphorylation of glycans • Affect solubility, clearance and biological activities • Other post-translational modifications • Y-carbonxylation • Lipidation • Phosphorylation

In spite of its dominance as the production vehicle for therapeutic proteins, the mammalian cell system does have some shortcomings in its process characteristics. Compared to microbial systems, mammalian cell systems have a slow growth rate and a relatively low achievable cell concentration. The product titer is also substantially lower than that of extracellular protein produced using fungal systems. Finally, the optimal range of growth environments for mammalian cells is much narrower than the range for either plant or microbial systems. After years of research effort, the low productivity that used to be associated with the low cell and product concentrations has largely been overcome.

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Tissue Plasminogen Activator (tPA)1 • Single polypeptide chain (70 kDa) or proteolytically cleaved at ARG276.

• Multiple N-linked carbohydrates: ASN117 (high mannose), ASN184 (50% complex multiantenary, 50% unoccupied), THR61 (O linked fucose). • Contains 35 cysteine residues, 17 pairs of disulfide bonds. CYS83 can form a disulfide with other free thiols depending upon the growth medium and buffer composition. • May form high molecular weight aggregate (complexes with protease inhibitors) and proteolytically cleaved tPA.

Erythropoietin

• Contains 40% carbohydrate, only 2 disulfide bonds. • 3 N-linked ASN (24,38,83), 1 O linked (SER126) glycosylation sites. • O-linked site not essential for in vitro or in vivo activity. • Sialic acid residues (average 10 moles/mole Epo) responsible for preserving pharmacokinetic behavior. Muteins lacking 2 or 3 N-linked sites are poorly secreted. • N linked glycosylation and sialylation is critical to optimal secretion, structure, in vivo potency.

Through cell adaptation and media development, the complex nutritional requirements for mammalian cell growth have been greatly simplified. Now, the relatively low tolerance of mammalian cells to their chemical and physical environment has not prevented highly stressed conditions from being used in the final production stage. What has been lagging is our ability to control the quality, notably the glycosylation profile, of the product.

The mammalian cell system is chosen for protein production, almost invariably for its capability of posttranslational modifications on the product (such as the formation of multiple disulfide bonds of tPA and the glycosylation of Factor VIII and EPO). Major posttranslational modifications commonly seen in protein therapeutics, such as disulfide bond formation, N- and O-glycosylation, and phosphorylation, all involve extensive enzymatic reactions in the endoplasmic reticulum or in the Golgi apparatus. The level of those enzymes, as well as the supply of precursors and co-factors, affects the outcome of those reactions. The enzyme levels vary with cell clone and growth stage, while the supply of precursors and cofactors change with the chemical environment. These variations cause fluctuations in the glycans attached to N(asparagin) sites or to O- (serine or tyosine) sites.

For a given glycoprotein, regardless of whether it is produced in culture or present in circulation, the glycans attached to different molecules are not identical. Rather, they are a mixture of different, but related, forms. In fact, most glycoproteins in blood circulation also have hetergeneous glycans. The structure of glycan and the extent of glycosylation on the protein molecule affect the blood circulation halflife of the protein. In some cases, the glycan structure even affects the protein’s biological functions. Thus, confining glycan distribution to an acceptable range is important for the quality control of the product. The glycosylation pathway is long and complex, and takes place in multiple compartments in the cell. Producing a glycoprotein product with a defined range of glycan structures throughout

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Table 14. In Process Structural Alternations to Mammalian Protein Biologics Glycoform Site occupancy Altered sialic acid content Uncapped galactosyl residue, High mannose

Possibly caused by stochasticity of glycosylation process

Glycan distribution out of range Amino acid alterations in protein Error rate of amino acid incorporation during translation (1/1000)

Mis-incorporation (codon misreading) Deamidation (Asparagine) Loss of terminal amino acid • lysine in C-terminus of heavy chain IgG, enzymatic cleavage • cyclization of N terminus glutamine

Most likely occurred in culture fluid, may be affected by process conditions, or even product titer

Glycation (addition of reducing sugar (glucose) to amino acids) Protein aggregation

May be caused by folding in ER or agglomeration in culture or in down stream processing

a product’s life cycle is still

a challenge.

Glycosylation may affect the folding of the protein molecule, but it does not affect its structure. Other post-translational modifications may affect protein structure. Failure to form a disulfide bond or the mispairing of a disulfide bond both give rise to an altered protein structure or the improper cross-linking (multimer formation) among different molecules. A lack of γ-carboxylation or phosphorylation also drastically changes a protein’s properties.

Errors in protein synthesis caused by amino acid misincorporation have been reported. Many production cell lines have multiple copies of the product gene; a non-silent mutation (i.e., a mutation causing a change of an amino acid in the protein) in one of those genes will inevitably result in the presence of some fraction of mutated protein molecules. An alteration of the amino acid structure may also result from chemical modifications after being secreted into the medium. After being secreted into culture medium, the product protein molecules are also subjected to modification by enzymes released by cells, which are either actively secreted or released from lysed cells. Extracellular proteolytic cleavage can give rise to degradation of Factor VIII, or can alter the ratio of single chain/ double chain molecules of tPA and Protein C. Also, the sialic acid moiety in glycans may be cleaved by sialidase released from lysed cells. Table 14 summarizes some more commonly-seen alterations in protein molecules in cell culture processes.

In the past decade we have seen the productivity of recombinant cells reaching or even exceeding the production rate of professional secretors in our body (such as liver cells or antibody- and insulinsecreting cells). We have also seen the product titer in the bioreactor approaching the concentrations of antibody in ascites fluid. As the productivity and product concentration of cell culture processes approaching its “natural” biological counter parts, we must also be cautious and ask ourselves whther we are pushing cell’s protein folding and processing machinery to operate at its limit.

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The unprecedented high productivity is achieved by operating the reactor at conditions that are neither optimal for growth nor the natural homeostatic state. Rather, they are often highly stressed to favor producing the product of our design. In today’s production cultures, both the intracellular and the extracellular environments are extremely harsh. While protein synthesis and secretion is boosted to a nearly unprecedented level, the cellular machinery for protein quality control may not be operated at the same level of stringency as it is for optimal growth. Process technologists must bear in mind that quality consistency and process robustness must be the highest priority when pushing productivity higher.

Concluding Remarks Over half a century, cell culture processes have evolved from tissue and small-scale cell culture for vaccine production to large scale manufacturing process for protein production. Therapeutic proteins, especially antibody and antibodybased proteins, are the dominant products. The continuing pressure to meet increasing demand for products has led to many process innovations and refinements over the past two decades. The cell and product concentrations in today’s process are nearly two orders of magnitude higher than they were at the dawn of the recombinant protein era. The success of this technology has also shifted the focus from production quantity to product quality.

Cell culture processes now aim to provide optimal growth conditions for cell expansion, while often employing highly-stressed conditions for the final production stage. All must be accomplished without compromising the quality of product produced. Achieving those aims through process innovation will be critical in the next phase of the technology, wherein follow-ons or biosimilars will have an increasing presence. Cell culture engineering efforts in the past quarter century have transformed bioprocess technology. The advances made in cell culture technology will greatly facilitate the development of the emerging stem cell and other cell therapy.

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Cell Biology for Bioprocessing Cells: Source, Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Cell Source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Cell Composition and Chemical Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Cell Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Cytoplasm and Organelles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Transport Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Major Mechanism of Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Extracellular Matrices and Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Movement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Growth, Death and Senescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Cell Cycle and Growth Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Senescence and Telomeres. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Cells: Source, Composition and Structure Cell Source Table 1. Cells Commonly Used in Bioprocessing Species W1 - 38 MRC - 5 FS - 4 HEK 293 Vero MDCK NS/SP2/0 CHO BHK

Human Human Human Human Monkey Dog Mouse Chinese Hamster Syrian Hamster

Fibroblast Fibroblast Fibroblast Epithelial Epithelial Epithelial Lymphoid

Tissue Isolated Lung Lung Foreskin Kidney Kidney Kidney Myeloma

Epithelial

Ovary

Epithelial

Kidney

Cell Type

The cells commonly used for the production of biologics are derived from different tissues of different species. Thus, they can vary widely at the genomic level. Their differences are even visible microscopically, with various numbers of chromosomes. However, at a physiological and transcriptome level, cells from the same tissue of different species are strikingly similar. Their similarity is much greater than different cell types from the same animal. For example, chicken embryo fibroblasts look morphologically very similar to human fibroblasts from the lung or foreskin, while the epithelial MDCK cells look rather different from dog fibroblasts even though they are both derived from the same species.

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• Among the ~200 different types of cells, fibroblasts, epithelial cells and myeloma cells are most frequently used cell types in biologics production • Cells in culture bare closer physiological and morphological characteristics of the tissue they were derived from than the species • Cells in vivo may be in a quiescent state or in a proliferative state, but are all adapted to rapid proliferation in culture

Even though there are about two hundred types of cells in a vertebrate animal, most cells that are used for the production of biologics are either epithelial or fibroblast in nature. These two cell types are more amenable to isolation from tissues and to in vitro culture, as demonstrated during the early explorations on tissue cell isolation more than half a century ago. NSO and CHO are the two prominent host cell lines used for therapeutic recombinant protein production. They exhibit different behaviors and were derived from two different tissues and two different species. CHO cells were isolated from the ovary of a Chinese hamster; NSO cells were isolated from a mouse myeloma. Cells used for recombinant protein production are primarily epithelial and lymphatic.

Both fibroblasts and epithelial cells are frequently used for viral vaccine production. These cells differ in both their functions and tissue locations. Epithelial cells line the “boundary” of tissues, while fibroblasts make up a larger part of the connective tissue. Epithelial cells form tightly connected sheets, which often get damaged, die, and are replenished by “new” ones. Thus, many of them are constantly growing in vivo. Conversely, fibroblasts are mostly quiescent. They migrate into wounds and begin to grow only when they are stimulated by various cues. Lymphatic cells, especially the terminally-differentiated plasma cells (from which myeloma cells are derived), are needed to secrete antibodies against a particular antigen, but only for a limited period of time after the host’s exposure to the antigen. They undergo apoptosis days after their differentiation into active antibody-secreting cells, so that the host does not continue to have unnecessary or maybe even harmful antibody molecules in circulation. Such native characteristics are often still evident in culture.

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Cell Composition and Chemical Environment Table 2. Typical Composition of a Cell E. coli

Mammalian Cell pg / Cell Wet weight Dry weight Protein Carbohydrate Lipid DNA RNA Water

Range

3,000

3,000 ‑ 8,000

600 250 150 120 10 25

300 ‑ 1,200 200 ‑ 300 40 ‑ 200 100 ‑ 200 8 ‑ 17 20 ‑ 40

Volume

4 x 10‑9 cm3

Diameter

18 μm

%

%

10‑20 1‑5 1‑2 0.3 0.7 80 ‑ 85

15 2 2 1 6 70

0.5-2 μm

Table 3. Cellular and Extracellular Fluids Ion Concentration Plasma (mmole / l) Interstitial (mmole / l)

Intracellular osmolality (mmole / l)

140

14

K+

4

4

140

Ca++

1

1

10-4

Mg++

0.8

0.7

20

Cl

110

110

50

Na+

-

• >10 fold concentation difference for K+ and Na+ across the plasma membrane • Opposite direction of concentration gradient for K+ and Na+ • Extremely low concentration of Mg++ in intracellular fluid • Total osmolality ̴ 280 mOsm

Most cells in culture have a diameter of about 12 – 18 µm. Some types of stem cells are rather small and have only a small amount of cytoplasm. In contrast, liver cells (i.e., hepatocytes) in some species are rather large, with an average cellular diameter of 20 µm. A typical cell has nearly 80% of its mass as water. Proteins make up the next largest portion of cell mass, after water.

Other than water and proteins, the other cellular constituents are present in much smaller amounts and rarely exceed 10% of the total dry mass. Lipids make up various membranes of the cell, including the cytoplasmic membrane and the membrane enclosing all organelles. Lipids, thus, constitute a significant portion (about 5-8% of total dry mass) of cell mass.

Carbohydrates (such as glycogen) are used to store energy in some cells. However, not all cells have a large amount of free carbohydrates. Carbohydrate molecules that serve as energy sources are quickly metabolized to become intermediates in energy metabolism. Most carbohydrate moieties that remain in their carbohydrate forms exist as part of nucleotides or are conjugated to proteins or lipids. The size of a haploid genome in a typical mammalian cell is about 3 Gbp. That equates to about 5 pg of DNA for a diploid cell. However, DNA is not the most abundant nucleic acid in the cell. RNAs are far more abundant than DNA in a cell and include messenger RNA (mRNA), ribosomal RNA (rRNA), and others. Ribosomal RNA, which is a major constituent of the cell’s protein synthesis machinery, constitutes over 90% of all RNA in the cell.

Since water constitutes the largest fraction of all cell materials, the chemical species that are present at a high concentration in the cytosol are also major cellular constituents. Combined, all minerals contribute a significant (~5%) proportion of the dry mass.

The concentrations of some ions are vastly different inside the cell versus outside the cell. Maintaining these concentration gradients is critical for cell functions. The concentration ratio between intracellular and extracellular K+ and Na+ is in the range of 15 to

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30. Conversely, their direction of concentration gradient is opposite: the concentration of K+ and Na+ should be far higher inside and outside the cell, respectively. The solutes in a solution exert osmotic pressure, which is typically quantified by osmolality. The osmolality of cellular fluid is about 280 mM (or mOsm). A typical medium has its osmolality at the same level as in cells, to avoid incurring osmotic stress.

Cell Membrane Lipid Bilayer Composition Phospholipids • Constitute the majority (35-70%) Glycolipids • Neutral glycolipids (e.g. galactocerebroside) Gangliosides • Have sialic acids Four types of phospholipids • Three have glycerol as backbone,Phosphotidyl ethanol amine, Phosphotidyl serine and Phosphotidyl choline

• Serine as backbone

Cultured mammalian cells have long been thought as being extremely fragile to mechanical stresses because their cellular materials are surrounded only by cytoplasmic membrane; the only thing preventing the cellular content from dissolving into the aqueous environment is that lipid bilayer. Yet in a modern manufacturing plant, these tiny cells thrive in bioreactors of tens of cubic meters in volume under such highly turbulent conditions. The membrane surrounding a cell is not merely a double-layer of lipids, and the integrity of a cell is not merely dictated by its membrane wrapping. The lipids which make up the lipid bilayer are amphipathic. They have a hydrophilic head group, and a hydrophobic tail group made of fatty acids. When suspended in an aqueous solution, amphipathic molecules can form micelles. In such micelles, the hydrophilic.

Fig.2.1: A phospholipid molecule with glycerol as backbone, with an ethanol amine, a saturated and an unsaturatted fatty acid.

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Lipid Bilayer

Characteristics of a Lipid Bilayer • The lipid bilayer is a fluid • As temperature decreases, the bilayer transitions from a fluid state to a gel state • The degree of fatty acid “unsaturation” affects the transition temperature of membrane from a fluid state to a gel state • The magnitude of diffusion of various solutes in the cell membrane resembles that of a liquid

A lipid bilayer membrane behaves like a fluid. If the lipid molecules in a specific location are labeled with a fluorescent dye, the fluorescence disperses shortly thereafter due to molecular diffusion (instead of staying in the same place as in a solid). The lateral diffusion coefficient of a phospholipid molecule in a bilayer membrane is about 10-8 cm2/s. A lipid molecule does not flip-flop (or change its side of a lipid bilayer) without the aid of membrane-bound phospholipid translocator. Gas species diffuse about equally fast in a lipid bilayer as they do in water. Even large protein molecules diffuse in a lipid bilayer membrane.

Fatty acids make up the hydrophobic tail. At very mild temperatures these acids undergo a phase transition from a fluid to an ordered structure. Thus, lipid bilayers also undergo phase transition to form a “liquid crystal” at a relatively moderate temperature. This tightly-packed, ordered structure acts as a very good barrier to keep most molecules from freely passing in or out of the cell. The permeability of most biological molecules across a lipid bilayer membrane is rather low. Even the smallest nutrient, such as glucose and simple amino acids, cannot pass by fast enough to support cell growth.

Fig. 2.2: Lipid bilayer membrane at a crystaline state and fluid state

All major biological macromolecules (e.g., DNA, proteins, and polysaccharides) are biopolymers made of covalently-bonded monomers. A lipid bilayer membrane is a not a polymer, rather, it is an assembly of phospholipids. The non-covalent nature of phospholipids within the cell membrane allows it to be very dynamic: expanding, shrinking, breaking, and fusing rapidly. The lipid bilayer also envelops various organelles to compartmentalize regions in the cell for specialized functions. Many of those organelles are in a constant dynamic process of membrane budding and fusion. For example, in trafficking between organelles and in protein secretion, the “cargo” is carried inside membrane vesicles while transiting from one organelle to another. This process occurs without the need to break up and re-form a larger number of covalent bonds. Three types of lipids make up a lipid bilayer membranes in cells and organelles: phospholipids,

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Characteristics: • One saturated, one cis-unsaturated (C14-C24)

typically constitute the tail of the phospholipid.

• Fatty Acids (the tail group) on the lipid affect the packing of lipids in bilayer membrane. Saturated fatty acids allow more dense packing; double bonds in unsaturated fatty acids creates kinks, reduce packing, increase fluidity. • Cholesterol has a small head polar group linked to a rigid planar region of steroid rings followed by a more flexible non-polar tail. They interact with phospholipids to stabilize the region closer to the head group as well as to make the lipid bilayer less inclined to become crystalline. Overall, they increase the membrane permeability to small compounds, and make the membrane less fluid. • Depending on temperature and the degree of hydration, lipid bilayer is in gel state or in liquid crystalline state. The temperature of bilayer phase transition from the crystalline lipid bilayers to fluid bilayers is affected by fatty acid and cholesterol composition.

Cholesterol in a Lipid Bilayer

Fig. 2.3: Schematic drawing of a cholesterol molecule interacting with two phospholipid molecules in one leaflet of a lipid bilayer

glycolipids, and gangliosides (phospholipids being the most common). There are also different types of phospholipids, with either glycerol or serine as the backbone, with the former being the most abundant type. Glycerol has three hydroxyl groups attached to its three carbons and one of them has a phosphate group, to which an ethanolamine or serine is attached. The phosphate moiety has a strong negative charge, thus making this end of the molecule the highly hydrophilic head group.

The other two hydroxyl groups of glycerol are linked to two fatty acids through an ester bond. Typically, one of those two fatty acids is saturated and the other is unsaturated, with a cis double bond in-between C14 and C24. The degree of unsaturation affects the packing of the lipid bilayer. Saturated fatty acids allow more dense packing, while the double bonds in the unsaturated fatty acids create kinks, which reduce packing and increase the membrane fluidity.

A lipid bilayer can be in a gel state or in a liquid crystalline state depending on the temperature and degree of hydration. A lipid bilayer’s phase transition temperature is affected by its composition of fatty acid and cholesterol. As temperature decreases, the lipid bilayer changes from a liquidcrystalline state to crystalline (or gel) state. A higher content of shorter, unsaturated fatty acids increases the fluidity of the lipid bilayer and decreases its phase transition temperature. Another molecule playing a key role in the membrane properties of animal cells is cholesterol. Cholesterol has a small polar head group linked to a rigid planar region of steroid rings that are further linked to a more flexible non-polar tail. Cholesterol interacts with phospholipids to stabilize the region closer to the head group and to make the lipid bilayer less inclined to become crystalline. Overall, cholesterol increases the membrane permeability to small compounds and makes the membrane less fluid. Cholesterol content varies in different lipid bilayer membranes. Its level in the cytoplasmic membrane is higher, but

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Membrane Proteins

in the membrane of many organelles it is very low.

• A typical biological membrane has ~50% proteins by mass; in terms of molecules, lipid:protein = 50:1 • Metabolically active mitochondrion has 75% protein in its membrane. • Na+/K+ ATPase acts as a pump, using ATP to pump 3Na+ out and 2K+ into the cell. • The electric protential across the plasma membrane is about -80mV.

Table 4. Biochemical Composition of Hepatocyte Plasma Membrane Total Lipids

Total Protein

Protein/ Lipid mass ratio

Cholesterol/ Phospholipid molar ratio

Cholesterol in total lipids

30-40% 50 -60% 1-2 0.4 - 0.8 12 - 20% (by (by mass) mass) Adapted from The Liver: Biology & Pathology, 4th Ed., p. 78 (2001)

Phospholipids in total lipids 50 - 70%

A typical biological membrane has ~50% lipids and ~50% proteins, by mass. In terms of molecules, however, the lipid:protein ratio is actually about 50:1, since proteins have much higher molecular weights than lipids. The protein content of a membrane is greatly affected by the tissue of origin and by the membrane’s function in the cell. The mitochondrial membrane, through which many molecules (e.g., amino acids, pyruvate, various ions and many other proteins) pass at a high flux, has a high protein content of about 75%, by mass. On the other hand, the myelin membrane, which serves as a protective sheath between the nerve cell and its surroundings, has a low protein content of about 25%. Lipid bilayer membranes separate cellular content from their surroundings and divide the organelles from the cytosol. Not only do they create a barrier for the physical retention of a cell’s contents, but they create a rather different chemical environment across membranes. For example, cells maintain about an 80 mV electric potential across the plasma membrane and about 140 mV across the mitochondrial membrane. The ER membrane separates an oxidative environment (inside the ER) from a reduced one (in the cytosol).

The maintenance of various chemical, electrical, and redox potentials across a membrane is accomplished by various membrane proteins. Rat small intestinal enterocyte has about 150,000 Na+ pumps per cell, which collectively allow each cell to transport about 4.5 billion Na+ ions out of the cell, each minute. The sodium and potassium membrane gradients generated by those pumps, as well as the electric potential across cytoplasmic and mitochondrial membranes, are fundamental to cellular bioenergetics.

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Membrane Dynamics

Cellular membrane is in a dynamic state contributed by: • Lipid turn-over • Inter-organelle shift of membrane vesicles • Secretion, endocytosis

Homeostasis of cellular membranes • Professional secretory cells in the body can add 0.5% per minute of their plasma membrane due to the fusion of secretory vesicles with the plasma membrane; they must be recycled to maintain a balance • Phospholipids in the membrane are subject to turnover

The cellular membrane is in a dynamic state; membrane constituents are continuously being added and removed. This is not only for membrane expansion and cell growth, but also for turnover and for vesicle trafficking. Like other cellular components, the turnover of the cellular membrane is necessary to replace lipid molecules that have been oxidized or damaged, or to allow cells to change their membrane composition to adapt to new environments. The turnover rate of a cell membrane varies widely. Phospholipids are said to have a half-life of three hours, while the half-life of cholesterol is about two hours. Cellular membrane proteins are also turned over. Their half-life ranges from a couple of minutes to a couple of days, whereas macrophage membrane proteins are turned extremely rapidly.

Inter-organelle trafficking and the secretion of proteins into the extracellular environment also contribute to a membrane’s dynamic state. Protein molecules that are destined for export are carried from organelles to the cytoplasmic membrane by vesicles. Upon reaching the inner surface of the cytoplasmic membrane, those vesicles fuse with the cytoplasmic membrane and release their contents outside of the cell.

In the liver, each hepatocyte synthesizes ~120 x 103 albumin molecules per min (translating to about 15 pg/cell/day). All of those molecules are wrapped in 280 – 400 nm of vesicles and delivered to the basal plasma membrane of the cell. The infusion of those membrane vesicles would cause the membrane surface to expand at a rate of 0.5%/min. However, since hepatocytes are typically in a G0 state (i.e., not dividing), the size of their cytoplasmic membrane does not need to increase to accommodate cell growth. Therefore, the lipid molecules that are added to the cytoplasmic membrane must be recycled back into the intracellular organelle (Golgi bodies) to maintain the cytoplasmic membrane in a homeostatic state.

Similarly, cells active in endocytosis can internalize up to 0.8%/min of a plasma membrane. The loss of lipids from membrane caused by endocytosis must be replenished to maintain the size of cell’s outer envelope.

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Cytoplasm and Organelles Total protein concentration in cytoplasm

150 g / L (~4 μM)

Total protein concentration in plasma

90 g / L (1.2 μM)

Albumin (MW 69,000)

45 g / L (0.65 μM)

Globulins (MW 140,000)

25 g / L (0.18 μM)

Fibrinogen (MW 400,00)

103 g / L (0.0075 μM)

The cytoplasm and nucleus are both enclosed by cytoplasmic membrane in the cell. The cytoplasm can be largely divided into two groups: various organelles and the highly-viscous cytosol. The cytosol has a very high concentration of proteins (100 – 300 mg/mL). For comparison, the protein content in blood plasma is only 90 mg/mL. The cytosol also contains the inorganic solutes, building blocks, and intermediates and metabolites of metabolic reactions.

The cytosol is not only full of soluble components. It also contains large assemblies (or aggregates) of particles. The ribosome is the main machinery for making proteins; it is a complex particle consisting of many ribosomal proteins and ribosomal RNAs (rRNA). Each cell contains thousands of ribosomes of ~30 nm in size. Many ribosomes are located on the cytosolic surface of the endoplasmic membrane and appear as a black spot, when viewed under an electron microscope. Some enzymes also form large complexes that can be seen under electron microscope, such as pyruvate dehydrogenase complexes.

• Cytoplasm is not a simple solution • Some protein complexes (like pyruvate dehydrogenase and ribosomes) are aggregated • Cytoskeletal network is interspersed in cytosol

Also rich in the cytosol are the fiber-like structures of the cytoskeleton. These large protein particles, enzyme complexes, cytoskeletal proteins, and organelles make the cytoplasm of a cell very crowded and render its solution phase very dense in mass. Under light microscopy, an animal cell appears to be primarily cytoplasm, wrapped in a membrane, with a nucleus sitting near the center spanning over half of the cell’s diameter. Other than the nucleus, various organelles include the mitochondria, the endoplasmic reticulum, the Golgi apparatus, peroxisomes, endosomes, etc., and are visible only by electron microscopy.

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Nucleus endocytosis endosome lysosome

rough endoplasmic reticulum golgi apparatus secretory vessel

smooth endoplasmic reticulum

chromatin plasma membrane

nuclear envelope (nuclear membrane) nucleus

nucleolus

mitochondria

Fig. 2.4: Organelles in an animal cell

In bacterial cells, DNA molecules are roughly localized at the center of the cell. Both DNA and RNA synthesis occur in the nucleoid region that is adjacent to the chromosome formed by the large DNA molecule. The nucleoid occupies a distinctive part of the cytoplasm and the DNA is tightly coiled and is bound by many proteins. If completely extended, a DNA molecule of an E. coli cell is nearly 1-mm long. In contrast, a eukaryotic cell’s genome is separated into a number of DNA molecules, which each form a chromosome. Then, the DNA molecules are segregated into nuclear compartments. The average genome of a mammalian cell is about three orders of magnitude larger than that of E. coli. If stretched, it extends to about 1-m in length. This large amount of DNA is packed into a small space by forming DNA-protein (histone) complexes.

DNA/RNA synthesis and ribosome assembly occur in the nuclear compartment and are segregated from the metabolic processes and protein synthesis in the cytoplasm. Ribosomes are assembled in nucleoli and are subsequently exported into the cytoplasm to participate in protein synthesis. The complex tasks of sorting out which segments of DNA, or which genes, are to be transcribed into RNA at a given moment occur in the nucleus. A large array of transcription factors and other transcription regulators are synthesized in the cytoplasm and then imported into the nucleus where they bind to specific genetic loci to perform their role in transcription.

Thus, there is a large volume of material trafficking between the nucleus and the cytoplasm. Components of the ribosome, nucleotides/deoxynucleotides, nuclear structural proteins, and transcription factors need to be imported into the nucleus. The RNAs (mRNA, tRNA, and some non-coding RNA) are exported into the cytosol for protein synthesis. A double-layered membrane separates the cytosol and the nucleoplasm. The nucleus and the mitochondrion are two organelles in the cell that have double membranes, instead of only a lipidbilayer membrane. Much of the trafficking occurs

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through nuclear pores on the surface of the nuclear membrane (also known as nuclear envelope).

Mitochondria Mitochondria are.... • The most abundant organelle in a cell (about 1,700 per cell) • Take up to 20% of cell volume • In the catabolism of glucose to carbon dioxide, the oxygen atom in CO2 is contributed from water molecules. The oxygen reacts with H in NADH, FADH2, to form water in mitochondria • Active mitochondrion has a negative 140 mV electric potential across its inner membrane, and 1.0 units of pH gradient (inside mitochondria pH is higher [H+ concentration is lower] and pH is pumped against concentration gradient) • The membrane potential cannot be charged up too much. Therfore the homeostasis of mitochondria is critical. • Cells meet long-term energetic needs by biogenesis of mitochondria.

The mitochondrian is the most common organelle in a cell. With about 1,700 per cell, they make up 20% of the cell’s volume. Mitochondria are about the size of bacteria and are thought to have originated from bacteria-like structures that were acquired by primitive eukaryotes. Mitochondria serve as the cell’s power plants. The most reactive reactions in the cell (e.g., oxidizing nutrients and generating energy through electron transfer and oxidative phosphorylation) take place in the mitochondria. Cells with different energy needs have different numbers of these power plants. In a high-energy demanding cell, there can be as many as 3,000 mitochondria.

The main ATP-generating process occurs via electron transfer, across the inner mitochondrial membrane. The total surface area of all mitochondrial inner membranes in a cell is greater than that of the cytoplasmic membrane. At the mitochondrial inner membrane, reactive electrons in electron transfer react with oxygen to form H2O. Mitochondria are thus rich in potentially damaging free radical species. By confining these reactions to the mitochondria, the cell can potentially reduce unintended cellular damage.

The mitochondrion resembles a bacterium, not only in size but also by having its own genome in the form of a circular DNA molecule. Each mammalian mitochondrion contains one or more mitochondrial genomes of about 18 kbp. The control of mitochondrial DNA replication is separate from the regulation of genomic DNA replication. The biogenesis (i.e., the replication) of mitochondria is independent of cell division.

An active respiring mitochondrion has a negative 140 mV electric potential and pH of 1.0 across its inner membrane. The pH inside a mitochondrion is higher, as the H+ ion concentration is lower inside, so pH is pumped against the concentration gradient. The pH gradient and the electric potential are critical

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for cells. The electric potential and pH gradient are created by pumping protons out of mitochondria. This occurs while transferring electrons at a high energetic state in NADH to a low energetic state that can be received by oxygen to form water. In other words, the chemical potential energy in the high energetic electron is transformed and “stored” in the electric potential and proton gradient.

The membrane potential cannot become too high; it can burst the organelle. It is important to maintain a homeostatic condition in mitochondria. When the energetic need of a cell is high over a long period, cells respond by increasing their number of mitochondria. The flux of energy (primarily pyruvate) into mitochondria is tightly controlled. Fig. 2.5: Proton and electric potential (charge) gradient across mitochondrial inner membrane. The direction of fluxes of major species are indicated by and arrow. Note NADH oxidation coupled electron transfer pumps protons against proton and charge gradient, while the movement of proton to drive ATP synthesis is in the direction of proton and charge gradient.

Mitochondria (along with other organelles) cannot be generated merely from the genetic content in the nucleus. A cell must have mitochondria at its origin in order to make more mitochondria as it proliferates. The mitochondrial genome encodes a number of mitochondrial proteins and RNA molecules, while other mitochondrial components are encoded by cellular genomic DNA and imported into the mitochondria.

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Endoplasmic Reticulum

Smooth ER • Function varies with tissue, in liver cells it detoxifies; in ovary and testes it makes hormones

Rough ER • Proteins for some organelles, integral membrane proteins and secreted proteins are folded in ER • Professional secretors in the body, such as pancreatic beta cell, hepatocyte and antibody secreting plasma cell, all have abundant ER. As B cells differentiate to become plasma cell, ER and Golgi apparatus expand drastically, at least by 15 fold

Some characteristics of ER • In hepatocyte, surface of ER is about 63,000 μm2 per cell, or about 40 times of plasma membrane • ER lumen very viscous, gel-like. Diffusion coefficient of fluorescent probe is 9 - 18 times lower than in water • ER has much higher oxidative environment than cytoplasm, appropriate for disulfide bond formation • High free Ca2+ environment • Many proteins are present at very high concentrations (PDI, GRP94, GRP74) • A major site of protein folding, other post-translational processing, Ca++ homeostasis, cholesterol synthesis

Protein processing occurs in ER • Cleavage of signal peptide • Addition of high mannose core oligosaccharide to Asnx-Ser / Thr N-linked glycosylation site • Trimming of terminal glucose and mannose residues from initial glycan • Fatty acid addition

The endoplasmic reticulum (ER) is largely classified into the smooth ER and the rough ER, based on morphology. The smooth ER is rich in enzymes involved in chemical transformation reactions. In liver cells, the smooth ER takes on the role of detoxification; in the ovaries and testes, it makes hormones. The rough ER is the site of folding and processing of proteins destined for some organelles, integral membranes, and secretion. It gains its name through the attachment of a large number of ribosomes to its cytosolic domain surface, thus appearing to be rough under the transmission electron microscope. Professional secretors in the body have abundant ER, such as the pancreatic beta cells that secrete insulin and the antibody-secreting plasma cells. As B cells (non-antibody secreting) differentiate to become plasma cells, the ER and Golgi apparatus expand drastically, more than 15 fold.

Hepatocytes secrete many proteins, including albumin, which are coagulation factors that constitute many of the blood’s protein components. In the liver, some hepatocytes specialize in protein secretion, while others play major roles in oxidative detoxification. These hepatocytes have distinctive ERs. Those involved in protein secretion have an abundance of rough ER, while those more specialized in xenobiotic metabolism have an abundance of smooth ER.

ER is also a major site of protein post-translational modifications, and is involved in Ca+2 homeostasis and cholesterol synthesis. The ER lumen is rich in proteins that facilitate protein folding and catalyze the formation of intermolecular disulfide bonds.

• Disulfide bond formation

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The classical view of the Golgi apparatus is as a stack of flattened sacks. More recently, the Golgi apparatus is viewed as a dynamic region where many key reactions occur. Many proteins are modified in Golgi bodies after they are folded in the ER.

Golgi Apparatus and Protein PostTranslational Modification

Protein Processing Occurs in Golgi • Addition glycoform modification • Sulfation of tyrosines or carbohydrates • glycosylation • Peptide proteolytic cleavage • Gamma-carboxylation of glutamic acid • Beta-hydroxylation of aspartic acid

Protein Secretion Through ER and Golgi Apparatus

Some Characteristics of Golgi • Compartmentalized into functionally distinct regions: Golgi stack (consisting of cis, medial and trans cisternae), and trans Golgi Network (TGN) • Proteins, lipids are sorted in Golgi for delivery to different cellular locations • From here proteins go to secretion (exocytosis) or other organelles • During mitosis the Golgi apparatus breaks down and reassembles after mitosis • Different molecules of the same secretory protein spend different amounts of time inside the cell, i.e. there is a distribution of “holding time” in the cell • Translation of a protein molecules takes only seconds, but the secretion process takes tens of minutes to hours

The Golgi apparatus is loosely divided into four compartments: cis, medial, trans, and the transGolgi network (TGN). The protein cargo from the ER is transferred to the cis Golgi and then to other Golgi compartments through membrane vesicles. The enzymes in the four Golgi compartments are not identical. Thus, different reactions may take place in different compartments.

For a high-producing industrial cell line, the secreted recombinant product constitutes a very large fraction of all of the total protein synthesized. Cells devote a large portion of their protein processing capacity to the secreted protein product. It is therefore useful to review this process of protein secretion. In a professional secretor, approximately 30% of all cellular proteins are destined for organelles, membranes, and secretion and are processed through the ER. Although some proteins are translocated into the ER post-translationally, most (including typical recombinant DNA protein molecules) are translocated as nascent protein molecules.

Proteins destined for secretion have a leader sequence at the amino terminus that serves as the signal peptide. After translation initiation, the signal peptide of the nascent protein is recognized by signal recognition particles (SRPs). This halts translation and docks the nascent protein (which has only the beginning segment of the entire sequence) to a receptor on the ER membrane. It thus prevents the protein molecule from being elongated in the cytosol. The nascent polypeptide is then transferred to a translocon on the ER membrane. Subsequently, translation elongation resumes and the elongating polypeptide passes through the channel of the translocon into the ER lumen. Folding of the polypeptide starts immediately upon

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Fig. 2.6: Physiological changes incurred during the transition from B cell to plasma cell

The Becoming of Plasma Cells -

A Hint to the Creation of a Super Secretor

• Overloading of secretory protein molecules induces unfolded protein response (UPR), which triggers the differentiation process of B cells to become nondividing plasma cells. • B cells differentiate into plasma cells (or memory cells) upon antigen stimulation, along with helper T cells, increasing their size significantly and their ER by at least 15 fold (in 4 days). They also increase metabolic machinery significantly. • Xbp1 codes for XBP-1 (55KDa). XBP-1 is posttranscriptionally modified upon UPR, and activates transcription of many ER proteins. Increase in XBP-1 coincides with an increase in antibody production (day 4 ), but lags in ER expansion. • ER appears to expand by increasing its abundance, not merely selectively increasing some ER proteins. The expansion starts before mass antibody production. ER is a very oxidized environment (unlike cytoplasm that is highly reduced). Disulfide bridges are formed in ER and catalyzed by protein disulfide isomerase (PDI). PDI and four oxidoreductase level increases in ER as the B cells differentiate. Many of the redox balance enzymes in cytosol and mitochondria are also upregulated. There is also evidence to show that Golgi increases along with ER. Note: the drastically increased antibody secretion is through biogenesis of ER and other protein secretion machinery, NOT merely by faster “throughput.”

translocation into the ER lumen. The signal peptide on the elongating polypeptide in the ER lumen is cleaved upon entry into the ER. The protein concentration in the ER is estimated to be 100 mg/mL, a concentration at which proteins would otherwise aggregate and fall out of solution. A class of ER chaperones and other proteins that facilitate protein folding act on the nascent protein molecules to prevent aggregation and assist in folding. Their actions require cellular energy (ATP). An important member of the ER luminal chaperones, BiP, is also a component of the translocon complex. In addition to BiP (also known as GRP78) major ER luminal chaperones include calnexin, calreticulin, and protein disulfide isomerase (PDI).

As will be discussed later, extensive glycolyation occurs along the secretion process, starting in the ER and continuing into Golgi bodies. In the ER, glycosylation also serves as an indicator of correct protein folding. Protein molecules that have completed the folding process are exported from the ER by inclusion in membrane vesicles. Vesicle fusion, fission, and trafficking are the main forms of molecular transfer from the ER to different organelles. Secretory proteins in the vesicles are taken from the ER to the cisGolgi, which, along with the trans-Golgi, comprises an array of tubules and vesicles on the opposite side of the medial-Golgi. The medial-Golgi, typically containing three to seven stacks of cisternae, is the main site of glycan elongation for glycoproteins. There are two different views on how protein cargos are transported outward towards the TGN and eventually to other organelles or secreted out of the cell. The vesicle diffusion model hypothesis states that the cargo from an earlier compartment is transported to the next compartment by the membrane vesicles. The cisternae maturation model views the cargo as stationary inside the stack, once they enter the compartment. The cisternae (including the cargo) then moves outward, with its enzyme constituents changing along the way, and the cargo protein molecules becoming “mature”.

Eventually, the cargo at TGN is transported through the vesicles to its final destination, be it the plasma membrane (for secretion) or to other organelles. As

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the contents of the early compartment translocate to the later compartment, they need to be recycled after the cargo is delivered. Thus, there are vesicles for retrograde transfer, in addition to anterograde transfer. The ER, Golgi, lysosomes, and endosomes are all part of the secretory network. They communicate through the dynamic trafficking of membrane vesicles.

An estimated 100 to 200 glycosyltransferases, transporters of various nucleotidesugars, are membrane proteins that constitute the majority of Golgi enzymes.

Fig. 2.7: Secretion time of IgG heavy chain. Intracellular proteins were completely labeled with C14N15 - arginine, then switched to unlabeled medium. ~50% heavy chain is secreted in two hours.

Table 5. Secretion time of liver proteins

Transferrin Ceruloplasmin Anti-trypsin IgG

Half-life in ER Half-life in Golgi (min) (min) 110 45 80 30 30 - 40 10 Total ~120

• Secretory proteins spend different amounts of time in the ER and Golgi apparatus and in different proportions

After translation, it takes a finite amount of time to process protein molecules before they are excreted. For an average protein of about 350 amino acids in length, the translation takes only tens of seconds. However, the time required for synthesized proteins to be secreted depends on the nature of the protein, and can thus range from 30 minutes to a few hours. For example, the α1-protease inhibitor is among the fastest secreted proteins, with a halflife of about 28 min. Transferrin, in contrast, takes around two hours to be secreted. Even for the same protein, the secretion time is not uniform for all molecules. Rather, we observe distribution between shorter and longer “holding times”.

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Retrograde transport

TGN

Trans Golgi

AAAA A

AA

AA AA A

A AA

Anterograde transport Medial Golgi

Endosome Cis Golgi

Signal peptide

Endocytosis

SRP

Bip

Endoplasmic reticulum

Translocon SRP

AAAAA

Nucleus

Fig. 2.8: Synthesis and secretion of proteins to an extracellular environment

Protein Secretion • Nascent protein molecules destined for ER have a special ER signal sequence being synthesized in organized polysome. They are recognized by SRP (signal recognition particle), a ribonucleoprotein. • SRP binding transiently arrests elongation, directing the ribosome/nascent polypeptide complex (RNC)to the receptor on ER membrane and transfer the growing polypeptide to translocon. • SRP is released from the ribosome/nascent polypeptide complex. • The nascent polypeptide begins to pass through translocon and elongate into ER lumen. • Signal peptide on the elongating polypeptide is cleaved. • Protein folding and post-translation modification begins as polypeptide continues to elongate. • Major ER luminal chaperons: BiP, calnexin, calreticulin and PDI. • Ribosome is released once the translation is complete. • Folded protein (with inner core of glycan if it is a glycoprotein) concentrate at exit site of ER and is thought to bud into vesicles and translocate to Golgi as pre-Golgi intermediates. • Golgi apparatus is in a dynamic state. There is also retrograde transport (its own proteins need to be recycled) and anterograde transport. • After reaching trans Golgi network (TGN), secretory proteins are packaged into post Golgi vesicles and move along cytoskeletal network through cytoplasm to fuse with plasma membrane and be secreted. • Different molecules of the same secretory protein spend different amounts of time inside the cell, i.e. there is a distribution of “holding time” in the cell. • Translation of a protein molecules takes only seconds, but the secretion process takes tens of minutes to hours.

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Other Organelles

In addition to the nucleus, the mitochondria, the ER and the Golgi apparatus, a number of other organelles are also in the cytoplasm.

Lysosome: • Low pH • Site of degradation of cellular materials destined for degradation and endocytosed material • Part of secretory pathway

Peroxisome: • Site of fatty acid oxidation • Rich in oxidative enzymes

Endosome: • In endocytosis the invaginated plasma membrane forms small organelles • They move inward along the microtubule network • There is extensive cargo distribution and sorting • Some material sent to lysome • Some recycle to plasma membrane

A lysosome is an organelle with a low pH in its interior. It is the site of degradation of ingested materials or cellular materials that are no longer needed by the cell. Most cellular materials have a useful life span, regardless of whether they are catalyzing chemical reactions or playing structural or mechanical roles.

Occasionally, a catalyzing enzyme can be improperly “locked up” in its transition state, resulting in an amino acid being modified to lose its catalytic capability. Even in the cellular environment, some amino acids in the protein may get oxidized. The accumulation of such “damages” may render a protein nonfunctional. Thus, most proteins have finite life span. Proteins that need to be turned over are tagged by ubiquitin and sent to the proteosome for degradation. Proteosomes are a complex of protealytic enzymes that are capable of degrading proteins.

Through a process known as endocytosis, eukaryotic cells can take up external particles by wrapping the particles within cellular membranes and taking them up as vesicles enclosed in lipid bilayers. This process is not seen in prokaryotes. Some cells in higher organisms are specialized “scavengers” that engulf foreign particles or dead cells. Lysosomes are the sites within those cells where engulfed particles are degraded. Lysosomes contain a large number of digestive enzymes. They have proton pumps in their membrane to maintain a low interior pH (pH = 5.0).

In fat cells, the catabolism of lipid occurs in peroxisomes. The reactions involved in such metabolism generate large amounts of reactive oxygen and, thus, need to be contained within these specialized organelles.

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As discussed earlier in this chapter, cells are not merely a droplet-like structure with its constituents enclosed by a lipid bilayer. They form various shapes and sustain mechanical forces by transmitting and responding to mechanical force stimuli within, and also between, cells. A class of proteins makes up the cytoskeleton, accounting for their shape and their ability to transmit and exert force. The three major components of cytoskeleton are microtubules, actin fibers, and intermediate filaments.

Cytoskeleton

Microtubules • Long, hollow tubes of polymerized subunit tubulin (MW 50 kD), about 25 nm diameter, more rigid than actin filaments . • Typically long and straight, many have one end (-) attached to a single microtubule organizing center (centrosome). • Form and break down rapidly. • α- and β-tubuline (GTP) form heterodimers; the dimer assembles in a head-to-tail fashion into chains called protofilaments, 13 of which make up the microtubule wall. • Microtubules also play a key role in intracellular organelles and small vesicle transport. • For secretory protein, the movement of postGolgi vesicle to plasma membrane is mediated by microtubules.

A microtubule is an assemblage of hollow tube structures formed by polymerized α- and β-tubulin molecules. The polymerization and de-polymerization occurs at the ends of the microtubules, allowing them to extend and shrink their length rapidly. The hollow organization enables cells to use a smaller amount of material to give a longer protrusion with a high structural rigidity, like the hollow legs of aluminum ladders sold in hardware stores. If the same amount of material were made into solid legs, the legs would be rather thin and would easily deform when subjected to stress. Being tubes, microtubules are relatively straight and do not bend in sharp angles or high curvatures.

Microtubules can be extended to protrude from some region of the cell, and can rapidly shrink to retract a part of the cell. They are also used as a “train track” in the cytoplasm to transport “cargos”, such organelles and membrane vesicles, to different parts of the cell.

When two ends of a microtubule are attached to different objects, they can also pull them together or push them apart. For example, during mitosis, multiple microtubules work in coordination to separate a pair of chromosomes, thus allowing the daughter cells to each receive one copy.

Fig. 2.9: Structure of a microtubule molecule and its cellular organization

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Intermediate Filaments

• Play a structural role, stable, transmit mechanical force • The subunit is not a globular protein, but fibril, different from the tublin and actin • 10 nm diameter, has a head, a tail and a α-helical rod; can be made of a wide variety of proteins in various tissues.

The main role of intermediate filaments is to transmit mechanical force, like the cable holding a suspended bridge in place. Each fiber is made of subunit proteins oriented in the same direction. The tail end of a subunit is locked into the head of the next subunit. Each intermediate filament fiber is made of multiple fibrils, which are in turn made of a series of subunit proteins.

To increase the structural integrity, the tail-head “lock” positions of different fibrils are at different locations in the multi-fibril filament. These intermediate filament fibers are flexible, capable of absorbing energy exerted by external force and transmitting it to other regions of the cell. In a tissue or in interconnected cells in culture, intermediate filaments also help to transmit forces between cells. Their deformable nature allows them to act like a shock absorber and to reduce the deformation of cells upon stress.

Fig. 2.10: Structure of an intermediate filament molecule and its cellular organization

Actin Filaments

Actins are two- or three-stranded filaments that often form web-like bundles underneath the plasma membrane. Like ropes that are woven into a net, actin fibers are twisted strings of filaments. This geometry enhances their structural integrity while maintaining a high degree of flexibility.

The actin-rich region immediately underneath the cell’s plasma membrane often has a high concentration of actin, where it forms a gel-like structure, called the cortex. They are like a mesh of thin nets underneath the lipid bilayer membrane. They act as the first absorber of external mechanical perturbations and give the lipid bilayer local shape.

Fig. 2.11: Structure of an actin filament molecule and its cellular organization

Cells extend their body and spread flat on a surface, both in tissues and in culture. The edge of an adherent cell has an irregular shape, much like a fried egg. In the protruded regions, actin fibers localize in the lamellipodia and filopodia. In stationary cells, the

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• Two stranded filaments (F actin) of helical polymer of actin (G actin)(50 kD) • 5-9 nm flexible structures, organized into linear bundles, 2-D networks and 3-D gels • Distributed all over the cell, but concentrated in the cortex beneath the plasma membrane • The subunit, G actin, is a globular protein • Projections from cells, like microvilli, lamellipodia, microspikes and filopodia are maintained by rigid bundles of actin filaments • In non-motile cells, actin filaments form bundles called stress fibers, loose meshwork of filaments underlies cell membrane.

actin fibers form stress fibers throughout the cell, but in moving cells, these visible fiber structures tend to localize at the moving ends of the cell.

Actin filaments, along with the other two major components of the cytoskeleton, require many other component proteins to be present for their polymerization, dissociation, cargo translocation, and other functions.

• In actively moving cells, stress fibers disappear and actin filaments concentrate at the leading edge. • There are many actin-related-proteins which affect the polymerization and motor functions of actin filaments • Actin is involved in motor functions. Best example is muscle contraction.

Transport Mechanisms The lipid bilayer membrane separates cells from their environment. It presents a barrier to keep most compounds outside the cell and to prevent those inside from leaking out. It has a very low permeability for large molecules, like proteins and polysaccharides. Even polar or charged small molecules cannot pass through easily. Fig. 2.12: Order of magnitude estimation of the permeability of various molecules across lipid membrane

Among the nutrients and metabolites, only oxygen, fatty acid, and ethanol pass through the membrane at a fast enough rate to meet growth requirements. Specialized transport mechanisms mediate the movement of the vast majority of nutrients and the excretion of metabolites. Cells have a large number of transporters (sometimes called permeases) that allow molecules to cross the cytoplasmic membrane and membranes of various organelles. Such transporter-mediated transport is used to pass small molecular weight compounds (up to about 1 kDa) across the cell membrane (e.g., sugar, oligosaccharides, amino acids, oligopeptides, nucleotides, cholesterol, ions, organic acids, etc.). Macromolecules are transported across membranes by membrane fusion (in the secretion process through the ER and Golgi apparatus), pinocytosis,

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or exocytosis. Specific receptors, such LDL and transferrin receptors, may be involved in pinocytosis.

Major Mechanism of Transport The cellular transport of solutes (as opposed to macromolecules) is grossly divided into two categories: transport along or against the concentration gradient of the solute. The former is thermodynamically favorable, whereas the latter (called active transport) requires energy input to make the process possible.

Three Classes of Transport Processes • Channel-mediated diffusion: Molecules or ion specific; once channel is open, very fast flux. • Facilitated difussion: Provides molecule specific opening in the membrane; barrier for molecular diffusion. • Active transport: Moves molecules up against a concentration gradient. Requires ATP or ion gradients of ion (H+, Na+) as an energy source to drive the transport.

The energy source of active transport can be derived from coupling to a chemical reaction, such as the hydrolysis of ATP. Active transport of a species may also be coupled to the diffusion of another solute along its concentration gradient. Thus, the driving force to transfer the second solute along its gradient is used to “push” the first solute to move up against concentration gradient. Transport along the concentration gradient can be mediated by carrier (transporter) proteins or by channel proteins.

Channel proteins open into a duct-like structure across the membrane that is specific for a specific solute, such as water, Na+ or K+. Channel proteins exist either in an open state or a closed state. Once the channel is open, the transfer is very fast in the direction of the concentration gradient. The flux is affected by the number of channel protein molecules on the membrane and by the time period that the channel is open. Carrier-mediated transport is also called facilitated diffusion. It entails, first, the diffusion of solute from the high concentration side of the membrane into the transporter, followed by the translocation of the solute to the low concentration side of the membrane. Once on the low concentration side, the solute is free to diffuse away.

Under normal culture conditions, amino acids and glucose are transported by facilitated diffusion. A ubiquitous transporter for glucose is the glucose transporter 1 (GLUT1). The rate of transport by a transporter is dependent on the concentration of the solute. More precisely, it depends on the concentration

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difference of the solute across the membrane. Its mechanism is similar to a typical enzymecatalyzed conversion of a substrate to a product. The dependence of transport rate to solute concentration can be described by MichaelisMenton enzyme kinetics. At low concentrations, the transport rate is in proportion to the solute concentration, while at high concentrations, the rate is constant as the transporter becomes saturated. Fig. 2.13: Three types of transport processes across the cell membrane

The half-saturation constant (km) for GLUT1 is about 0.1 mM. In the 0.01 – 0.1 mM range, the glucose import rate of the cell increases with increasing glucose concentration. In the range typically used in cell culture media (1 – 10 g/L, or 5.5 mM to 55 mM), the rate is not affected by glucose concentration at all.

Transporters for facilitated diffusion and active transport can also be categorized according to the number of solutes each carries and the direction of solute flow.

General Types of Transporters • Uniporter: Transfers a single molecule (e.g. glucose, fructose), usually uncharged. • Bispecies-transporter (co-transporters): Requires stoichiometric exchange of two species simultaneously; important in change balance. • Symporter:Two species transported in the same direction. • Antiporter:Two species transported in the opposite direction.

Uniporters transfer a single solute from a high concentration side to a low concentration side, for example the GLUT1 transporter for glucose and the GLUT5 transporter for fructose.

Symporters and antiporters transfer two solutes, simultaneously. If the two solutes move in the same direction, the transporter is called symporter. Conversely, antiporters transfer two solutes in opposite directions. Collectively, symporters and antiporters are called co-transporters. Co-transporters are often used to transport charged organic molecules. Dissociable solutes exit with a counter ion to maintain electric charge neutrality. When a charged solute moves from one side of the membrane to the other, the charge neutrality must be maintained. Otherwise, the net charge will accumulate across the membrane and create impudence for further transfer of the solute. For example, lactic acid exists as lactate in an aqueous solution. If it is removed from the cell, a negative charge will be moved along with it. After a while, the cell membrane will be negatively charged outside, creating a negative voltage. The negatively charged outside will prevent further

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excretion

of

the

negatively

charged

lactate.

In order to prevent the buildup of a charge across the membrane, charged solutes are transported by co-transporters. Two mechanisms are commonly seen: 1) co-transport with a counter ion (such as H+ for lactate) by a symporter, and 2) co-transport with an ion of the same positive or negative charge, but in the opposite direction (such as Cl- for HCO3-).

In co-transporter mediated transfer, the transport rate is not only affected by the concentration difference of the solute, but also by the concentration gradient of the co-transported ion. Thus, the transport of lactate by the monocarboxylate transporter (MCT) is not only affected by the concentration of lactate, but also by pH.

Fig. 2.14: Three types of transporters categorized by the solute being transported

Co-transporters may also be involved in active transport. One such case involves an ion species being transported along its concentration gradient. The tendency of the ion to “push” across the transporter is used to “drive” the transport of a solute against its gradient.

For example, the Na+/glucose transporter in the epithelium of the intestine can take up glucose from the digestive track, even when glucose is lower than in the cell. This is accomplished by using the Na+/ glucose transporter to transport two Na+ atoms from the lumen of the intestine into the cell, where the Na+ level is very low. As the sodium ion is transported, a glucose molecule also binds to the transporter and is transported simultaneously. The propensity of Na to move across the membrane is so high that it can drive glucose to move against a large concentration gradient.

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Transport of Nutrients

• There are 12 different facilitative glucose transporters (GluT) in animal cells. Different transporters are expressed in different tissues. • GluT 1: the major transporter in all cells • Amino acid transporters have overlapping amino acid specificity. Some amino acids compete for the same transporter. Many have alternative transporters. • MCTs transport lactate, pyruvate together with H+

Major nutrients like glucose, other sugars, amino acids and oligopeptides, are taken up by cells through facilitated transport. The excretion of lactate, ammonium, and some non-essential amino acids are also transported by the same mechanism.

A large number of glucose and amino acid transporters are present in the mammalian genome. Different glucose transporters are expressed in different tissues for different cellular needs, but GLUT1 is present in all cells. GLUT4 is responsive to insulin and is expressed only in some tissues. Upon insulin stimulation, the intracellular GLUT4 molecules are translocated to the plasma membrane to take up glucose.

The number of amino acid transporters in a mammalian genome is also large. Some transport entire classes of amino acids that share a common property, such as the neutral amino acid transporter for uncharged amino acids. Others are specific for one or a small number of amino acids.

The monocarboxylic acid transporter (MCT), the transporter for lactate, also transports pyruvate. A number of MCTs are expressed in different tissues. In a cell, different MCTs are located at the cytoplasmic membrane and others at the membranes of some organelles. Another class of transporters for active transport is the ATP-binding cassette (ABC) transporter, which transports some hydrophobic compounds by utilizing ATP. After prolonged exposure to methotrexate, some cancer cells develop drug resistance by pumping the chemical out of cells using ABC transporters.

Ion Transport

Bulk ion species (H+, Na+, K+, PO4-3, Cl-) are present at very different concentrations across the cell membrane. For instance, Na+ and K+ have opposite directions in their concentration gradients across the plasma membrane. The intracellular concentration of K+ is 20 – 50 times higher inside than outside the cell, while the Na+ concentration is 10 – 15 times higher outside the cell versus inside the cell. Along with the concentration gradients of major ions,

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cells also maintain an electric potential gradient of -80 mV across their plasma membrane. This electric potential is fundamental to the transport of many compounds across the membrane.

Because of this concentration gradient across the membrane, sodium ions have a natural tendency to flow into the cell, wherever they are allowed to pass. The -80 mV membrane potential further enhances the propensity of Na+ to influx, as the negative charge in the inner surface of the membrane draws Na+ to move across the membrane. The combined concentration and electric potential gradients are used as a driving force in the Na+-dependent glucose transporter, which will transfer glucose from the lumen of the digestive track into intestinal epithelial cells, moving against a glucose concentration gradient.

The Na+/K+ ATPase transporter, which is present in the cytoplasmic membranes of all animal cells, is important for establishing sodium and potassium gradients across the plasma membrane. ATPase is an integral cell membrane protein that has multiple subunits. It simultaneously transports two K+ ions into the cell and three Na+ out of the cell.

Fig. 2.15: Transporters involving the transfer of ions and ABC transporter

Although the cytoplasmic membrane is relatively impermeable to ions, it does allow a small but finite diffusion of Na+ and K+ along their concentration gradient (i.e., a net influx of Na+ and a net outflux of K+). It should be noted that the membrane permeability for K+ is higher than Na+. Intracellular K+ also leaks out through potassium channels. Overall, through diffusion across the membrane and transport by channel proteins, there is a net movement of ions into the environment from the cytosol. This balance is maintained by the action of the Na+/K+ ATPase.

Na+/K+ ATPase has three binding sites for Na+ and two for K+. The Na+ binding sites on the cytosolic side of the ATPase have a Km for Na+ in the range of submillimolar concentrations. Because the cytosolic Na+ concentration is about 10 mM, virtually all three Na+ binding sites exposed to cytosol will be occupied. Na+/K+ ATPase also has a binding site for ATP. Hydrolysis of ATP results in the phosphorylation of the protein subunit and release of ADP. This allows two K+ ions to bind to the protein on the extracellular

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• The concentrations of other major ion species (H+, Na+, K+, Ca2+, PO4-3, Cl-) across membrane are polarized. Na+ and K+ have opposite direction in their concentration gradient across the plasma membrane, which is about ten fold difference. For Ca2+ the intracellular concentration is so low that the gradient is extremely steep. • Na+-K+ ATPases play a major role in maintaining Na+ and K+ gradients. ATPase utilizes the hydrolysis of ATP as the energy source. • Iron is extremely reactive and participates in many redox reactions. In biological systems, it exists as “bound” form. In its free form, it catalyses the formation of peroxide and peroxidizes unsaturated fatty acids. In cell culture, it is supplied as transferrinbound or bound by other chelators and taken up via transferrin receptors. • Another class of ions are transported into the cells via binding to proteins and internalized through specific transportors. One example is iron transport by transferrin. Iron is extremely reactive, participates in many redox reactions. In biological systems, it exists as “bound” form. In its free form, it catalyses the generation of peroxide and peroxidize unsaturated fatty acids. In cell culture, it is supplied as transferrin bound or bound by other chelators and taken up via transferrin receptor.

side, while simultaneously exposing the Na+ binding sites to the extra-cellular solution. At the external side of the enzyme, the Km of Na+ is at a higher value. As a result, Na+ is released to the outside environment. After the release of Na+, the phosphate is released from the protein and K+ releases to the intracellular environment. The Na+/K+ ATPase then resets, ready for another round of reactions.

The net result is that ATPase uses ATP to pump three Na+ ions out and two K+ ions into the cell. With its 3:2 stoichiometric ratio of sodium to potassium, a prolonged operation of ATPase without any balancing action will inevitably generate a large electric potential across the membrane. To counter this, cells also have chloride ion pumps to pump negatively charged Cl- out of the cell. In some cells, Na+ and Clchannel proteins also facilitate the maintenance of the membrane electric potential in the correct range.

Extracellular Matrices and Cell Movement ECM

The vast majority of cells in the body are embedded in tissues in an acelluar tissue structure. When cultured in vitro to plastic or glass surfaces, they secrete materials onto the surface after adhering. Those excreted materials to which cells attach are collectively called the extracellular matrix (ECM).

The ECM is made of proteins, proteoglycans, and glucosaminoglycans. Different types of cells excrete somewhat different ECM components. Among the prominent members of ECM proteins are collagens, laminins, and fibronectin. The role of ECM is not merely to provide a surface appropriate for cell adhesion. It is also important for cell-surface signaling and growth control. Receptors on the cell membrane establish cell adhesion complexes with ECM components and a tension force is then transmitted through cytoskeletal fibers and

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ECM Proteins

• Collagen



• Laminin



• Firbronectin

the cell’s internal signaling pathway to allow cell cycle to proceed. The cell adhesion receptors exhibit a wide range of diversity. For example, integrin receptors have a variety of α and β components and form a large number of combinations of integrin complexes that have different affinity for different ECM components. As a result, different cell types often have different ECM requirements for adhesion and growth.

Proteoglycan

• Chondroitin sulfate

Glucosaminoglycan

• Heparin



• Hyaluronic acid

• The extracellular matrix is rich in electrocharges, allowing growth factors, cytokines etc. to be “stored” inside them • The extracellular matrices are the substrate for adhesion of many cell types, and provide cues for growth, differentiation and development

Cell Movement

Many ECM components are highly negatively charged. This allows many protein growth factors to be adsorbed to the ECM and released to surrounding cells, perhaps even serving as chemoattractants. Therefore, they also play a role in providing cues for cell migration and differentiation.

The vast majority of cells are capable of movement on surfaces. In general, cell movement can be the result of an attraction to chemicals, or unidirectional random movement.

• The filopodia extend as a result of microtubules growing at the cell front, and establish a “grip” on the surface • The subsequent dissociation of cell-substrate contact at the rear of the cell allows the cell’s center of mass to move forward

Cell movement is a coordinated series not unlike walking. It involves a restructuring of the cytoskeleton, a protrusion of the membrane, the establishment of surface adhesions on one side of the cell, and the detachment of the cell membrane from adhesion complexes in the rear end of the cell.

The actin fibers and plasma membrane of moving cells extend the cell to become more elongated in one direction. The extended regions form lamellipodia, which may also contain microspikes, or filopodia, which are active even within a few minutes.

Cells moving in an “open” surface (i.e., not crowded) move randomly. They exhibit locomotion contact inhibition, meaning when two moving cells encounter each other, both will move away in opposite directions. Furthermore, the two daughter cells of a dividing mother cell move away from each other when cell division is complete. Cell migration is a regulated by many factors, including growth factors. For example, epithelial cells

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respond to hepatocyte growth factor by moving away from each other and become more scattered, instead of forming cell clusters typical of epithelial cells.

Fig. 2.16: A cell moving on its substrate. Lamellipodia extend in the leading edge on the right side. A few filopodia are extruding out.

Cell migration is not intentionally controlled or manipulated in cell bioprocessing. However, in some cases, when seeding cells into threedimensional matrices for tissue engineering applications, efficient cell migration into the interior of matrices is important for subsequent growth.

Growth, Death and Senescence Cell Cycle and Growth Control Positive and Negative Cues

Cell Cycle • G1, S, G2 and M constitute four consecutive phases of cell cycle. S stands for DNA synthesis and M for mitosis. They are separated by two gaps. • The duration of S and M phases is relatively constant. Cells grow at different rates and spend differnt amounts of time in G1 and G2. For mammalian cells, the duration of S phase is 5-8 hours and that of M phase is 1 hour. • The progression from G1 to S, and from G2 to M phase is tightly controlled. Only when a cell is “ready”, will it proceed to the next phase. • Before M phase, chromosomes and all organelles, ribosomes and other cellular contents are all duplicated from time 0 (immediately after cell division).

Cell growth is the manifestation of a delicate balance between positive and negative regulations that respond to signals both outside and inside the cell. Positive signals stimulate cell growth and proliferation and suppress the cell death mechanism, while negative signals suppress those events and promote cell death. External signals from the environment tell cells the availability or absence of nutrients necessary for DNA replication and biomass synthesis. External signals from other part of the body allow cells to coordinate their response to the need of the organism. The internal signals modulate cellular programs to increase cellular component content, to divide, or to die.

Eukaryotic cells progress through four stages in their procession to grow in cell number: G1, S, G2, and M phases. G1 and G2 refer to the gap phase. S and M phases derive their designation from DNA synthesis and mitosis, respectively. The four stages constitute a cell cycle and this cycle is repeated every time a single cell becomes two daughter cells. Cells that are in a long period of quiescence, such as terminally differentiated cells, divert from G1 and enter G0 stage indefinitely.

Checkpoints are present between the different stages of cell cycle control. After mitosis, cells increase in size and mass. They only enter the S phase from G1 if cellular conditions are right. Similarly, they only enter mitosis from G2 if cellular components are ready.

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Additionally, the decision to enter the S phase is subjected to the regulation of external positive mitogenic factors, such as insulin, insulin-like growth factors, and fibroblast growth factors. Anchorage-dependent cells also receive growth stimuli by establishing contacts between the surface receptors and the ECM, and thereby maintaining tension in the cytoskeletal network.

Countering the actions of the mitogenic factors are those factors that provide signals to cause growth arrest. Cell-cell contact, for instance after reaching a confluent state, causes growth to cease. Such contact inhibition of growth has been noted for more than five decades. Only recently have researchers found that it is the interference of the adherent junctions between cells that disrupts a cell’s internal signaling networks to cause growth arrest.

Fig. 2.17: Phases of cell cycle and approximate duration of each phase

Cyclins and CDKs

Whether cells divide and grow or self-destruct is the outcome of a balancing act of a network of external and internal positive and negative factors. Loosening the controls may lead to unscheduled proliferation and transformation of cells to their malignant derivatives.

The progression through each of the four phases of the cell cycle (G1, S, G2 and M) is positively regulated by cyclins and cyclin-dependent kinases (CDKs) and negatively controlled by CDK inhibitors (CDI), which deactivate cyclin-CDK complexes.

Each of these regulatory proteins displays a characteristic periodic dynamic profile throughout the cell cycle. Each protein’s profile is the result of interactions of the other cell cycle regulatory components with their expression, activation, inactivation, or degradation corresponding to a specific time frame. An important cell-cycle checkpoint occurs along the transition from the G1 to S phase. The pivotal player in the G1/S phase transition is the CDK4/6-cyclin D complex. The activated CDK4/6-cyclin D complex can phosphorylate the regulatory protein retinoblastoma

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Growth Control • Growth, the increase in biomass and its contents (i.e. organelles and cytosol) as well as the increase in cell number of almost all cells (except those which have been adapted in vitro) is regulated by growth factors, cytokines. • The regulation balances positive factor (mitogenic) and negative factors. which prevent cell death or apoptosis. • The most common growth factors for cells of bioprocess interest are insulin or insulin-like growth factors (IGF). • IGF and insulin have different receptors on cell surface. Insulin regulates glucose metabolism and has a mitogenic effect. IGF, which is used in much lower concentrations and also has a mitogenic effect.

(pRB). pRB, in its unphosphorylated state, binds to and inhibits the transcription factor E2F. Upon phosphorylation, pRB dissociates from E2F, leading to the activation of cyclin E transcription by E2F. E2F activation positively regulates the transcription of genes involved in cell cycle progression.

Inputs from growth factor signaling and cell adhesion-mediated signaling are prerequisites to the G1 phase. These two pathways are not independent of each other. On the contrary, they have rather extensive crosstalk. In normal untransformed cells, all the important growth factor signal transduction cascades are regulated by integrinmediated cell adhesion. As a result, adherent cells rely on attachment to the ECM for growth.

Except for vaccine production, where normal diploid human fibroblasts are employed, virtually all cells used for protein production are continuous cell lines, including CHO, BHK, HEK 293, and mouse myeloma cells, such as NS0 and Sp2/0. All of these cell lines have lost their normal growth control. Their cell cycle checkpoint controls have been compromised and their entry into a quiescent state in the absence of mitogen has been relaxed.

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Fig. 2.18: Schematic representation of the interaction between cell cycle and apoptosis pathways. CDK Cell cycle-dependent kinase, IRF-1 interferon-regulatory factor 1, pRb phosphorylated retinoblastoma, ERK extracellular signal regulated kinase, FADD Fas associated death domain protein, FLIP FLICE-inhibitory protein, Cdc42 cell division cycle 41, EIF4E eukaryotic translation initiation factor 4E, Cyc Cyclin, XIAP cross-linkied inhibitor of apoptosis proteins, Apaf-1 apoptotic peptidase activating factor – 1, BAX Bcl-2 associated X protein, BAK Bcl-2 homologous antagonist/killer, Ub ubiquitination, cyt C cytochrome C

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Apoptosis • Cells, under some conditions, commit suicide, undergo programmed cell death; this is different from cell death caused by injuries (necrosis). • Necrosis may entail cell swelling, rupture and leakage of cellular materials. In vivo, necrosis may cause inflammation of surrounding tissues after encountering cell debris.

Apoptosis is the process of regulated cell death in response to developmental cues or to accumulating non-lethal stresses, such as nutrient depletion, growth factor deprivation, virus infection, and/or metabolite accumulation.

This process of programmed death is marked by specific cell morphological changes: DNA condensation, chromatin shrinkage, and membrane bulging (also called blebbing). The final intracellular event involves a series of cascades leading to cellular • Apoptosis can be caused by the lack of positive signal/ growth factors (e.g. withdrawal of IGF) or the imposition destruction. These final acts of self-destruction of negative signals. are similar in all apoptosis mechanisms; however, • Two general pathways for apoptosis: one, induced by the initiating “signal” can differ. The two major specific signals, occurs during cell development; another, apoptosis signaling pathways are the death induced by a variety of stress conditions. receptor pathway and the mitochondrial pathway.

• Apoptosis entails cell shrinkage, mitochondria breakage and release of cytochrome C, DNA fragmentation, and release of phosphatidylserine from phospholipids which causes phagocytotic cells to engulf the cell fragments.

Death Receptor Pathway

In many developmental events, individual cells serve their function only for a finite period of time. Beyond this period, their existence may interfere with, or even imperil, the well being of the organism. In those cases, cells are built to die after their functional duration by endowing an individual cell’s survival to depend on the presence of positive factors or the absence of negative effectors.

In the event that a cell survival signal is absent or a cell death signal is present, a cell undergoes self-destruction and ceases to serve its function. Developmentally related apoptosis is largely regulated by death receptors on the cell surface. The death receptor pathway is mediated by binding, or a lack of binding, of ligands to death receptors. For example, immature neurons die in large numbers during early brain development because neuronal cells require positive survival signals. The lack of such positive survival signals leads to neurodegenerative disorders.

Ligand binding to death receptors initiates the recruitment of an adaptor molecule, Fas-associated death domain (FADD), to the cytoplasmic end of the receptors. The presence of FADD causes caspase 8 or 10 to associate with the receptor, forming a death inducing signaling complex. The caspase is then proteolytically activated, triggering

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the activation of a series of downstream effector caspases (3, 6 and 7). The activation of these effector caspases leads to the final stages of cell destruction.

Mitochondrial Pathway

Mitochondrial Apoptotic Pathway Non-Apoptotic State: The balance of anti-apoptotic and pro-apoptotic factors • At non-apoptotic state anti-apoptotic factors hold pro-apoptotic factors in check • Apoptogenic factors are held in inter membrane space of mitochondria

Apoptotic State: Upon insult of apoptotic inducing agents or environment factors • Anti-apoptotic factor(s) had conformational change (exposing BH3) • Membrane disruption releases cytochrome C and other pro-apoptotic and apoptogenic factors • The releases of procaspase3 form apoptosome with the adaptor (Apaf -1), becomes capase-3. • Caspase-3 converts procaspase-9 to caspase-9 which becomes the executioner caspase and starts the apoptotic cellular event

In addition to its role in energy metabolism, mitochondria also sequester pro-apoptotic proteins in the space between their outer and inner membranes. These pro-apoptotic factors are released in a controlled manner in stressed cells to initiate apoptosis. Mitochondrial cytochrome C, a major component of electron transfer, is also a signal for apoptosis.

As in the control of cell growth, the components of the mitochondrial apoptosis pathway also involve positive proapoptotic and negative anti-apoptotic factors. The Bcl-2 family that consists of over 20 pro- or anti-apoptotic proteins is a major player in the mitochondrial apoptosis pathway. The proapoptotic subfamily includes Bax, Bak, and Bok, which all contain BH1, 2, and 3 homology domains.

Upon exposure to death signals, Bax undergoes conformational changes and translocates to the mitochondria, where it inserts into the outer mitochondrial membrane and forms channels. These channels allow the leakage of cytochrome C and other pro-apoptotic molecules. Cytochrome C proceeds to form a complex with Apf-1, pro-caspase 9, and dATP, known collectively as the apoptosome. In the apoptosome, the inactive pro-caspase 9 is activated and the active enzyme subsequently activates downstream caspases.

Two anti-apoptotic proteins, Bcl-2 and Bcl-xL, counter the actions of the pro-apoptotic components. Bcl-2 is localized on the mitochondrial membrane and inhibits the release of pro-apoptotic molecules from the mitochondria by maintaining membrane integrity. Bcl-xL is localized in the cytoplasm and binds to pro-apoptosis members of the Bcl-2 family. The involvement of multiple protagonist and antagonist factors ensures tight control of apoptotic event. This scheme also provides an amplification

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of the desired signal. An excellent example of this is the cascade of caspases. When cell destruction is needed, the signal is greatly amplified through a series of steps where one caspase activates another caspase in an exponential fashion.

Fig. 2.19: Mitochondrial pathway mediated apoptosis

Senescence and Telomeres

Fig. 2.20: “life” of normal cells from isolation to senescence and occurrence of cell line

The vast majority of animal cells isolated from tissues require surface adhesion in order to multiply, since they are anchorage-dependent cells. Cells are typically isolated from tissues by an enzymatic dissociation of the tissue. After dissociation and the removal of large chunks of undissociated debris, cells are plated on a compatible surface overlaid with media. Under the microscope, cells in media suspension can be seen to attach to the surface or out-grow from the remaining tissue chunks. Subsequently, they extend their body length, spread and begin to multiply. Those cells derived from normal tissues generally possess two sets of chromosomes and are diploid cells.

Eukaryotic cells enter an exponential growth phase, similar to microbial cells. The growth rate slows as they begin to cover the entire surface area to form a “monolayer”. Upon reaching confluence, they stop dividing. While the cell bodies of neighboring cells may cross each other, their nuclei never overlap. This is called contact inhibition of cell growth. Cells can be dissociated from a surface after being treated with trypsin (i.e., trypsinized) or with other proteases. They can then be plated on a larger surface area for continued growth. This process can then be repeated to expand the population. Each round of detachment and expansion is called a “passage”. Non-immortalized cells cannot be grown in culture indefinitely by simple repeated passage in culture. Normal diploid cells from animals (except stem

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tissue dissociation Plating in nutrient medium

In a historical experiment carried out half a century ago, the continued passaging of mouse fibroblast cells beyond crisis gave rise to a small fraction of survivors. These cells eventually grew, expanded, and could be cultured continuously in vitro without a limited life span. They were given the name 3T3 because the cells were passaged every three days by expanding the surface area three times more. These cells appear normal and are subjected to contact inhibition of growth under typical culture conditions.

Growth Replating to larger surface area

cells) have a limited life span in culture. Fibroblasts (a cell type from connective tissue) isolated from a mouse embryo can be cultured in vitro for about 60 doublings. As that limit approaches, the cells begin to fail to reach confluence. Eventually, high passage cells will cease to grow. This is referred to as having reached “crisis”. Such a limit in the proliferating potential is called “Hayflick’s phenomenon”. It is a common phenomenon for all normal diploid cells obtained from vertebrates.

Contact inhibition

Cell Dissociation

Fig. 2.21: Anchorage dependency and contact inhibition of cultured normal diploid cells

However, although the parental mouse fibroblast cells had diploid set of chromosomes before reaching crisis, 3T3 cells have abnormal number of chromosomes. Cells that succumb to Hayflick’s constraint (e.g., those that are diploid and have a limited life span) are called “cell strains.” The cells that reestablish after crisis and can grow in culture indefinitely, but are aneuploid (do not have a normal set of chromosomes), are referred to as “cell lines.”

Cells derived from cancer also give rise to cell lines. Cell lines can also be established by “immortalization” through viral or oncogene transformation. These transformed cells are also often aneuploid. They are capable of growing beyond the monolayer, since they are not subject to contact inhibition of growth.

Fig. 22: Telomere at the end of a chromosome

Normal diploid cells, thus, appear to “count” their number of doublings. They achieve this by using their telomeres. Telomeres are special repetitive sequences at the end of chromosomes. They are not replicated by DNA polymerase during DNA replication, but are synthesized by telomerase. The reaction is not precise for accurately reproducing

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the number of tandem repeats of the sequence, thus there can be much variation in telomere length among cells. As the number of passages increases, telomeres decrease their length unless they are repaired by telomerase. For instance, in stem cells, telomerase activity is high to maintain the telomere length. Unlike cell strains, stem cells do not exhibit senescence.

Concluding Remarks In this long chapter we provided a condensed overview of knowledge in cell biology that is essential for biotechnologists to practice cell culture bioprocessess. The structure and make-up of cells that gives them their functional versatility, also constrains their capability. In practicing biotechnology, while exploiting their biological versatility, we also must understand cell’s structural and functional constraints and biological limits. In the meantime, we must also keep in mind that our objectives are frequently different from scientists studying the biology of the cell. To fully harness a cell’s biological potential, we do not necessarily need

to be bound by the nature of cells; rather we should employ means to “adapt” them to serve our goals better. For example, most cells used for biologics production were anchorage-dependent originally and are now cultivated not only in suspension, but also in highly turbulent flow conditions. Most of the adaptation processes in the past two decades were conducted empirically. At a molecular level, what causes those cells to have the adapted behavior is poorly understood. By equipping ourselves with a better knowledge of cell’s capability and limits, we will be able to push the technological boundary further.

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Cell Physiology for Process Engineering Overview of Central Metabolism of Cultured Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Glucose and Energy Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Oxidation of Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Pentose Phosphate Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Lactate Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Carbon Flow and Reaction Intermediates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 NADH Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Role of Transport and Transporters in Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Glucose Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Lactate Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Transport Across Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Regulation of Glucose Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Isozymes and Differential Allosteric Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Signaling Pathways and Regulation by Growth Control . . . . . . . . . . . . . . . . . . . . . 77 Metabolic Homeostasis and Lactate Consumption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Glutamine and Its Relation to Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . 80 Amino Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Lipid Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Lipid Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Fatty Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Acetyl CoA shuttle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Cholesterol & Its Biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Glycan Biosynthesis and Protein Glycosylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Importance of Glycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Protein Folding and Glycosylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Glycan Extension in Golgi Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Glycan Types and Microheterogeneity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Synthesis and Transport of Nucleotide Sugar Precursor. . . . . . . . . . . . . . . . . . . . . 93 Glycan Diversity Among Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

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Overview of Central Metabolism of Cultured Cells Glucose Oxidation C H O + 6O 6

12

6

6CO + 6H O

2

2

2

Glucose Anaerobic Metabolism 2CH6 $ CHOH $ COOH

CH O 6

12

6

Glutamine Oxidation CO (NH ) CH CH CH (NH ) COOH + 4.5O 2

2

2

2

2

2NH + 5CO + 2H O 3

2

2

Cells in culture take up sugar, amino acids, lipids, and nucleosides from their growth media. They metabolize these components to derive energy and use them as building blocks to generate more cell mass, create more cells, and produce products. The processes of making more cell mass and protein product are very energy intensive. Proteins constitute over 50% of the dry mass in a typical cell; they are essentially amino acids connected by peptide bonds. The synthesis of each peptide bond costs at least 3 ATP, which is nearly 1/10 of the amount of energy that can be obtained by oxidizing one glucose molecule. One high-producing recombinant cell produces over 40 pg per day of IgG protein. Since an average cell has about 400 pg of cell mass (or about 200 pg of cellular proteins) it is easy to see that producing the protein product is a major energetic load for cells.

A classical cell culture medium contains 1 – 5 g/L of glucose, and somewhat lower levels of amino acids (about 0.8 mM, or 1 g/L). The sum of the nutrients, together, typically generates only about 1 – 3 x 109 cells/L, or approximately 0.1 – 0.3 g/L of cell dry mass. The efficiency of producing cell mass from glucose and other nutrients is rather low.

Fig. 3.1: Lactate and ammonium profiles in manufacturing runs with high product titers (blue) and low product titers (red). Lactate profile correlates to productivity, but not ammonium.

Glucose is the most important source of energy for most cells. Even when another sugar such as galactose or fructose is used as the sole carbohydrate source, it still enters the metabolic pathway that has evolved for glucose (called glycolysis) to get catabolized.

The complete oxidation of one glucose molecule consumes six O2 and generates six H2O and six CO2. For cells in culture, however, the majority of consumed glucose is not completely oxidized; it is converted to lactate and excreted. By converting to lactate instead of completely oxidizing to CO2, much less energy is derived from each mole of glucose. This is the root cause of the low efficiency in conversion from glucose to cell mass. For some cells, especially transformed cell lines, each mole of consumed glucose produces almost two moles of lactate, which is the theoretical maximum of the glucose to lactate conversion.

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Glucose, Glutamine Major Carbon Source

• Both glucose and glutamine consumed in excess to what are need to grow biomass • Most glucose consumed, converts to lactate • Excess glutamine consumption, results in ammonium excretion

Lactate Acid and Fedbatch Culture Productivity

• Lactate is produced in exponential growth phase • In stationary phase, some cultures continue to produce lactate, others switch to lactate consumption • Lactate consumption correlate to sustained higher viability and high productivity

This type of “wasteful” metabolism is common to almost all vertebrate cells in culture. For bioprocessing, the accumulation of these byproducts inhibits cell growth and impedes productivity.

Cells invariably produce lactate from glucose when growing rapidly. However, under some conditions, such as the stationary phase of fed-batch culture, lactate may also be consumed. It is not unusual that under the same operating conditions, different culture runs have different metabolic outcomes. In some runs, lactate production in the rapid growth phase continues into the stationary phase. In others, the transition from lactate production to lactate consumption occurs in the stationary phase. When production data from a manufacturing plant were analyzed, it was found that the top productivity runs switched from lactate production to lactate consumption, while low productivity runs remained in lactate production mode throughout the culture. This is an indication that cell metabolism plays a key role in determining productivity.

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Glucose and Energy Metabolism

Glucose Oxidation • Main metabolic pathways in energy metabolism: • Glycolysis • TCA cycle (tricarboxylic acid cycle)(Kreb cycle) • Pentose phosphate pathway (PPP)

Oxidation of Glucose

Glucose is mainly catabolized through three pathways: glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid cycle (TCA) cycle. In glycolysis, one mole of glucose is converted to two moles of pyruvate. In this segment of catabolism, only a small fraction of the chemical potential energy of glucose is converted to the “usable” form of chemical potential energy in the cell, i.e., ATP. Two moles of ATP are generated per one mole of glucose. Pyruvate may enter the TCA cycle for further oxidation, or it may become a shunted product as lactic acid (at a neutral pH it exists as lactate). Through the TCA cycle, the carbon skeleton of glucose is finally broken down to CO2 and H2O. The PPP is a shunt from glycolysis. It generates five-carbon sugars for nucleoside synthesis and supplies NADPH for many biosynthesis reactions and to maintain a redox state in the cell.

In eukaryotic cells, glycolysis and PPP take place in the cytosol, while the further oxidation of pyruvate to CO2 occurs in the mitochondria. It is in the mitochondria that the majority of the chemical potential energy of glucose is converted to ATP for use in cellular synthesis and other energy-dependent cellular processes.

In glycolysis, glucose is broken down to two pyruvates and generates two ATP and two NADH. Pyruvate may then be further converted to lactate as a final product, instead of entering the TCA cycle. Thus, when glucose metabolism is terminated at glycolysis, it produces two lactate molecules and two ATP, but no additional NADH.

Although overall glycolysis generates high-energy compounds (e.g., ATP and NADH), its first segment actually consumes ATP. Two ATP are used to add a phosphate group to each end of the glucose molecule. The two phosphate groups pull their surrounding electron clouds toward the two ends of the molecule, thereby making the carbon-carbon bond

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Glycolysis - Each Mole of Glucose (6 Carbons)

• Consumes two moles ATP (to activate to fructose, 1, 6-bisphosphate) • Produces two moles NADH, 4 moles ATP • Net: Becomes 2 Pyruvate Produces 2ATP, 2NADH

TCA cycle

• Pyruvate enters mitochondrion • Pyruvate loses CO2, becomes acetyl CoA (2 carbons) • Acetyl CoA enters TCA cycle, • Becomes 2 CO2 • Produces NADH, FADH2, which stores energy • Never reacts with oxygen directly

NADH, FADH2 enter electron transfer pathway passes its high energy electron down, also its proton • As electron passes on energetic ladder, it pumps protons out of mitochondrion • create a high pH inside mitochondrion • also a negative charge of ~120 mV across mitochondrion inner membrane • The electron and proton, at the bottom of energetic ladder, react with oxygen to form water.

Oxidative Phosphorylation Pathway

• The higher concentration of proton in cytosol and the negative charge inside mitochondrion drives proton to move into mitochondria • Those protons moves into mitochondrion by passing through ATP synthase; as they pass through ATP synthase, ADP is converted to ATP inside mitochondrion • There are a lot of fluxes across mitochondrial membrane, inclusind • going in: pyruvate, ADP, phosphate, H+, • going out: ATP, CO2

in the middle susceptible to enzymatic cleavage.

After cleavage, the six-carbon skeleton becomes two three-carbon compounds: glyceraldehyde-3phosphate (G3P) and dihydroxyacetone-phosphate (DHAP). These two compounds are interconvertable through a reversible reaction. The continued reaction of glyceraldehyde-3-phosphate (G3P) effectively draws DHAP toward G3P and moves it further downstream in glycolysis. The conversion of two G3P to the end product of two pyruvates also converts two NAD+ and four ADP to two NADH and four ATP. The net energetic consequence of the conversion of glucose to two pyruvate in glycolysis is the generation of two ATP (because two ATP are consumed to activate glucose) and two NADH.

The further oxidation of pyruvate takes place in the mitochondria, where it is first converted to acetyl CoA, releasing one CO2 and generating one NADH. Acetyl CoA is then fed into the TCA cycle where it is broken down to two CO2. The pathway is cyclic, with four- to six-carbon skeletons cycling in a loop. At the beginning of the cycle, the four-carbon oxaloacetate (OAA) takes in acetyl CoA to become citrate. Citrate has three carboxylic acid groups; hence the name “tricarboxylic acid cycle”. The TCA cycle is also known as the citric acid cycle and the Krebs cycle.

As noted before, molecular oxygen does not react with the carbon compounds in the reactions from the TCA cycle. CO2 is released, through decarboxylation reactions, from the carbon skeleton without the participation of molecular oxygen. In two of the reactions catalyzed by pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, the energy from the breakup of the C-C bond is preserved in the high-energy compounds acyl CoA (acetyl CoA and succinyl CoA, respectively) and NADH. In the other case, one of the three carboxylic acid groups in citrate is released and one NADH is generated.

If the carbon-carbon bond is broken by directly reacting with oxygen, such as the case for combustion, a very high temperature is necessary to provide the activation energy. As we all know, it takes a fire to burn wood. Furthermore, the

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Figure 3.2. Major pathways in energy metabolism. Glucose and glutamine uptake, glycolysis, lactate excretion, TCA cycle, and oxidative phosphorylation. Symbols of metabolites in energy metabolism: Glc: Glucose; g6p:Glucose 6-phosphate; f6p:Fructose 6-phosphate; f16bp (f16p2): Fructose 1,6-bisphosphate; f26bp (f26p2): Fructose 2,6-bisphosphate; gap: Glyceraldehyde 3-phosphate; Dhap: Dihydroxyacetone phosphate; 1,3bpg: 1,3-bisphosphoglycerate; 3pg: 3-phosphoglycerate; 2pg: 2-phosphoglycerate; pep: Phosphoenolpyruvate; pyr: Pyruvate; lac: Lactate; NADH: Nicotinamide adenine dinucleotide (reduced); NAD: Nicotinamide adenine dinucleotide (oxidized); NADPH: Nicotinamide adenine dinucleotide phosphate (reduced); NAPD: Nicotinamide adenine dinucleotide phosphate (oxidized); Gln: glutamine; Glu: glutamate; Asp: aspartate; ala: alanine; Mal: malate; αKG: α-ketoglutarate; OAA: oxaloacetate; SucCoA: succinyl CoA; 6pg: 6-phosphogluconate; ru5p: ribulose 5-phosphate; r5p: ribose 5-phosphate; xyl5p: xylulose 5-phosphate; e4p: erythrose 4-phosphate; s7p:sedoheptulose 7-phosphate Symbols of enzymes and transporters in energy metabolism: GLUT: Glucose transporter; HK: Hexokinase; GPI: Glucose phosphate isomerase; PFK: Phosphofructokinase; PFKFB: 6-phosphofructo-2-kinase/ fructose-2,6 bisphosphatase; ALDO: Aldolase; TPI; Triosephosphate isomerase; GAPD: Glyceraldehyde 3-phosphate dehydrogenase; PGK: Phosphoglycerate kinase; PGM: Phosphoglycerate mutase; ENO: Enolase; PK: Pyruvate kinase; LDH: Lactate dehydrogenase; PYRH: Pyruvate mitochondrial transporter; G6PD: Glucose 6-phosphate dehydrogenase; 6PGD: 6-Phosphogluconate dehydrogenase; RPE: Ribulose phosphate epimerase; RPI: Ribose phosphate isomerase; TK: Transketolase TA: Transaldolase; PRPPS: Phosphoribosylpyrophosphate synthetase; PYRH: Pyruvate mitochondrial transporter; MCT: monocarboxylate transporter

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chemical potential energy would have been released as heat. Cells utilize decarboxylation reactions to form CO2 and to preserve energy in NADH.

Oxygen is then used to extract chemical potential energy from NADH and FADH2, in order to generate ATP that can be used in cellular work. The participation of oxygen in the oxidation of NADH/FADH2 generates the six O2 molecules required to oxidize one glucose, as shown in the stoichiometric equation of glucose oxidation. Fig. 3.3: Structure of key compounds in glycolysis.

Fig. 3.4: Structure of key compounds in TCA cycle and glutamine metabolism.

Extraction of the chemical potential energy of NADH and FADH2 takes place through an electron transfer chain residing in the mitochondrial inner membrane. The high-energy electrons of NADH and FADH2 enters the electron transfer chain down the energy ladder, mediated by electron carriers such cytochrome C. The energy released is then used to trigger a proton pump to drive H+ out of the mitochondrial inner membrane. In the last step of the electron transfer chain, the electron reacts with oxygen and H+ to form water.

The export of H+ from the mitochondria creates a single unit pH difference across the membrane, as well as about -120 mV of electric potential. Because of the higher pH (lower proton concentration) and excess negative charge inside the mitochondrial membrane, there is a propensity for the proton ions outside the mitochondria to cross the mitochondrial membrane. They enter the mitochondria through an ATP synthase embedded in the inner membrane of mitochondria. Through the process, the act of a proton passing through ATP synthase brings an ADP and a phosphate together to synthesize ATP. The electron transfer and the generation of ATP are often referred to as “oxidative phosphorylation”.

The amount of ATP generated per mole of glucose varies somewhat among different species because of their variable expression of ATP synthase. However, in general, the number of ATP generated per mole of glucose is about 30 – 32 for mammals. The older literature tends to list the number as 36 moles of ATP / mole of glucose. Under some physiological conditions, the electron transfer chain

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and oxidative phosphorylation are uncoupled. Instead of generating ATP, the energy from NADH is released as heat to maintain body temperature.

The amount of energy, two ATP and two NADH (or the equivalent of six ATP, since one NADH in the cytosol can be roughly considered to be two ATP), produced from splitting glucose into two moles of pyruvate is only about 1/6 of what can be generated from completely oxidizing glucose to CO2 and H2O. In glucose oxidation, the majority of energy conversion in glucose metabolism therefore occurs in the hundreds of mitochondria in the cell and not in the cytosol.

Pentose Phosphate Pathway

PPP

• Two segments: • Oxidative: remove 1 CO2 from glucose-6- phosphate, • Generate 5 carbon sugar phosphate for synthesis of nucleotides and other compounds • produce 2 NADPH • Molecular transformation • Interconverts 5 carbon sugar phosphate to 3 carbon and 6 carbon • To allow NADPH and 5 carbon sugar to be produced at different ratios • NADPH is important in biosynthesis and in neutralizing ROS

PPP is an important shunt from glycolysis that supplies five carbon sugars and NADPH. PPP is divided into two segments: an oxidative segment and a monosaccharide transformation pool.

In the first segment, glucose-6-phosphate from glycolysis is oxidized and then decarboxylated to form the five-carbon ribulose-5-phosphate and two NADPH. The five-carbon sugar phosphate is used in nucleotide (such as ATP and dATP) synthesis to supply the building units for RNA and DNA.

Cells use two different nicotinamide-adenine dinucleotides as reductive chemical potential energy carriers: NADH and NADPH. NADH is used to store chemical potential energy in glycolysis, the TCA cycle, and lipid catabolism. Eventually, NADH is used to derive ATP in the mitochondria. NADPH, on the other hand, carries a chemical potential that is used in biosynthetic reactions, such as in the synthesis of lipids, nucleotides, etc. NADPH is also used to reduce oxidized glutathione and to regenerate it. The reduced form of glutathione is important in maintaining the cell’s reductive environment and in the suppression of reactive oxygen species (ROS).

The second segment of PPP is a molecular conversion pool that allows a two-carbon aldehyde unit or three-carbon keto units to be translocated among a number of three-carbon to five-carbon aldoses.

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This allows the interconversion of carbohydrate molecules that are three to seven carbons in length. This “mixing pool” enables five-carbon sugars from the first segment of PPP to be connected to glycolysis through six-carbon fructose-6-phosphate or three-carbon glyceraldehyde-3-phosphate.

Lactate Formation Aerobic Glycolysis • Cultured cells and cancer cells undergo glycolysis and produce lactate even at high oxygen concentration • This propensity toward lactate production is not for lack of oxygen (anaerobic glycolysis) • At high glycolysis flux, not all NADH can be oxidized by electron transfer in mitochondria • Lactate production serves to regenerate NAD • continuous supply of NAD is crucial for glycolysis to proceed

The first segment of PPP generates five-carbon ribulose and NADPH at a molecular ratio of 1:2. However, cells do not always need those two compounds at a 1:2 proportion. The molecular conversion in the second segment allows the net flux from glycolysis to PPP to vary to meet the cellular demand of ribose and NADPH at different proportions.

Under anaerobic conditions, some bacteria and yeast produce ethanol or lactate. In the absence of oxygen, the electron transfer chain does not operate because no oxygen is available to receive the electron from NADH. When NAD is not regenerated by NADH oxidation, the TCA cycle ceases to operate. The accumulated pyruvate from glycolysis is then excreted as lactate or ethanol. (Note that we denote NAD+ as NAD in text without the superscript “+” associated with its positive charge).

Mammalian cells in culture take only a small portion of pyruvate generated in glycolysis into their mitochondria, for further oxidation to CO2. They appear to have a limited capacity to translocate pyruvate into the mitochondria. The rest of glucose is converted to lactate. This occurs in spite of the presence of sufficient oxygen. The phenomenon is, thus, different from anaerobic fermentation in bacteria or yeast, and is referred to as “aerobic glycolysis”. Not all cells in our body convert a large portion of the glucose they take up into lactate. The vast majority of cells in our body are in a quiescent (nonproliferating) state. They consume less glucose than proliferating cells. The contrast in cellular glucose metabolism, known as the Warburg effect, was first observed between normal tissues and cancer cells. While normal cells have a lower glucose flux, cancer and proliferating cells consume a larger amount of

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glucose and convert much of the glucose to lactate.

Energetic Yield of Aerobic Glycolysis 1. Oxidation

Glu cos e + 2ADP + 2Pi + 2NAD+ + 2ATP + 2NADH

2Pyruvate

2. Reduction [oxidizing pyruvate to lactate (or ethanol as in yeast)]

2Pyruvate + 2NADH

2Lactate + 2NAD +

Net Reaction:

Glu cos e + 2ADP + 2Pi

2 Lactate + 2ATP

Lactate synthesis is catalyzed by lactate dehydrogenase. This reversible reaction takes one pyruvate and one NADH to become one lactate and one NAD.

In glycolysis, two ATP and two NADH are generated, along with two pyruvate. Continued glucose metabolism through glycolysis requires continued supplies of both ADP and NAD as reactants. ATP is used by cells to perform many tasks, such as synthesis, maintaining osmotic balance, etc. It is continually being consumed in various cellular reactions and converted back to ADP to resupply the reactant for glycolysis.

NADH is converted back to NAD through the electron transfer chain in mitochondria. To be regenerated in the electron transfer chain, cytosolic NADH must first enter the mitochondria and the regenerated NAD must be exported out of the mitochondria. A reaction with lactate dehydrogenase allows NAD regeneration from NADH to be carried out into the cytosol, thereby enabling glycolysis to continue at a high flux.

Cells in culture convert about 90% of their glucose to lactate. Most of the other 10% of glucose is converted to CO2. At the completion of glycolysis, two ATP are generated while about 30 ATP are generated, following complete oxidation. The 90% of glucose converted to lactate generate 1.8 ATP (2 ATP x 0.9), while the other 10% generate 3 ATP (30 ATP x 0.1). Aerobic glycolysis of proliferating cells generates a significant amount of total energy to allow for proliferation.

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Carbon Flow and Reaction Intermediates

Pyruvate is a controlling node • Generation rate of pyruvate is balanced by entry into the mitochondrion and conversion to lactate • Reduction of pyruvate to lactate recycles NAD+ for glycolysis to continue • Lactate dehydrogenase (LDH) is reversible • Transport of lactate by monocarboxylate transporter which is coupled to proton gradient

Lactate dehydrogenase reaction Pyruvate + NADH Lactate + NAD+

Among all of the pathways in the cellular metabolic reaction network, glycolysis has the highest flux in terms of moles of substrate flowing through it. For cells in culture, carbon flux (based on the number of moles of carbon atoms, or the number of carbons in the compound multiplied by the number of moles of the compound) or molar flux (based on the number of moles of each compound) of glycolysis is normally a few times higher than that of the TCA cycle. PPP flux usually constitutes only about up to 5% of glucose intake.

Glycolysis and the TCA cycle also supply precursors to build cellular components. Culture media do not necessarily supply cells with the right balance of all of the components that they need to synthesize cell mass. The three main pathways for energy metabolism also serve as key distribution centers for carbon skeletons needed for other cellular functions. Glycolysis supplies glycerol phosphate, which is for the synthesis of phospholipids. Glucose-6-phosphate and fructose-6-phosphate are both a source of nucleotide sugars for glycan synthesis, such as UDP-galactose, UDP-glucose, and GTP-mannose. Except for liver cells (hepatocytes), cells in culture have little gluconeogenesis activity; that is, they cannot make hexose from lactate or amino acids. So, even if cells can derive energy from lactate and amino acids, they will still need hexose to synthesize ribose and glycans. Cells in culture take up a large quantity of amino acids, especially glutamine. The intake of amino acids exceeds what is needed to make cell mass and product. The surplus of nitrogen is either excreted as ammonia or transferred to pyruvate to form alanine, and excreted. Since alanine is much less growth inhibitory than ammonium, pyruvate has some moderating effect on ammonium toxicity.

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NADH Balance • While glucose is oxidized in Glycolysis and TCA cycle, its carbons never react directly with O2 • The energy is preserved to NAPH/FADH2, which is then reacted with O2 in oxidative phosphorylation in mitochondria to generate ATP • Altogether 12 reducing equivalents (NADH/FADH2) are generated to react with 6O2, generating 6H2O • 2 of the 12 reducing equivalents, two are generated in cytosol (in glycolysis) 10 in mitochondria

Glucose 6 Phosphate NAD NAD NADH

NAD

Pyruvate

Lactate malate NAD aspartate shuttle

reducing equivalent NADH

cytosol mitochondrion

oxidized NAD

Election transfer chain

Pyruvate

Equal moles of Pyruvate and NADH reducing equiavlent enters mitochondria

NADH- Pyruvate Balance

Fig. 3.5: NADH/NAD balance in cytoplasm. NADH generated in glycolysis is recycled back to NAD via lactate production and malate-aspartate shuttle to transfer the reducing equivalent into mitochondria.

A total of 12 moles of reducing equivalent (10 NADH and 2 FADH2) are produced when 1 mole of glucose is oxidized to CO2. The 12 mole reducing equivalents consume 6 moles of O2 in oxidative phosphorylation, consistent with the stoichiometry of glucose oxidation (1 glucose/6 O2). Among the 12 NADH/FADH2, 10 are produced in the mitochondria and the other 2 NADH are produced in cytosolic glycolysis. The two reducing equivalents produced in the cytosol must then be transported into the mitochondria where they drive the reaction that consumes the 6th molecule of O2.

NADH does not pass through the inner membrane of mitochondria. Rather, it passes its reducing potential through a carrier system called the malateaspartate shuttle. This system takes the reducing equivalent into mitochondria through an exchange of molecules between the mitochondria and the cytosol. On the cytosolic side, NADH is oxidized to NAD and transfers its reducing equivalent to malate by reducing oxaloacetate (OAA). Malate is then transported across the mitochondrial membrane. Once inside the mitochondria, the reducing equivalent in malate is transferred back to NADH by being oxidized to again become OAA.

The net result of the transfer must be that only one NADH in the cytosol can become one NADH in the mitochondria. To maintain that balance, cells employ two transporters: one for transporting malate and the other for transporting aspartate (hence the name “malate-aspartate shuttle”). To maintain the charge balance in the transport process, each of the two transporters transfers a pair of compounds in opposite directions (malate and α-ketoglutarate; aspartate and glutamate). In this fashion, there is no net change in total carbon or nitrogen on either side of the mitochondrial membrane. On each side of the membrane, the same glutamateOAA to α-ketoglutarate-aspartate amino-transfer reaction occurs, but it is in the opposite direction. The transfer of the reducing equivalent of NADH into

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the mitochondria is therefore not only dependent on the NADH concentration but also linked to amino acid metabolism and the activities of the TCA cycle, through the concentrations of associated carriers. Glycolysis and the TCA cycle are thus connected by a cytosolic balance of pyruvate and NADH. The flux of both pyruvate and NADH are stochiometrically linked in glycolysis, as well as through the LDH reaction. As a result, the fluxes of pyruvate and NADH entering the mitochondria are also stoichiometrically related. Regardless of whether cells are at a lactate-producing or -consuming state, the molar ratio of pyruvate flux to NADH flux is always one.

1,2-Bisphospho glycerate

Glyceraldehyde 3-Phosphate

Glucose

NAD+

Pyruvate

NADH

NAD+

QAA

Lactate Malate-Aspartate Shuttle Aspartate

Mitochondria

Glu

Aspartate Glu

αKG

Malate

αKG

Malate

NAD+ Pyruvate

NADH

OAA

Oxidative Phosphorylation ATP

Fig. 3.6: Detailed reactions of malate-aspartate shuttle.

• • • •

Each enzyme reaction/co-transportation is given in the same color Direction of reaction in NHDH reducing equivalent transport is indicated by an arrow Net transport of one reducing equivalent-1 NADH in cytosol, and +1 NADH in mitochondria Transport of 1 malate + 1 glutamate into mitochondria and 1 αKG + asparate in cytosol

Fig. 3.7: Transfer of NADH reducing equivalent from cytoplasm into mitochondria requires continuous transport of four malate, aspartate, glutamate and α-ketoglutarate.

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Fig. 3.8: Biochemical reactions in glycolysis, TCA cycle and Pentose phosphate pathway

Energetic Yield of Oxidation of Glucose Cytosol

glu cos e

2pyruvate + 2NADH + 2ATP

Mitochondrion

2pyruvate 2acetylCoA

2acetylCOA + 2NADH + 2ATP + 2CO2 6NADH + 2FADH2 + 2GTP + 4CO2

• The overall energetic yield is ~30ATP, considering the cost of ATP transport out of mitochondria • The NADH generated in cytosol (Glycolysis) is recycled back to NAD+, either through reduction of pyruvate to lactate, or by NADH/NAD+ shuttle into mitochondria for oxidation.

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Role of Transport and Transporters in Metabolism

Glucose Transporters Two Main Types of Glucose Transporters • GLUT transporters mediate facilitative diffusion across plasma membrane. • SGLT, the sodium dependent glucose co-transporters are expressed primarily in small intestinal absorptive cells or renal proximal tubular cells. They use Na+-K+ ATPase pump for active transport of glucose.

Glucose Transporter Isoform • GLUT1 is highly expressed in all cells • Km is small for GLUT1. At culture glucose concentration it operates at maximum rate.

Energy metabolism takes places in multiple compartments, separated by lipid bilayer membranes. First, glucose must cross the cytoplasmic membrane to undergo glycolysis in the cytosol. The products from glycolysis (pyruvate and NADH, in the form of reducing equivalents) are transported into the mitochondria. This exchange of molecules across the membrane is mediated by membrane transporters.

Glucose transporters mediate the influx of glucose across the cytosolic membrane. Generally speaking, there are two types of glucose transporters: GLUT and SGLT.

The GLUT transporters are uniporters for facilitated transport, allowing glucose to move along its concentration gradient. They have twelve transmembrane regions and intracellular carboxyl and amino termini. According to common sequence motifs, they are divided into three subclasses.

GLUT1 is ubiquitous, found in almost all cells. It can transport glucose and galactose in a concentrationdependent manner that is described by MichalisMenten kinetics. The Km for glucose is very low (1 – 2 mM). At the glucose concentration used in culture medium, the flux of GLUT1 is at its maximum. In some cells, GLUT1 is under the regulation of the transcription factor HIF-1 (hypoxia inducible factor). Under hypoxic conditions, the expression of GLUT1 is up regulated to increase the uptake rate of glucose. The Km of GLUT1 for galactose is rather high. When galactose is used as the only sugar, even at a concentration of 25 mM, the uptake rate is so low that only a little lactate is produced.

Fig. 3.9: Michaelis-Menten kinetics plot for GLUT1 transporter

A few other notable GLUT transporters are: insulin responsive GLUT4 and fructose transporting GLUT5. In addition to GLUT1, cells in culture and in different tissues may express other GLUT transporters at different proportions. The expression of different transporters will give them different responses to the concentration of glucose or other sugars.

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Table 1: Glucose Transporters Tissue Expression ubiquitous

glucose, K m = 1 - 2 mM

Liver, pancreas, intestine, kidney

glucose, K m = 16 - 20 mM glucosamine K m = 0.8 mM glucose, K m = 0.8 mM

GLUT5

brain, neurons heart, muscle, adipose intestine, testis

GLUT7

intestine, testis

GLUT9

kidney, liver

GLUT11

heart, muscle brain, spleen, leukocytes testis, brain, liver liver, pancreas heart, muscle, prostate

GLUT1 Class 1

GLUT2 GLUT3 GLUT4

Class II

GLUT6 Class III

GLUT8 GLUT10 GLUT12

Affinity

glucose, K m = 5 mM fructose K m = 10 - 13 mM glucose, Km = 0.3 mM fructose K m = 0.1 mM fructose K m = ? mM fructose K m = ? mM glucose, K m = 5 mM glucose, K m = 6 mM glucose, K m = 0.3 mM not well known

Lactate Transport

Fig. 3.10: Monocarboxylate transporter for lactate and pyruvate

The second type of glucose transporter, SGLT, is a co-transporter with Na+. It transports two sodium ions and one glucose molecule into the cell. The Na+ concentration is low intracellularly but is high in the medium and in body fluid. The large sodium concentration difference and negative electric potential across the cytoplasmic membrane gives rise to a high propensity of Na+ to enter the cell. Thus, the chemical potential energy of the sodium gradient and electric potential is used to drive the uptake of glucose against a concentration gradient. SGLT transport is abundant in intestinal epithelial cells and is responsible for moving glucose from the gut into the intestinal epithelial cells. The glucose is then exported into the blood stream on the other side of the cellular barrier.

Lactate and pyruvate are transported by monocarboxylate transporters. These transporters exist in two forms: one on the cytoplasmic membrane and one on the mitochondrial inner membrane. Lactate and pyruvate are both negatively charged. Their movement across the cellular membrane will cause a charge unbalance and create an electric potential, unless measures are taken to counter that imbalance. The monocarboxylate transporters (MCT), which are responsible for their transport, are a family of co-transporters that couple the transport of lactate or pyruvate to the transport of a hydrogen ion in the same direction to neutralize the charge transfer. MCT is thus a symporter; its mechanism of transport is facilitated diffusion. Lactate transport is enhanced by a large difference in lactate concentration between intracellular and extracellular environments. pH also affect the flux of lactate through MCT, however, whether the

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Transport Across Mitochondria

effect is enhancing or retarding is dependent on the direction of the proton gradient. MCT allows for lactate transport in both directions, for excretion as well as uptake. Keeping medium pH at a lower level reduces lactate production during rapid growth period, but enhanced lactate consumption in the stationary phase.

Cells in culture typically channel about 1/20 of the carbons from their glucose intake to the TCA cycle • The chemical potential energy generated by pyruvate and oxidize them to CO2. The molar flux of pyruvate oxidation/TCA cycle, i.e. NADH and FADH2, is not necessarily all converted to ATP. The process can be into the mitochondria is thus about 1/10 of that of decoupled to generate heat instead of ATP, as occurs in the glucose consumption rate. Each mole of pyruvate hibernating mammals. entering the TCA cycle via acetyl CoA generates • The mitochondrion is also the main site of molecular about 15 moles of ATP. These are exported to the interconversion and degradation of amino acids and cytosol and require the import of equal moles of ADP the main source of acetyl CoA. The excess glutamine and PO 3- for their synthesis. Each mole of pyruvate 4 consumed enters the TCA cycle through α-ketoglutarate. generated from glycolysis or lactate consumption For some cells, asparagine acts as a sink (in addition to alanine), which is formed through oxaloacetate and is also accompanied by one mole of NADH, whose reducing equivalent is transferred into the aspartic acid. mitochondria through the malate-aspartate shuttle. Additionally, cells in culture consume glutamine at a high rate (approximately 1/5 to 1/10 of glucose in molar ratio). Nearly half of the glutamine enters the TCA via α-ketoglutarate. CO2 produced in the TCA cycle is then exported out of the mitochondria. Besides these major species, many other molecules (including amino acids and nucleotides) are transported into the mitochondria for DNA, RNA, and protein synthesis. As will be described later, the precursor for fatty acid and cholesterol synthesis, acetyl CoA, is generated in the mitochondria, while fatty acid and cholesterol synthesis occurs outside the mitochondria. Acetyl CoA is very reactive and does not get transported directly across the inner membrane of the mitochondria. Rather, it is transported out of the mitochondria as citrate. After cleaving off acetyl CoA, the remaining four carbons are returned to the mitochondria as pyruvate or malate. Thus, the citrate and malate flux across the mitochondrial membrane is also substantial to sustain lipid and cholesterol synthesis. The transport across the mitochondrial inner

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Regulation of Glucose Metabolism

membrane is dynamic and complex. Many compounds crossing the membrane are charged, yet their transport must not perturb the proton and electric potential gradient. The transport across the mitochondrial membrane must be tightly regulated. Our understanding of its regulation is still rather limited.

Under physiological conditions, the flux of glycolysis and the TCA cycle is not controlled by one or a small number of “rate-limiting” enzymes. Glucose flux is the result of mutual constraints of many enzymes in the pathway, through their feed-forward and feedback inhibition and activation. A large number of pathways are highly inter-connected and crosstalk with each other through shared common substrates or regulators. In mammals, different tissues serve different metabolic roles to maintain the overall homeostatic state of the organism. The partition of metabolic roles is largely accomplished by giving different tissues a different set of isozymes. Different isozyme sets allow cells to respond to environmental fluctuations or cellular cues differently.

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Isozymes and Differential Allosteric Regulation

• Cells express different isozymes in different tissues, under different conditions • different isozymes have different kinetics and regulation

Different isoforms (isozymes) of glycolytic enzymes catalyze the same reaction step, but have very different kinetic properties and are often subjected to contrasting regulations. Isozymes of four glycolysis enzymes, hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and phosphofructokinase/fructose biphosphatase (PFKFB), play key roles in controlling glycolysis flux. Their different allosteric regulations give them very distinct reaction characteristics.

The isoforms of those enzymes making up glycolysis are different in proliferating and quiescent cells. They are thought to be responsible for endowing the high glycolysis flux and high lactate production in proliferating and cancer cells and are thought to be related to oncogenic transformation. They also give tissues their metabolic capabilities.

Fig. 3.11: Allosteric regulations in glycolysis. Note strong activation of glycolysis flux by F16BP and inhibition by lactate.

PFK is pivotal in modulating the overall rate of glycolysis and is a key node in energy metabolism. Its activity is subjected to allosteric inhibition by ATP and citrate, and is activated by AMP. PFK has three isozymes: liver (PFKL), muscle (PFKM), and platelet (PFKP). Among the three isoforms of PFK, the muscle and the liver isozymes are activated allosterically by fructose 1,6-bisphosphate (F16BP). Muscle phosphofructokinase is inhibited by lactate, a characteristic which may facilitate reduced glycolysis flux at high lactate levels.

Fructose-2,6-phosphate is a shunted glycolytic intermediate that plays a key regulatory role in rapidly modulating the activity of PFK. All three PFK isozymes are activated by fructose 2,6-bisphosphate (F26BP). F26BP activates PFK1 by allosterically increasing its affinity for F6P, even in presence of inhibitors such as ATP, lactate, etc. The synthesis and degradation of F26P is catalyzed by a bi-functional enzyme, PFKFB (also known as PFK2); its kinase activity catalyzes the synthesis of F26P and its phosphatase activity catalyzes the hydrolysis of F26P to F6P. PFKFB has four isozymes, each with different kinase and phosphatase activities, allowing each to

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• F2,6P plays a key regulatory role • Key regulatory enzyme of glycolysis • PFK (PFK1) • PFK2 • PKM • Cancer cells (fast growing cells) and quiescent cells have different isozymes

respond differently to regulators. The brain isoform, PFKFB3, has the highest kinase to phosphatase activity and is expressed in several tumor cells. This suggests that PFKFB3 can be accountable for the glycolytic phenotype of reported cancerous cell lines by allowing them to have high cellular F26P levels.

The enzyme catalyzing the penultimate step of glycolysis, pyruvate kinase, has three isozymes in mammalian systems. The muscle isozyme is expressed as either of the two splice variants, M1 or M2. The M1 isoform is mostly expressed in adult tissues whereas the M2 isoform is expressed exclusively in rapidly growing tissues, such as fetal and tumor tissues, and also is thought to be a critical player in the transformation leading to cancer. In addition, the M2 isoform is known to be under positive feed forward regulation by F16P. Isozymes are often named after the tissue in which they are the dominant isoform. However, it is important to remember that the expression of isozymes is not limited to one form in a cell type. Different isoforms are often expressed in the same tissue or the same cell. Different combinations of isozymes give rise to different kinetics and regulatory behaviors that may meet different physiological needs.

With the available genomic tools, we can easily determine the relative expression of different isoforms of the key enzymes of glycolysis, and further evaluate how to influence cellular metabolism.

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Signaling Pathways and Regulation by Growth Control

The regulation of cellular metabolism is tightly linked to the control of cell growth. The signaling pathways that regulate growth rate (involving key Signaling pathway/growth rate control of players like p53 and cMyc) also play regulatory roles Glycolysis in regulating glucose metabolism. Transformation • Insulin signaling that causes cells to switch from a quiescent state to a proliferating state also triggers metabolic changes • positively regulating growth rate • through AKT regulate glucose, amino acid metabolism to increase their glucose uptake and glycolysis flux. • P53 (tumor suppressor) supresses glucose uptake and p53 is a major tumor suppressor that plays key glycolysis roles in cell cycle arrest, senescence, and apoptosis.

Its role in the regulation of glucose metabolism and • Fast growing (tumor) cells have fast glycolysis (and oxidative phosphorylation was not well understood until only recently. It induces the overexpression of lactate production) TIGAR under mild oxidative stress conditions. TIGAR contains a fructose-2,6-bisphosphatase catalytic Regulation of Metabolism activity domain, which mediates the degradation of Glucose fructose 2,6-phosphate, leading to a decrease in PFK1 GLUT1,4 p53 activity and the attenuation of the glycolytic flux. • cMyc (proto-oncogene) stimulates glycolysis

Glucose

H2O

Glucose-6TIGAR Phosphate

Fructose-2,6Bisphosphate

PPP

NADPH

ROS

Fructose-6Phosphate

PFK2

PFK1 Transcriptional Inhibition

Fructose-1,6Bisphosphate

Transcriptional Activation Allosteric Activation

p53

PGM

Lactate

Pyruvate NADH

SCO2

p53

COX ATP

Mitochondria

Fig. 3.12: Tumor suppressor p53 negatively regulates glycolysis flux.

Akt and Myc Regulation of Metabolism Glucose

Akt

Fructose-2,6Bisphosphate

GLUT1 Glucose HK Glucose-6Phosphate

Activation by phosphorylation or localization Transcriptional Activation Allosteric Activation

PFK2 Fructose-6Phosphate PFK1

Myc

Fructose-1,6Bisphosphate LDH Pyruvate

Malate

Lactate

GLS Glutamate Glutamine

TCA

ASCT2 SN2 Glutamine (Extraacell ular)

p53 can also modulate glycolytic rate by regulating the activity of PGM, GLUT1, and GLUT4 transporters. Furthermore it up-regulates mitochondrial oxidative phosphorylation by upregulating the expression of SCO2 (synthesis of cytochrome c oxidase 2), which mediates the assembly and activity of the cytochrome c oxidase complex.

Pro-oncogenic genes may also provide the link between increased glycolytic flux and oncogenic transformation. Akt is a pro-oncogene which, when deregulated, affects many cellular functions including metabolism, cell proliferation, and protein synthesis. In tumor cells, constitutively activated AKT is sufficient to shift the cells from a primarily oxidative state to a primarily glycolytic state.

Myc is another proto-oncogene whose pleiotropic regulatory roles include energy metabolism. Glycolytic enzymes have Myc canonical E-boxes in their promoter and are deregulated when Myc is overexpressed.

Mitochondria

Fig. 3.13: Signaling kinase, AKT and transcription factor, Myc, positively regulate energy metabolism.

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Metabolic Homeostasis and Lactate Consumption Cells in culture, including those used in bioprocessing, have all been selected for their capability to proliferate. Their particular set of glycolytic isozymes directs their metabolism to a high glucose flux and a high rate of lactate production. However, even the exact composition of isozymes is not monolithic among different cells. Most cells express multiple isoforms of the same enzyme in glycolysis and their proportion is not identical. Consequently, even though the • Metabolism is a balancing act of many constraining metabolism of all cultured cells share the common trait of a high glucose flux and high lactate production, reaction nodes their metabolic behaviors are not all identical. • Glycolysis flux influenced by: Lactate accumulation in culture inhibits cell growth • Regulation (feed back and growth) and hastens the decline of cell viability in the • Pyruvate/NADH flux into mitochondria stationary phase. A key to achieving a high cell • LDH (NAD recycle rate) concentration and high productivity is to direct cell • Lactate export metabolism to minimize the accumulation of lactate. • Glucose intake One may aim to alter the cells’ metabolism to reduce or eliminate lactate production. However, it is important to keep in mind that virtually all rapidly proliferating cells produce lactate. Such a metabolic state might be a default state that is “essential” for cell growth and cannot be easily altered over a long period without affecting growth behavior. It is also important to keep in mind that through in the course of culturing cells for manufacturing, the duration of the actual production period is only a very small fraction of the total process lifetime. The long duration of the process time is mostly for expanding cell numbers to reach a production scale. Altering the metabolism from the cell’s “default” state may have an unforeseen effect on cells. Another approach for alleviating lactate inhibition is to tamper with the cell metabolism only in the production scale or in the final stages of production. Increasing evidences show that the switch from lactate production during the growth stage to lactate consumption in late stages is correlated with a high productivity. One may seek to alter cell metabolism to change lactate consumption in the last stage to be more reproducible.

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The LDH reaction that catalyzes pyruvate to lactate is reversible. The direction of the flux depends on the concentrations of NAD, NADH, pyruvate, and lactate. Those concentrations, in turn, are related to fluxes of reactions that produce or consume them, including pyruvate entry into mitochondria and lactate export (or import). Further observations related to lactate consumption:

Cycle

Lactate consumption occurs in post-rapid stages of growth, when the glucose consumption rate is low. The pyruvate flux into the mitochondria is rather limited. When cells are at a high glucose flux, the excess pyruvate must be converted into lactate to regenerate NAD+. Therefore, a high glucose flux state is always linked to a lactate production state. Furthermore, rapid proliferation is generally associated with high glucose flux. Lactate consumption occurs only when the growth rate is slow. Experimental observation has demonstrated that the absence of rapid growth and a low glucose consumption are necessary conditions for lactate consumption.

Glucose is still being consumed while cells consume lactate. While lactate is being taken up by cells, its specific consumption rate is relatively small. Lactate is converted to pyruvate, and then it enters the mitochondria for further oxidation and energy generation. Even though energy is being derived through lactate oxidation, cells still need many constituents derived from glucose for glycan synthesis, including NADPH (from PPP), glucosamine, and galactose. These compounds cannot be derived from pyruvate in most culture cells, since they lack key enzymes for gluconeogenesis. They must be supplied through glucose. The glucose consumption rate in the lactate consumption stage is small, but it is never zero.

Cycle

Fig. 3.14: Metabolic fluxes in lactate production and consumption metabolism.

The conversion of lactate to pyruvate by LDH does not occur in isolation. It is coupled to the reduction of NAD to NADH (also catalyzed by LDH), and linked to the reverse transport of lactate and H+ by MCT from the medium to the cytosol. The propensity and rate of lactate consumption is affected by the pH of the media. In addition to transporting pyruvate

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formed from lactate into mitochondria, NADH has to be transported from the malate-aspartate shuttle into the mitochondria for oxidation.

Glutamine and Its Relation to Energy Metabolism

For most cultured cells, glutamine is the second highest consumed nutrient. Its molar consumption Glutamine rate is about 1/5 to 1/10 of that of glucose for most • Consumed by growing cells at a high rate cells. Glutamine is a major amino acid constituent • Non-essential in vivo. Glutamine synthesase decreases of cellular proteins. It supplies amino groups for the upon in vitro culture synthesis of purine and pyrimidine bases, which are • Supply TCA cycle intermediates the backbone of nucleic acids. However, the amount • causes NH3 release of glutamine consumed by cells far exceeds what is needed for synthesizing cellular components. • used in nucleotide and protein synthesis Glutamine is not an essential amino acid for mammals; it is only essential for cultured cells. Cells in some tissues, like the liver, synthesize glutamine by the incorporation of an ammonium into glutamate at the expense of an ATP using glutamine synthase. The transcript level of glutamine synthase in cultured cells is low. It actually decreases by nearly two orders of magnitude when liver cells are put into culture.

A large portion of glutamine is converted to glutamate by glutaminase, and then to α-ketoglutarate before entering the TCA cycle. Through α-ketoglutarate, glutamine is a major contributor to central metabolic flux. There is increasing evidence that glutamine is needed to drive the TCA cycle for faster growth.

Fig. 3.15: Entry of glutamine into TCA cycle. Glutamine is converted to glutamate by glutaminase. Glutamate is then convert to α-ketoglutarate by either transamination reaction (transfer its α-amino group to pyruvate or oxaloacetate) or via oxidation by glutamate dehydrogenase (converting NADP to NADPH and releasing ammonium).

In the course of being converted to glutamate and α-ketoglutarate, the nitrogen is released as ammonium or transferred to an amino acid, such as alanine or asparagine, and excreted. The ammonium that is released from glutamine contributes to the waste metabolite accumulation.

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Amino Acid Metabolism Mammals can synthesize only fewer than 10 of the 20 amino acids used in the translational synthesis of proteins. 11 or 12 (depending on the species) essential amino acids that they cannot synthesize must be acquired through diet.

Fig. 3.16: Major amino acid transporters

• Amino acids are transported with overlapping transporters. • All IgG antibodies produced by mammalian cells are glycoproteins, with an N-linked oligosaccharide attached to each heavy chain in the hinge region at Asn-297

Amino Acid Degradation • Amino acids are consumed at a rate of three to five times what is needed for making biomass and product. • Excess amino acid consumed must be excreted. • The nitrogen (amino group) is removed from the carbon skeleton by transamination, oxidative or non-oxidative deamination. The excess nitrogen is excreted as ammonium ion or as excreted amino acids (e.g. alanine, proline, asparagine) • The carbon skeleton mostly enters the TCA cycle to be converted to excreted non-essential amino acids or to be converted to pyruvate and lactate, or to contribute to acetyl CoA in cytosol through citrate. • Large excess of glutamine consumed by cells in culture is converted to glutamate in the cytosol, or is transported into the mitochondria and then converted to glutamate. Glutamate is then converted to α-ketoglutarate and enters carbon metabolism. • Glutamine (amide group) is used as an amino group donor in adenosine (AMP), guanosine (GMP) and Cytosine (CTP) biosynthesis. • Aspartic acid and glycine are also used in nucleic acid synthesis • Methionine is methyl group donor. Tryptophan is used in NAD synthesis • Glutamate participates in a large number of reactions. The flux of its synthesis or supply is expected to be high.

Cells in culture have more essential amino acids requirements than the organism; a number of non-essential amino acids become essential for in vitro culture, including glutamine, tyrosine, serine under some growth conditions, proline for some cells. All essential amino acids must be supplied in medium, while non-essential amino acids can be derived from other amino acids. Amino acids are taken up by cells through a large number of amino acid transporters. Most amino acid transporters transfer a family of amino acids with similar characteristics, such large neutral (uncharged side chain) amino acids, cationic or anionic amino acids. The rate of transfer for particular amino acids is thus not only dependent on the concentration difference of itself between intracellular environment and extracellular medium, but also on the competition with other amino acids for the same transporter. One amino acid may be taken up via more than one transporter, albeit with different affinity.

The amino acids taken up by cells are not likely to be in the “right” stoichiometric ratios to meet their need. Excess amount of amino acids is converted to the non-essential amino acids which are not supplied in sufficient quantities, or is degraded. The interconversion and degradation of amino acids takes place through the intermediates of glycolysis and TCA cycle.

Before the carbon skeleton of an amino acid enters carbon metabolism pathways its amino group or other nitrogen containing functional groups is first removed. Glutamine and glutamate become α-ketoglutarate, asparagine and aspartate become oxaloacetate; all are catabolized through the TCA cycle. Alanine becomes pyruvate, while leucine, isoleucine and others enter carbon catabolic pathway through

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acetyl-CoA. Although the degradation of amino acids yields energy, it is not a major energy source.

Alanine Cysteine Glycine Serine Threonine Tryptophan Pyruvate

CO2

Acetyl-CoA

Acetoacetate

Isoleucine Leucine Lysine Threonine

Citrate

Asparagine Aspartate

Oxaloacetate

Aspartate Phenylalanine Tyrosine

Isoleucine Methionine Valine

Isoleucine Leucine Lysine Threonine

Citric acid cycle

Fumarate

The excess nitrogen from amino acid degradation is excreted as ammonia or as the amino group of non-essential amino acid, especially as alanine or proline. In some cell culture processes ammonium accumulates to growth inhibitory levels. Although the adverse effect of ammonium on cell growth is usually not as severe as lactate accumulation.

Isocitrate CO2 α-Ketoglutarate

Succinyl-CoA CO2

Arginine Glutamate Glutamine Histidine Proline

Fig. 3.17: Entry of amino acids into catabolism

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Lipid Metabolism Functions of Lipids • Contributes to the membrane fluidity • Storage of precursors metabolized to second messengers (diacylglycerol, inositol triphosphate) • Lipids (such as, polyphosphoinositide) involved in protein traffic and membrane fusion events • Anionic lipids (such as, PS) involved in attachment of cytoskeletal proteins to membranes • Cholesterol and sphingolipids form microdomains or ‘rafts’: enriched in specific subsets of membrane proteins

Subcellular Localization of Lipid Metabolism in Animal Cells Cytosol

• NADHP synthesis (pentose phosphate pathway, malic enzyme) • Isoprenoid and early cholesterol synthesis • Fatty acid synthesis

Mitochondria:

• Fatty acid oxidation

Lipids play key roles in many physiological functions critical to cellular properties of particular interest to bioprocessing. In addition to forming the bilayer membrane, lipids are also involved in cell signaling. Lipid content in bilayer membrane affects membrane fluidity and permeability. Lipid bilayer membrane partitions various organelles from the cytosol. Secreted recombinant proteins are processed first in endoplasmic reticulum (ER) and the Golgi apparatus. They are excreted via membrane vesicles. For optimal protein secretion capacity, the membrane homeostasis and biogenesis among organelles, secretory vesicles and plasma membrane is critical. Not all lipid bilayer membranes are the same. The lipid composition of ER, mitochondrial and plasma membrane differ from each other. The plasma membrane of hepatocytes is enriched in cholesterol; however, the amount of cholesterol in the rough and smooth inner ER is lower. There is very little cholesterol in the inner mitochondrial membrane.

• Acetyl-CoA synthesis • Ketone body synthesis • Fatty acid elongation

Endoplasmic Reticulum: • Phospholipid synthesis

• Cholesterol synthesis (late stage) • Fatty acid elongation • Fatty acid desaturation

Peroxisome:

• Cholesterol precursors on synthesis • Final steps of cholesterol synthesis also occurs in peroxisome

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Lipid Transport Lipid Transport Processes Intramembrane Transport: Transbilayer movement of lipid molecule • Transbilayer movement at eukaryotic ER: relatively nonspecific, ATP-independent process • Three classes of transport: • Aminophospholipid translocases (also called flippases) • Bidirectional transporter or scramblases

• ATP Binding Cassette (ABC) pumps Intermembrane Transport: Movement of lipid molecules from one distinct membrane domain to another Possible mechanisms: • Monomer solubility and diffusion (for molecules such as free fatty acids) • Soluble carriers such as lipid transfer proteins • Carrier vesicles

Although some cells can be cultured in lipid-free medium, most cell culture media contain some fatty acids and lipids. Cells readily take up fatty acids, phospholipids, and cholesterol from the medium and incorporate them into cellular lipids.

Lipids can be supplied as serum lipoproteins in the form of a complex with albumin or liposomes, or as solubilized conjugates, such as sorbitol-fatty acid esters. The cellular uptake of fatty acids is a passive, non-energy dependent process. After being taken up by cells, fatty acids quickly become esters; the intracellular levels of free fatty acids are quite low. Cholesterol is complexed to low density lipoprotein (LDL) in the body and is taken up by cells through the LDL receptor. For cells in culture, cholesterol is often supplied as a conjugate with serum albumin, or as complexes with cyclodextrin.

• Membrane apposition and transfer • Membrane fusion

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Fatty Acid Metabolism

Most cells have the capability of synthesizing various fatty acids. Under starvation conditions, cells also perform β-oxidation to degrade fatty acids into acetyl CoA in the mitochondria or peroxisomes. Acetyl CoA then enters the TCA cycle to generate energy. Typically, cells in culture do not need to derive energy from fatty acid oxidation. Fatty acids are synthesized from acetyl CoA in the cytosol. The first step of fatty acid synthesis involves adding a CO2 to acetyl CoA to form malonyl CoA, which then reacts with acetyl CoA to become a four-carbon fatty acyl CoA. It is noted that even though CO2 is a catabolic product, it is also an essential nutrient for biosynthesis. Fatty acid synthesis, thus, involves the step-wise elongation processes of using three-carbon malonyl CoA to add a two-carbon unit to fatty acyl-CoA in each cycle. NADPH is also consumed in this process. There are a number of fatty acid synthetases that can synthesize fatty acids to different lengths. The fatty acid products from elongation reactions are all saturated fatty acids. Double bonds are then synthesized by unsaturation reactions after saturated fatty acids are made.

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Acetyl CoA shuttle Acetyl CoA shuttle On mitochondria inner membrane

• Citrate transporter: transport citrate into cytosol • Malate, α-ketoglutarate transportor: transport into mitochondria • Pyruvate transporter: transport into mitochondria

In Cytosol • Citrate lyase: citrate +CoA+ATPOAA+acetylcoA • Malate dehydrogenase: OAA+NADH+Malate +NADH+ • Malic enzyme: malate+NADP Pyruvate +NADPH+CO2

In mitochondria matrix

• Pyruvate carboxylase: Pyruvate+CO2+ATPOAA+ADP • Malate dehydrogenase: malate+NAD+OAA+NADH+ • Citrate synthase: OAA+acetylCoACitrate +CoA

CO2 Pyr

NAD Malate NADPH NADP

Pyr CoA

NADH OAA

CO2

acetyl CoA ADP+Pi Citrate

Acetyl CoA is the building block of fatty acids. It is generated primarily through the oxidative decarboxylation of pyruvate in the mitochondria. However, fatty acid synthesis takes place in the cytosol. Acetyl CoA does not pass through the bilayer membrane. Rather, it is exported to the cytosol via an indirect process. Citrate, formed by condensation of OAA and acetyl CoA in the TCA cycle, is then transported into then cytosol, where it is split into oxaloacetate and acetyl CoA. Oxaloacetate gets reduced to malate at the expense of one NADH. Malate is then converted to pyruvate, losing one carbon and consuming one NADPH. Pyruvate then recycles into the mitochondria. The process of making fatty acids using acetyl CoA is thus energetically expensive.

Fatty acid cholesterol synthesis

ATP CoA

acetyl CoA OAA

Citrate αKG

Malate Fum

Suc

Glu NHg

Glu

Gln

NH3

Fig. 3.18: Citrate and acetyl CoA shuttle.

Cholesterol & Its Biosynthesis

Fig. 3.19: The structure of cholesterol

Cholesterol is a 27-carbon molecule. Mammals require cholesterol as a constituent of membranes and as a precursor for the synthesis of steroid hormones, bile acids, and lipoproteins. Cholesterol is relatively insoluble and resides exclusively in various cell membranes. Its regulation is particularly important since excess cholesterol forms solid crystals, leading to cell death. Proper cell function requires that cellular membranes have the appropriate composition,

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• Cholesterol metabolism is a tightly regulated pathway subjected to stoppage of cholesterol biosynthesis in the presence of excess cholesterol. • Animal cells obtain cholesterol from both de novo biosynthesis and receptor mediated uptake.

Cholesterol •

Component for membrane biogenesis and precursors for the synthesis of steroid hormones, bile acids and lipoproteins.

•

Cholesterol resides exclusively in cell membranes and its regulation is particularly important since excess cholesterol forms solid crystals leading to cell death.

•

Cholesterol is ~10% of dry weight of plasma membranes. Precise lipid composition of the plasma membrane has been controversial since it is difficult to isolate high yield of pure samples from tissues or cultured cells.

•

Cells obtain cholesterol by de novo synthesis or through receptor-mediated uptake of plasma lipoproteins.

including cholesterol, in order to maintain bilayer fluidity, impermeability and other characteristics specific to different organelles. Cholesterol constitutes ~10% of the dry weight of plasma membranes, and plasma membrane cholesterol accounts for 65% to 80% of total cellular cholesterol. Cells in culture obtain cholesterol either by de novo synthesis or through receptor-mediated uptake of exogenous low-density lipoproteins. Cholesterol is synthesized from acetyl CoA, which is condensed by 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) to form HMGCoA. HMG-CoA is converted to mevalonate by HMG-CoA reductase (HMGCR). This enzyme is the target of statins, the class of drugs that suppress cholesterol synthesis in patients. Further synthesis of mevalonate to farnesyldiphosphate takes place in peroxisomes. Subsequent condensation of two molecules of farnesyl diphosphate to form squalene, lanosterol, lathosterol and finally cholesterol occur in the ER.

Out of the 18 key enzymes taking part in cholesterol biosynthesis, 5 enzymes reside in the peroxisome and 13 reside in the ER. HMGCS is upstream of HMGCR and is found in the cytosol. Thus, there are at least three different sub-cellular compartments involved in cholesterol biosynthesis.

Although cholesterol in mammals is synthesized primarily in the liver, most cells have the capability of synthesizing cholesterol for their own growth requirements. NS0 cells lack an enzyme, 17-β-hydroxysteroid dehydrogenase, which converts lanosterol to lathosterol. In NS0 cells, 17-β-hydroxysteroid dehydrogenase is silenced through methylation of a CpG island upstream of its promoter, leading to the cell line’s dependency on cholesterol for growth.

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Fig. 3.20: Cholesterol biosynthesis takes place in mitochondrion, peroxisome and endoplasmic reticulum.

Cholesterol Biosynthtic Pathway • Cholesterol is synthesized from acetyl CoA, which is condensed by 3-hydroxy- 3-methylglutaryl (HMG)-CoA synthase (HMGCS) to form HMG-CoA. • HMGCS exists in a mitochondrial form in hepatic tissue and is present in cytosol as 53kD protein for other tissues. • HMG-CoA is converted to mevalonate by HMG-CoA reductase (HMGCR) • HMGCR exists as a 97-kDa glycoprotein in the endoplasmic reticulum (ER) and is exemplified as the rate determining the enzyme of the biosynthesis pathway. • Mevalonate is further metabolized to farnesyl-diphosphate by a series of peroxisomal enzymes as shown in the figure. • First committed step to cholesterol byosynthesis is typified by condensation of two molecules of farnesyl diphosphate to form squanlene by farnesyl diphosphate farnesyl transferase-1 (Fdft1), or (squalene synthase) a 47-kDa protein residing in the ER • Squalene is converted to the first terol, lanosterol by the action of squalene epoxidase. • Conversion of lanosterol to lathosterol involves a series of oxidations, reductions and demethylations. It is a 17

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Glycan Biosynthesis and Protein Glycosylation

N-linked Glycosylation attachment of oligosaccharide to the protein through the amine group of an asparagine

O-linked glycosylation attachment of oligosaccharide to the protein through the hydroxyl group of a serine or threonine All IgG antibodies produced by mammalian cells are glycoproteins, with an N-linked oligosaccharide attached to each heavy chain in the hinge region at Asn-297

Importance of Glycan

Effect of Glycan • Facilitate protein folding in the ER • Increase solubility • Affect biological activities • Fucose for ADCC activity • Affect half-life in circulation and pharmacokinetics

Heterogeneity In Glycoforms • Macroheterogeneity: when multiple sites of glycosylation are present in a protein, the occupancy on different sites differs on different molecules • Microheterogeneity: the structure of the glycan occupying the same site differs among different molecules

A vast majority of recombinant therapeutic proteins are glycoproteins. The extent of glycosylation and the structure of the glycans on those glycoproteins may have a profound effect on their activities and circulatory half-life. Glycans are classified as O-linked or N-linked glycans. O-glycans attach to the polypeptide through the -OH group of serine or threonine. N-glycans link to protein through the amide group of asparagine. For N-linked glycan, the asparagine is in an Asn-X-Thr/Ser recognition sequence, where X indicates no specificity.

N- and O-glycans attached to proteins are structurally heterogeneous. The glycans attaching to the same attachment site of different glycoprotein molecules are often structurally different. Such heterogeneity is called microheterogeneity.

Multiple glycosylation sites are often present on a protein molecule. Not all glycan attachment sites on a protein molecule may be occupied. Different protein molecules may have different combinations of occupied and free sites; such difference in the occupancy on different attachment sites is called macroheterogeneity.

Glycosylation starts while the protein is still being translated and undergoing protein folding in the ER. The presence of glycans affects the solubility, aggregation, and the stability of the folding protein.

The glycan structure affects the half-life of blood circulation and immunogenicity of a therapeutic protein. The presence of carbohydrates delays clearance from blood, as demonstrated by a comparison of glycosylated protein and its nonglycosylated counter-part. Higher sialic acid content increases the circulation half-life of erythropoietin (EPO). Under-sialylated glycoproteins are thought to be cleared by a higher liver uptake via the hepatic asialoglycoprotein binding protein. It was also postulated that the glycosylated recombinant proteins are better trapped by the extra-cellular matrix, thus having a longer bioavailability in vivo than their unglycosylated variant.

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Protein Folding and Glycosylation

Fig. 3.21: Glycosylation of secretory proteins

Fig. 3.22: N-glycan processing of glycoproteins in endoplasmic reticulum

Glycan on glycoprotein may also affect its biological activities. Many therapeutic antibody IgG molecules facilitate killing of target cells through antibody dependent cellular cytotoxicity (ADCC). ADCC activities of those antibodies have been reported to be affected by its glycan structure. Molecules without a fucose on its mannose core have a fifty fold higher ACDD activity than those with a fucose. O-glycosylation initiates in either the ER or the Golgi. Overall, our understanding of O-glycosylation is far less than N-glycosylation. N-glycosylation is initiated by the transfer of a preassembled oligosaccharide (Glc3Man9GlcNAc2, an oligosaccharide of three glucose, nine mannose, and two N-acetylglucosamine) to an asparagine in the recognition sequence of a nascent protein in the ER lumen. The addition of glycan occurs during the process of protein synthesis, prior to the completion of protein folding.

The first part of N-glycan synthesis involves the assembly of a high mannose backbone on a membrane anchored dolicol molecule, on the outside surface of the ER. The glycan is linked to the dolicol carrier through a pyrophosphate group in an activated form. After the seven-sugar backbone is formed (with five mannose and two N-acetyl glucosamine), it flips over to the interior of the ER. No transporters are needed for the transport of backbone glycan; rather, a flippase catalyzes their translocation into the ER lumen. Inside the ER, the backbone acquires an additional four mannose and three glucose to become a mature core. The mature core is then transferred to a binding site (Asn-X-Thr/Ser) on a nascent protein molecule. However, not all glycan binding sites may be occupied. This could be due to competition between protein folding and core glycan transfer. Hence, different permutations of site occupancies in different protein molecules exist, giving rise to a macroheterogeneity of glycosylation patterns. After being synthesized, chaperones surround protein molecules to facilitate their folding process. The three glucose molecules on the glycan core serve as a quality control signal for the proper folding of these glycoprotein molecules. Upon

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completion of folding, the three glucose molecules are removed from glycan, signaling the protein’s readiness to be transitioned to the Golgi apparatus. Proteins that are not folded properly, with glucose still in their glycan, may be recycled through the calnexin pathway for refolding. Some unfolded proteins are sent to the proteosome for degradation.

Glycan Extension in Golgi Apparatus

Fig. 3.23: N-glycan extension in Golgi apparatus

The well-folded glycoprotein molecules are enclosed in membrane vesicles of ER and then bud from the ER and translocate to the Golgi apparatus. Once there, they fuse with the Golgi body membrane and the glycoprotein cargos are released into the lumen of the Golgi apparatus.

Inside the Golgi, mannose is trimmed from the N-glycan core, effectively reducing the number of mannoses from nine to three, before extension takes place. Three main carbohydrate molecules constitute most of the extended glycan: N-acetyl glucosamine, galactose, and sialic acid. Different glycosyltransferases add a different monosaccharaides to the growing core glycan on the protein. The incoming monosaccharide provides the activated carbonyl group and the receiving carbohydrate moiety on the growing glycan on the protein may have more than one hydroxyl group available for extension. The glycosyltransferases recognize different incoming monosaccharaides and catalyze different glycosidic bond formations. However, a number of glycosyltransferases do allow for some flexibility in glycosidic bond formation. Intermediate glycans on the protein have more than one sugar that can be extended and each sugar may have more than one hydroxyl group that is available for extension. Importantly, the extension reaction does not take place on all of the available reaction sites. Each growing glycan, thus, has multiple available reaction paths for extension. The glycans can grow into different numbers of branches, creating structures such as biantennary and triantennary glycans. In some cases, the reactions of adding those sugars to different branches of the glycan may occur in different sequential orders, but lead to the same product. In other cases, the

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addition of a particular glycosidic linkage may hinder the reaction of the others; thus, the reaction, itself, leads to different glycan structures.

Consequently, a very large number of glycan structures can be formed in the N-glycosylation pathway; however, the number of glycosyltransferases for the extension reactions expressed in a cell or tissue is relatively small (only a dozen or so). However, diverse patterns of glycosylation are often seen throughout many glycoproteins.

Glycan Types and Microheterogeneity

Fig. 3.24: Different types of N-glycans in glycoproteins

The web of glycan extension reactions forms a complex network which, when drawn out graphically, indeed resembles a network of diverging and converging paths leading to a number of different fully-extended N-glycan structures.

N-glycan structures are generally classified into three principal categories: high mannose, complex, and hybrid. All of them share a common trimannosyl (Man3GlcNAc2) core structure. The high mannose glycans have five to nine mannose (Man5GlcNAc2) sugars. Those with two GlcNAc’s attached 9 to the tri-mannosyl core are called “complex”. As its name implies, the hybrid types are a combination of high mannose and complex glycans, and have at least three mannose sugars but only one GlcNAc on one nonreducing mannose. This diversity of glycan sugar composition on each glycosylation site is referred to as microheterogeneity. N-glycan microheterogeneity arises through alternative reaction paths of extension in the Golgi apparatus, as described above. The Golgi apparatus consists of stacks of membranous compartments commonly grouped into cis, medial, trans, and trans-Golgi network (TGN) cisternae. These cisternae are not biochemically homogeneous. As the secretory glycoproteins traverse through these Golgi compartments, the glycan extension reactions are catalyzed by varying the composition of glycosylation enzymes in each compartment. Adding to this diversity, not all protein molecules spend an equal length of time in different Golgi compartments; some exit early while others linger. Some glycans

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Synthesis and Transport of Nucleotide Sugar Precursor

do not acquire a terminal sialic acid, while others have multiple sialic acid molecules on them.

Glycosidic bond formation is mediated by nucleotide sugars. The nucleotide (NTP) reacts with an activated sugar (glucose-1-phosphate, or glalatose1-phosphate, N-acetyl-glucosamine-phosphate, mannose-1-phosphate) to form an NDP-sugar. Uracil is used for glucose- and galactose-based sugars (e.g., UDP-glucose and UDP-galactose), guarnyl for mannose and fucose, and cytidyl for sialic acid. Mannose, galactose, and fructose are synthesized in branches of the glycolysis pathway. All three sugars are activated at their first carbon. Therefore, they link to glycans through the formation of (1→n) glycosidic bonds. For example, UDP-NAcGlc is added to a growing core by forming an N-acetylglucosamine β (1→n) mannose bond; there can be a number of possible positions on mannose (e.g., 2, 3, 4, or 6).

Fig. 3.25: Transport of nucleotide-sugar precursors into organelles

For sialic acid, the second carbon is activated; thus CMP-2-sialic acid will form a sialyl (2→n) bond with galactose. The synthesis of all the precursor sugars occurs in the cytosol, including a ninecarbon sialic acid and N-acetyl neuraminic acid. Similarly, all nucleotide sugars are formed in this way, except for sialic acid. The activation of sialic acid to CMP-sialic acid occurs in the nucleus.

The backbone of N-linked glycan is synthesized on the cytosolic side of the ER membrane through the membrane-anchored dolicol. The nucleotide sugars used in the formation of the backbone, GDPmannose, UDP-N-acetyl glucosamine, and GDPglucose are synthesized in the cytosol and directly react with dolicol or with the growing glycan backbone. The assembled backbone is then “flipped” into the ER and the subsequent reactions occur inside the ER. Transporters for these nucleotide sugars have been reported to be present in the ER. The nucleotide sugars for the extension reactions in the Golgi apparatus are also transported through transporters. These include CMP-sialic acid, GDP-fucose, UDP-N-acetyl glucosamine,

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• Glycan synthesis use nucleotide sugar as monomer unit • Nucleotide sugar highly charged, require specific transporter into organelles • Sialic acid synthesis occurs in cytosol, activation to CMPSialic acid to places in the nucleus.

UDP-galactose, and the activated sulfate donor (3’-phosphoadenosine, 5’-phosphosulfate). All of these transporters are antiporters, meaning that a stoichiometric exchange of nucleotide sugars with NMPs is responsible for the charge balance.

Fig. 3.26: Biosynthesis of precursors of glycans

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Glycan Diversity Among Species

Glycosylation • Enzymes are somewhat different among species • Possible immunogeneity (e.g. high mannose glycan from most yeast

Unless intended for vaccination, the immunogenicity elicited by recombinant proteins is a concern. An antibody elicited by and against the protein therapeutic can result in neutralization of the therapeutic protein and may result in an unintended drop in efficacy, thus causing serious adverse clinical effects.

The potential immunogenicity of recombinant therapeutics may arise from the aglycosylated protein core or from the glycan associated with them. There are at least two mechanisms by which glycans on a protein may affect the immunogenicity of a human therapeutic: 1) by being a foreign glycan structure, or 2) by shielding a segment of the protein that is otherwise antibody inductive.

Different recombinant human therapeutic proteins that are produced in different organisms are differently glycosylated (such as those from CHO versus yeast) or aglycosylated (such as from CHO versus E. coli). Comparison of those proteins indicates that the “shielding” effect of minimizing immunogenicity is affected by the nature of the protein, as well as by the source of the protein. The concern about the immunogenicity of different glycoforms of the rDNA proteins produced in insect cells and in transgenic plants has hindered those technologies’ application for rDNA therapeutic protein production. Glycosylated proteins produced in CHO and mouse myeloma cells are minimally immunogenic. The glycosylation pathway is highly conserved in mammals. Nevertheless, a divergence among different species does occur. Host cells derived from other species may possess a set of glycosylation enzymes that are different from humans. Human glycans have terminal N-acetylneuraminic acid (NANA), whereas other mammals have N-acetylglycolylneuraminic acid (NGNA). NGNA is the hydrolytic product of CMP-sialic acid hydroxylase, which is present in nearly all mammals but absent in humans. Thus, glycoproteins produced in CHO have some NGNA present among all sialylated glycans. Similarly, glycoproteins expressed in CHO

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CHO Glycan • Has NGNA instead of human NANA • Has only α(2,6) sialic acid, human has both α(2,3) and, α(2,6).

Concluding Remarks In this chapter, we presented a brief overview of broad areas of cellular metabolism. We explored the core of energy metabolism, the process of glucose utilization through glycolysis, PPP and the TCA cycle, and how all of these affect cell growth behavior productivity. Through interconnected pathways, the central corridor of energy metabolism also influences the synthesis, and even the glycosylation, of the product proteins. The excessive consumption of glucose and glutamine and the corresponding accumulation of lactate and ammonium in culture all contribute to growth inhibition and low productivity. Lactate consumption in the late stage culture has been positively associated with a high productivity. There are, therefore, ample incentives

cells have only terminal α(2,3)-linked sialic acids, in contrast to α(2,6) and α(2,3) seen in humans, due to a varied composition of sialyltransferases. Such differences in glycan composition have posed a concern; however, the antigenicity of recombinant proteins directly caused by glycans is still scant. to better understand cell metabolism and to possibly find different ways of manipulating cell metabolism to better redirect the process. In recent years, we have developed a better understanding of the link between glycolytic regulation and growth control. We have also established better tools to probe the relationship between metabolic flux distribution and other aspects of physiology that influence both productivity and product quality. With the benefit of global physiological perspectives, we continue to gain a deeper understanding of metabolism. Global views at a systemic level will significantly enhance our capacity to manipulate cell metabolism, and thus increase productivity and product quality.

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Medium Design for Cell Culture Processing Optimization of Cell Growth Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 A Guide for Medium Design—Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Types of Media and Classes of Medium Components. . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Types of Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Basic Components of Cell Culture Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Classes of Medium Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Components of Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Sugars and Energy Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Vitamins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Nucleosides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Optimal Concentration of Organic Nutrients. . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Fatty Acids and Lipid Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Non-Nutritional Medium Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 High Molecular Weight and Complex Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Medium for the Industrial Production Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Optimization of Cell Growth Environment Medium, like the food we eat, exerts a fundamental influence on the well-being of cultured cells, Industrial Cell Culture Process profoundly affecting their growth, metabolic 1. Cell expansion activities, and other biological capability. The 2. Production/differentiation question of how best to devise culture medium emerges whenever new in vitro cultivation• Cell expansion stages last much longer than production based science or technology is on the horizon, • Medium design for both stages as occurred three decades ago driven by the revolution in recombinant mammalian cell based biotechnology, and as is occurring now concomitant with the emergence of stem cell science. Cells are the heart of cell technology; however, without proper medium, cell cultivation cannot accomplish process goals. Most cell types share common basic nutritional requirements although their needs for growth factors and cytokines may

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• Classical Medium Design - Optimize Cell Growth • Optimization for production - squeeze cell’s last productivity out • Resurgence of media research in stem cell culture • opportunities in growth factors, antogonists, and signaling pathway manipulation

differ. In the nearly five decades since scientists began to isolate and cultivate cells, the focus of medium design has been to optimize cell growth, maintain growth potential and sustain the differentiated properties in cultures of differentiated cells.

With the growing importance of biologics, the focus of medium design has been extended to enhancing production characteristics, such as productivity or product quality. However, even with the focus shifting to production, optimizing medium for cell growth remains important. In fact, the time that cells spend in the production stage in a manufacturing reactor is a comparatively small portion of their life span. A cell spends the majority of its lifespan in growth, yielding progeny to generate a sufficiently large number of cells for producing product. Providing cells with an optimal medium during the expansion stage is critical.

Recent advances in stem cell science have spurred renewed interest in elucidating the nutritional needs of cells in culture. Although the fundamental aspects of nutritional requirements of a stem cell are not different from other cell types, their requirements for growth factors, surface matrices, and other microenvironmental factors makes medium design for stem cells far more complex than that for any cell lines used in protein biologics production. Furthermore, stem cell applications require that the stem cell progeny be directed to differentiate to specific lineages, for which the growth factor requirements pose an even greater challenge.

Regardless of traditional biologics-based cell technology or stem cell bioprocessing, the culture process will involve both cell expansion and product formation or differentiation. Medium optimization strategy for cell expansion and for production may be rather different. For expansion, the long term healthy state of a cell while proliferating must be safeguarded. In contrast, for production of biologics, the cells are approaching their final stage of utility, and after all products have been released into the medium, cells and product molecules must be separated. Hence, even conditions that might ordinarily hamper

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growth or harm cells, such as reduced temperature or increased osmolality, are sometimes used.

For stem cell processing, or for other cell therapy, the distinction between cell expansion and production is not as significant. Even though cells are no longer being expanded during the differentiation stage or other final stage of preparation for clinical applications, cells must not to be subjected to deleterious conditions. Since the final product is cell mass itself, their survivability and functional capability after the cell culture process is critical.

In the following sections we will focus on nutritional needs for cell expansion first, as that is best known. We will then discuss how “optimal” medium for production compares with that required for growth.

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A Guide for Medium Design—Body Fluids Table 1. Cellular Chemical Environment in vivo Approximate Concentrations in Cellular Environment

Interstitial Intracellular (mM) (mM) Na+

140

14

K

4.0

140

1.2

0.01

0.7

20

108

4

28.3

10

+

Ca

2+

Mg

2+

CI-HCO3

-

HPO4 , H2PO4

2

11

0.5

1

2

8

Lactate

1.2

1.5

Glucose

56

Protein

02

4

Total Chemical Species (mmole/L)

301.8

302.2

Corrected osmolar activity (mM)

281.3

281.3

3-

2-

SO43Amino Acids

To design an optimal medium for cell growth, it is instructive to examine the chemical environment of their natural niche. The ultimate objectives of medium design are cell expansion, differentiation, and production, not necessarily to reproduce their niche. Understanding their native chemical environment provides us with a starting point from which to devise an environment suited to process goals.

The vast majority of cells in the body are not in direct contact with blood, but are surrounded by interstitial fluid. The chemical composition of interstitial fluid, especially the protein and hormone content, varies with tissues. However, the general chemical composition of small molecular weight solutes in interstitial fluid and in intracellular fluid is similar. The low molecular weight solute composition of interstitial fluid bears a few important characteristics. The total osmolarity is around 280300 mM (or mOsm). A couple of percentage points of error notwithstanding, the osmolarity can be taken as the sum of molarity of all dissolved species in the fluid. The largest contributor to the final osmolarity is Na+, followed by Cl–. In addition to Na+, other inorganic species are present; notable are K+, Mg2+, and Ca2+. However, those positively charged ions are all present at low concentrations. Since the net charge in a solution must be neutral, the total molarity of positively and negatively charge ions must be equal. In general Cl– concentration is lower than Na+ because bicarbonate (HCO3–) is also present at ~30 mM contributing to the negative charge in the solution.

For many ion species the interstitial concentration and intracellular concentrations are strikingly different. Both Na+ and Cl– are present outside the cell at a tenfold higher concentration than inside the cell, as is Ca2+. In contrast, K+, Mg2+ and PO43– concentrations are much higher on the intracellular side. Cells can tolerate deviations from “optimal” conditions for some period of time. The estimated range of non-lethal physiological concentrations of key compounds vary considerably. Keep in mind

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Table 2. Non-Lethal Range of Medium Constituents Normal Range

Approximate Nonlethal Limits

Oxygen

35 – 45

10 – 1,000 mm Hg

Carbon dioxide

35 – 45

5 – 80 mm Hg

138 – 146

115 – 175 mmol / L

Potassium ion

3.8 – 5.0

1.5 – 9.0mmol / L

Calcium ion

1.0 – 1.4

0.5 – 2.0mmol / L

Chloride ion

103 – 112

70 – 130mmol / L

75 – 95

20 – 1500 mg / dL

Sodium ion

Glucose Body temperature

98 – 98.8 65 – 110 (18.3 – 43.3) (37.0) F° (C°)

pH

7.3 – 7.5

that the non-lethal range for the human body can be rather different from that for cultured cells. For example, cells in culture can tolerate low (80 mM) or high (140 mM) sodium concentrations, or high osmolarity (400 mOsm), for a period of days, whereas these extremes would not be tolerated by most aquatic animals for more than a few hours.

6.9 – 8.0

Types of Media and Classes of Medium Components Basal Medium • Sugar • Amino acids • Fatty acids, lipid precursors • Vitamins, nucleosides • Bulk salts, trace elements • pH buffer Supplements • Serum, hydrolysates • Growth factors • Carrier proteins

A complete cell culture medium often has two major categories of components: basal medium and growth supplements. The basal medium is the nutrient mixture consisting of the small molecular weight components including sugar, amino acids, vitamins, various salts, etc. The basal medium does not merely provide a nutritional source for deriving energy and making new cell mass and product, it also provides balanced salt concentrations and osmolarity to allow for cell growth.

However, most cells will not grow if provided with basal medium alone, as basal medium does not contain growth factors or other factors necessary for “optimal” growth conditions. Growth supplements that may be added to basal medium include growth factors, phospholipids, soy hydrolysate, serum, etc. These supplements may promote cell growth by providing constituent components for specific signaling pathways, or may supply special nutritional needs (such as delivering cholesterol), and may direct cellular differentiation

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or maintain cells at a particular differentiation state.

There are numerous formulations of basal media that are made into powder, packaged and sold for commercial or routine use. Sterile filtration is the standard means of sterilizing basal medium, but heat sterilization (as is used for microbial media) is also possible.

Types of Medium

Complex Medium vs. Chemically Defined Medium

Chemically Defined Media • Progress in this area will likely be accelerated by: • an urgency to demonstrate control over all aspects of production and downstream processing for licensing by the FDA • the availability of recombinantly produced (in E. coli) tissue culture supplements (i.e. insulin, EGF) • genetic engineering of cells to produce their own growth factors • development of small, synthetic peptides that can mimic the action of the larger, naturally occurring protein (i.e., RDG sequence) • acceptance of continuous bioreactor systems and the operation of these systems in a “maintenance” mode

Serum-Free and Animal Component-Free Medium

Traditional cell culture medium contains up to 15% animal serum in addition to basal medium. Serum is a highly complex fluid in terms of its chemical composition. Such a medium, containing a largely undefined chemical composition, is called a complex medium. Many supplements commonly used in industrial processes, e.g., plant hydrolysates, soy phospholipids, also fall into this category. Their use renders the chemical composition of the medium undefined.

A chemically defined medium contains only components whose chemical composition is known and characterized, and has all of its chemical species specified. It does not contain any mixture of components with unknown or undefined composition. For example, “lipids” or “phospholipids” are not well defined compounds, but are mixtures of a class of compounds and are not chemically specified. A chemically defined medium often contains growth factors, cytokines, and carrier proteins. Thus, a chemically defined medium is not necessarily protein-free.

A large number of industrial production processes of rDNA proteins has eliminated serum from the medium in the past decade. The use of serumfree and animal-component-free medium has become the industrial manufacturing norm, with the intent to minimize animal virus or prion contamination. Recently, serum-free medium has been increasingly used even during the cell line development stage to eliminate exposure of cells to serum and animal components throughout the

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Serum-Free Media • Serum-free medium consists of nutritionally complete basal medium supplemented with an empirically determined mixture of hormones, growth factors, attachment factors, attachment proteins and binding proteins. • Many serum-free media contain a complex mixture of undefined components, such as soy-meal hydrolysate, peptone or beef hydrolysate, or other plant hydrolysate.

entire cell banking and manufacturing process.

Nonetheless, the use of serum-free medium is not yet universally practiced. For example, bovine serum is still widely used in manufacturing viral vaccines. As well, it is often not an easy task to eliminate animal serum from processes involving the cultivation of primary differentiated cells.

By definition, protein-free medium contains no protein. Most protein-free media are, as well, chemically defined. However, a protein-free medium may contain undefined lipids or fatty acids, and thus, may not be chemically defined. Cells that grow well in chemically defined medium are likely to be highly adapted or transformed, which eliminates any dependence on mitogenic molecules or lipid sources.

Protein-Free Medium

Basic Components of Cell Culture Medium Classes of Medium Components Stoichiometric vs. Habitation-Conducive Basal Medium Components

• Stoichiometric: glucose, amino acids, vitamins, nucleotides, lipids, fatty acids, some growth factor, some salts • Habitation conducive: Bulk salts, such as sodium, chloride, and some proteins, albumin, some growth factors.

Medium serves two important roles: to provide a chemical environment in which cells can grow, and to supply the components cells need to generate energy and convert to cell mass and products. Some medium components are taken up by cells and “utilized” to make more cells and products; other components provide the chemical environment but are not appreciably taken up by cells. The majority of medium components, including glucose, amino acids, lipids, and vitamins, are consumed stoichiometrically. The more cells are made, the more of those components are consumed. In a most precise sense, nearly all medium components are utilized to generate more biomass. Even water is taken up by cells; as the cell volume expands water must be taken up along with other components that constitute cellular materials. However, in practical terms, many medium

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components are consumed in such small quantities that their consumption may not be measurable.

Stoichiometric medium components must be supplied in sufficient quantities to reach the cell, and must meet the product concentration target of the production. To reach a higher goal, more stoichiometric components must be supplemented. For the unconsumed components of the medium—components whose role is only to provide a conducive environment—the key issue is to maintain their concentration.

Examples of conducive, unconsumed medium components include chloride ion, sodium ion, and even phosphate ion. Under ordinary conditions, only a negligible amount of these components are taken up by cells. However, these normally unconsumed salts may be taken up in substantial quantities in fed-batch cultures where fortified feeding is used to add more glucose, amino acids, and other nutrients to grow cells to a higher concentration. In that case, these will also need to be replenished. Thus, unconsumed medium components may become stoichiometric components under high density cultivation conditions.

Example

• PO4-3 concentration in medium is typically 1 mM, while its cellular concentration is 11 mM. The intracellular ATP/ADP concentration is about 2mM, which represents around 6mM of phosphate. Total DNA and RNA contain another 35 mM equivalent of phosphate. A culture with cells of an average volume of 2 x 10-12 L and at a cell concentration of 1010 cells / L take up about [(11+6+35) mmole / L x 2 x 10-12 L / cell x 1010 cell / J= ~ 1 mmole / L of PO4-3. • Cell growth will certainly cause PO4-3 concentration in the medium to decrease drastically at such a cell concentration. • For instance, phosphate and trace metals are consumed in very small amounts, although their depletion may not lead to stoppage of growth immediately, they must still be supplied in stoichiometric quantities.

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Stoichiometric vs. Catalytic Macromolecular Components • Some high M.W. medium components are interalized and consumed • Others are not internalized when exerting their functions and are degraded only slowly

Cell culture medium often contains macromolecules such as insulin, fibroblast growth factor (FGF), serum albumin, etc. These constituents serve a variety of purposes; some are carrier proteins carrying ligands into the cell, while others are growth factors that bind to receptors on the cell surface. Molecules that are taken up by cells may need to be replenished to maintain their concentration, whereas molecules that transmit signals by binding to cell surface receptors may not need to be replenished as frequently during cultivation.

Many macromolecules are internalized, and some are degraded (consumed), while others are recycled. For example, the ferric ion carrier, transferrin, binds to transferrin receptor and is internalized. After being internalized, transferrin is translocated to lysosome, and after releasing ferric ion in the low pH environment of the lysosome, transferrin is recycled to the extracellular medium. As long as ferric ions are available (i.e., replenished), transferrin can continue its role as ferric ion carrier. Insulin, another commonly used growth factor, binds to the insulin receptor and triggers a signaling event that does not involve its own internalization. However, when present at a high concentration (a few microgram per ml), insulin is rapidly internalized by hepatocytes and degraded. Therefore, even though as a growth factor it is not consumed, its concentration does decrease.

An example of a consumable macromolecule is lowdensity lipoprotein (LDL). After an LDL particle binds to an LDL receptor on the plasma membrane, the receptor-ligand complex is internalized in a clathrin-coated pit that pinches off intracellularly to become a coated vesicle. The clathrin coat then depolymerizes, resulting in an uncoated (smooth surfaced) vesicle, often called an endosome. The endosome then fuses with an uncoupling vesicle that has an internal pH of about 5.0, which causes the LDL particles to dissociate from the LDL receptors.

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The LDL receptors are then recycled back to the plasma membrane. The vesicles containing the LDL particles fuse with lysosomes, in which the cholesterol esters are hydrolyzed to fatty acids and cholesterol. Cholesterol is incorporated into cell membranes.

Components of Basal Medium Water • Types of Contaminants in City Water: • Inorganics—heavy metals, iron, calcium, chlorine • Organics—by-products of plant decay, detergents • Bacteria—endotoxin or pyrogen • Particles—colloids or particles • A typical water preparation process involves filtration, Reverse Osmosis • WFI used to be employed, now mostly pure water.

Mammalian cells are exceedingly sensitive to the quality of water used for media preparation. City water, the usual source of water for medium preparation contains particulates, bacteria which are the source of endotoxin, trace organics, and various inorganic ions including harmful heavy metals. Typical water preparation processes include deionization through ion exchange, microfiltration to remove particulates and bacteria, and finally reverse osmosis to reduce conductivity (or increase resistance) to > 20 MΩcm. In some cell therapy applications, because the product (i.e., cells) is subjected to little purification process before administration to the patient, cell culture medium may be prepared using water for injection (WFI) to avoid the entry of any pyrogenic contaminants. WFI is prepared by low evaporation rate distillation, minimizing the chance of any water droplet in the evaporating stem from carrying over any solute or particle from the water.

Sugars and Energy Source

Glucose and glutamine are the primary nutrients that supply a cell’s energy needs in culture. The physiological concentration of glucose in blood is 0.8 g / L. In culture, glucose is typically present from 1 g / L (5.5 mM) to 5 g / L (27.5 mM). In the production reactor, sometimes a high level of glucose, as much as 15 g / L (82.5 mM), is used. In this case, glucose is a large contributor to the osmolarity of the medium, and adjustment of the composition of the medium must be made (by reducing sodium and chloride concentration) to

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maintain osmolarity in a growth permissible range.

Six-Carbon Sugars • Glucose:1-5 g / L, • Fructose, galactose, may also be used. • Galactose and glucose can both be transported by the GLUT1 transporter, which is present in most cells. • Fructose is transported by a different transporter (GLUT5); unless the transporter is expressed, the cell may not be able to use fructose efficiently. • The alternative sugar is often taken up by cells at a slower rate, which may reduce lactate production and be good for cell maintenance. • Pyruvate and ribose are sometimes supplied in small quantities, insufficient to supply cell's energy needs.

Glutamine Serves Two Roles: • source of amino acid for protein • nucleoside synthesis, energy source.

• Glutamine is consumed in large quantities, approximately 1/5 to 1/10 of glucose in molar amount. • Glutamine is spontaneously degraded in aqueous solution.

All cultured cells express the GLUT1 transporter at a significant level, and take up glucose readily. GLUT1 also transports galactose. Thus, galactose can be used as an alternative sugar to glucose. The galactose km for uptake is higher than for glucose. In the concentration range used for glucose, galactose is taken up by cells at a lower rate, resulting in lower lactic acid production in the culture. Fructose is also transported by the GLUT5 transporter. The Km for fructose transport by GLUT5 is also high. Thus, similar to galactose, the uptake rate for fructose is lower than for glucose unless a high concentration of fructose is used. At a low uptake rate, the use of fructose also results in lower lactic acid production compared to glucose.

Glutamine is an essential amino acid for cells in culture. Most cells in tissues express glutamine synthase, and make glutamine from glutamic acid. In culture, they consume glutamine at roughly 1/5 to 1/10 of the consumption rate of glucose. Glutamine supplies the amino group for nucleotide biosynthesis. It also supplies the carbon backbone of TCA cycle intermediates by converting to α-ketoglutarate. Through carbon 13 tracer experiments, it has been shown that glutamine contributes to lactate production during cellular energy metabolism in culture. Glutamine spontaneously degrades in aqueous solution, and releases ammonium. Consequently, to avoid degradation, glutamine is typically added to culture medium immediately prior to use.

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Amino Acids Table 3. Essential and Non-Essential Amino Acids Essential amino acids†

Non-essential amino acids

L-arginine*

L-alanine

L-cysteine*

L-asparagine

L-histidine

L-asperatic acid

L-isoleucine

L-glutamic acid

L-leucine

L-glycine

L-lysine

L-proline

L-methionine

L-serine

Amino acids are classified as essential or nonessential based on nutritional studies using animals or tissue culture cells. Cells lack the biosynthetic pathways for making essential amino acids, and rely on exogenous supply to meet growth needs. Some amino acids are essential only for cells in culture, but not for animals.

In animals, different tissues may cross feed each other; amino acids synthesized in one tissue (e.g., liver) may be transported to cells in other tissues. Some enzymes involved in amino acid biosynthesis are expressed at lower levels in cultured cells in vitro than in cells from their tissue of origin. Glutamine is non-essential for animals; it is synthesized from glutamic acid through glutamine synthetase. The expression level of glutamine synthetase decreases when cells are cultured in vitro. There is also a CpG island in the promoter region of glutamine synthetase, which may be methylated to cause glutamine synthetase to be silenced in some cultured cells.

L-phenylalanine L-threonine L-tryptophan L-tyrosine* L-valine L-glutamine* *Essential for cells in culture, not for animals

• Most culture media contain all twenty amino acids. • proline is required by mutant CHO cells; • serine is frequently required at clonal densities; • asparagine is required by certain malignant cells; • glycine sometimes needed in case of borderline folic acid deficiency or in the presence of folate analogues (methotrexate and aminopterin) • Small peptides can serve the same function as amino acids--some of these are more stable (e.g., glycineglutamine) or are transported more readily than their free amino acids counterparts. • Some non-essential amino acids are excreted into culture medium; alanine is most commonly seen. Asparagine and proline may also accumulate in medium.

Upon cell isolation for in vitro culture, expression of some amino acid synthesis enzymes is suppressed, but expression of some amino acid transporters is elevated, which allows for a faster transfer rate to meet growth needs.

Cell culture media developed in the 1960s and 1970s contained at least the 14 essential amino acids. Those media were designed to be used with serum supplementation, which also supplies some amino acids. Media designed for serum-free culture include all amino acids. In medium preparation, amino acid mixtures are often prepared as concentrated stock solutions organized into groups: neutral, acidic, basic, etc. Although the solubility characteristics of amino acids indicates that solubilizing them is feasible, the kinetics of their dissolution is slow. Some stock solution preparation methods rely on pH adjustment using acid or base to increase the rate of dissolution. Those stock solutions also introduce additional salts into the culture medium. Thus, when using such stock solutions, it is important to assess the osmolarity of culture.

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Vitamins • Ascorbic acid may be beneficial for cells that synthesize collagen. • Vitamin A can have a pronounced effect on growth and differentiation of some cell types. • Vitamin K is required for gamma-carboxylation and correct processing of vitamin K dependent proteins. • Vitamin E functions as an antioxidant. • Vitamin D regulates Ca+2 and is regarded by many as a hormone rather than a vitamin. Most toxic of all vitamins when present in excess. • Thiamine pyrophosphate catalyses the transfer of carboxyl group, transketolase, transaldolase. • Pyridoxal phosphate (pyridoxine vitamin B6) catalyses transamination.

The biological activities of vitamins vary. Even though they are classified as a common class of nutrient, their biological roles are diverse. They all share the common feature of being essential for the vitality of humans, and are needed only in minute quantities compared to glucose and amino acids. Some vitamins are cofactors involved in biochemical reactions, and are required by all cells. These include biotin, thiamine pyrophosphate (or its precursor), riboflavin and cobalamin. Some vitamins such as vitamin D, vitamin K, vitamin A, and vitamin C are required by only certain differentiated cell types.

• Biotin is a carrier of activated CO2, and is involved in pyruvate dehydrogenase, pyruvate carboxylase, and fatty acid synthesis. • Cobalmin (B12) is involved in free radical reactions of intramolecular C-C bond rearrangement, methylation, and conversion of ribonucleotides to deoxyribosenucleotides.

Nucleosides

Nucleosides are not included as essential components of basal media when serum is supplemented. Inclusion of small quantities of nucleosides is common in serum-free medium. The small quantities included reflect the low nucleoside content in cell mass: nucleic acids (RNA and DNA together) constitute only about 5% of dry cell mass. A purine source (adenosine or hypoxanthine) together with thymidine is beneficial when folic acid is in limited supply (e.g., in the case of methotrexate selection).

Table 4. Nucleosides in Basal Medium RNA

DNA

Adenosine

Thymidine

Cytidine

2’deoxyadenosine

Guanosine

2’deoxycytidine

Uridine

2’deoxyguanosine

Fatty Acids and Lipid Precursors

Lipids constitute a significant portion of biomass of mammalian cells. Lipid bilayer membrane forms vesicles which play key roles in protein secretion and virus replication. The composition of lipids in membrane affects its fluidity. However, the effect of lipid supply in medium is usually rather subtle.

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Unlike the supply of essential amino acids or sugar, whose deficiency cause observable effects on cell growth almost immediately (with only a delay of one doubling time), the effect of supplementing or removing lipid is not as easy to assess.

Fatty Acids • Cells can synthesize fatty acid, but can’t introduce double bond beyond C9. • Some cell lines benefit from cis-unsaturated fatty acids, such as oleic acid, linoleic acid and arachidonic acid (a precursor for prostaglandin formation)

Phospholipid Precursors • Choline • Ethanolamine

Inositol—precursor for phosphatidyl-inositol biosynthesis • Cholesterol—required by some cell lines (e.g. NS-0 myeloma)

Mammalian cells can synthesize almost all lipids required for their growth, including fatty acids, phospholipids and cholesterol except for auxotrophic mutants. Cells can make fatty acids of all different carbon length in membrane, including most unsaturated fatty acids, which constitutes almost half of the fatty acids in phospholipid. However, mammals do not introduce double bonds beyond C9 into fatty acids. Linoleate (18:2 cis-∆9, ∆12) and linolenate (18:3 cis-∆9, ∆12 ∆15) are thus essential fatty acids. Hence complete medium for serum-free culture usually contains oleic acid, linoleic acid, sometimes also arachedonic acid. However, the removal of essential fatty acids from culture medium, does not cause immediate cessation of growth, subtle changes in membrane properties is often masked by its residual amount in the cell and visible effect may take a long time to emerge. Ideally supplementing cells with phospholipids is a good practice. However, non-animal source of phospholipids with reproducible quality is not easily available. Often precursors of phospholipids (choline, ethanolamine, inositol) are supplied. Cholesterol is supplied to its auxotrophic mutants, such as NS0 cells. Cholesterol and fatty acids have very low solubility. Fatty acids are conjugated in serum albumin when serum supplement is used. In albumin-free medium fatty acids may mixed in with other hydrophobic supplements. Cholesterol can be supplied as derivatized ester with a organic acid attached to its hydroxyl group to increase its solibility, or can be supplied as cyclodextrin conjugated complex.

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Optimal Concentration of Organic Nutrients

Optimal

Growth Rate

Suboptimal

Inhibitory

Optimal range typically spans over 10 fold or more

Starvation Nutrient Concentration Fig. 4.1: Optimal range of nutrients for cell growth

Bulk Salts and Trace Elements

Upon determining the essential and beneficial components of medium, it is necessary to decide on the optimal concentration of those nutrients. The experimental determination of concentration range is complicated by cell growth and consumption that causes medium composition to change. Furthermore, metabolites accumulate as cell grow, making culture conditions change as time goes by.

This problem was resolved by using clonal growth as originally demonstrated with ells of human, chicken and other species.. Cells were seeded in small petri dishes at very low density of about 1050 cells/cm2 as opposed to 104 cell/ cm2 At such a low cell concentration, the amount of nutrients consumed is negligible compared to the amount present in the medium. It was further assumed that nutrient utilization is completely independent;

thereby allowing the effect of one component to be tested when all the other components are present in their “optimal” concentrations determined by the point of experimentation. After plating cells are dispersed as single cells on the surface. A couple weeks later, the extent of growth from all the cells initially plated is estimated by the total surface area covered by cells after cells on the dish is stained with a dye. The optimal concentration is one which gives largest cell growth area. The optimal concentration for almost all nutrients (amino acids and other small molecular weight molecules) span over a wide range of at least ten fold. One can thus conclude that for the purpose of cell cultivation, most cells have a wide range of optimal concentration for most organic basal medium components.

Mineral elements are also essential components of cell mass. Phosphate constitutes part of nucleotides and nucleic acids; magnesium is present in high concentrations in the cell as it is conjugated to ATP, which is present in the mM range; calcium, which is

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Table 5. Concentrations of bulk ions in basal medium (mM) DMEM/ F12 (1:1) Na+

William’s E

DMEM

RPMI

F12

150.31

143.71

155.12

137.74

144.03

K

4.18

5.37

5.37

5.37

3.00

Mg2+

0.71

0.81

0.81

0.41

0.60

Ca

1.05

1.80

1.80

0.42

0.30

126.66

125.33

118.48

108.03

134.83

1.02

1.17

0.78

5.63

1.17

HCO3

29.02

26.19

44.04

23.81

14.00

SO42-

0.41

0.81

0.81

0.41

0.00

0.85

0

282

288

+

2+

ClPO43-

NO3

-

Total

313

302

327

Role of Bulk Ion • Maintenance of membrane potential (Na+, K+) • Osmotic balance (Na+, Cl-, HCO3-etc.) contribute most of the osmolality of fresh medium. Optimal range is 280 310 mOsm / kg. • Biological roles: Mg+ conjugate with ATP Ca2+, Mg2+ for cell adhesion PO43- for nucleotides • Signaling (Ca2+ ) • Buffering (HCO-3, HPO3-4)

Table 6. Trace Elements in MCDB 104 (serum-free medium for human diploid cells) (mM) CuSO4·5H2O

1.0 x 10-6

FeSO4·7H2O

5.0 x 10-3

MnSO4·5H2O

1.0 x 10-6

(NH4)6Mo7O24·4H2O

1.0 x 10-6

NiCl2·6H2O

5.0 x 10-7

SeO2

3.0 x 10-5

Na2SiO3·9H2O

5.0 x 10-4

SnCl2·2H2O

5.0 x 10-7

NH4VO3

5.0 x 10-6

ZnSO4·7H2O

5.0 x 10-4

essential for signaling in some differentiated cells, is present in high concentrations in endoplasmic reticulum (ER) and in some organelles. Calcium and magnesium are also involved in cell-cell and cell-substrate adhesion. It must be noted that sodium and potassium, through their reverse abundance in cytosol and medium, balance membrane potential, which is fundamental to life. Many trace metals play key biological roles. Ferrous ion plays a key role in electron transfer complexes, as does copper ion. Zinc is present at high concentration in pancreas and is conjugated with insulin. Selenate serves as an antioxidant. These elements are required by cells in minute quantities; however, their long term deprivation is detrimental to cells. There is a wide range of concentrations of bulk ions that is conducive to cell growth. What is apparently most important for growth is the balance of osmolarity. The most important contributors to osmolarity are sodium ions, chloride ions, and bicarbonate; while the concentrations of these can be varied, the total osmolarity must be maintained in the range of 270-330 mOsm. When bicarbonate is not used in culture medium its contribution to osmolarity must be replaced by other salts. It is common practice to add a mixture of NaCl and KCl to maintain their molar ratio at about 30.

There is a wide range of conducible concentration for bulk ions. What is apparently most important is the balance of osmolarity during growth stage. The most important contributors to osmolarity is sodium and chloride ions, the concentration of both can be varied, so is bicarbonate. However, the total osmolarity must be maintain in the range of 270-330 mOsm.

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Trace Elements • Those clearly required by cultured cells are: iron, manganese, zinc, molybdenum, selenium, vanadium, copper. • Trace elements are ubiquitous contaminants of chemicals and supplements used in preparation of medium. • Some media contain rare trace elements such as rubidium, cobalt, zirconium, germanium, molybdenum, nickel, tin and chromium; may be needed for long-term growth in protein-free medium.

When bicarbonate is not used in culture medium its contribution to osmolarity must be replaced by other salts. It is common practice to add a mixture of NaCl and KCl to keep their molar ratio at about 30.

• What are the roles of heavy metal ions? The tables provide a reference of optimal starting

Non-Nutritional Medium Components Some components of medium are additives that make it operationally easier to grow cells. They can be removed from medium without harmful effect, and do not appear to be taken up by cells. However, their presence in the medium, especially under process conditions, can minimize operational deviations.

Sodium Bicarbonate Buffer

Sodium bicarbonate provides a pH buffer in our body fluid, and was used to buffer cell culture medium in the early days of in vitro culture development. However, the pKa of bicarbonate is 6.1 making it less than ideal as a buffer for neutral pH. The buffering capacity of bicarbonate derives from the equilibrium with soluble carbon dioxide as shown in the inset. The buffering action of bicarbonate requires the presence of CO2. With a given bicarbonate concentration, the pH is inversely proportional to the CO2 level; thus, as gas phase CO2 goes up, pH goes down.

Typical cell culture medium contains 14-44 mM NaHCO3, which at equilibrium requires 10% CO2 to maintain pH at 7.4. As pH decreases due to lactate production, the CO2 level in the gas phase can be reduced, which takes the proton to the left hand side of the equilibrium equation, to maintain pH. Conversely, as cells begin to consume lactate in the late stage of a fed-batch culture, the CO2 concentration in the gas phase is increased to maintain pH.

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Bicarbonate Concentration in medium 44mM in DMEM, 14 mM in F12, 26 mM in circulating blood. • It is necessary to use 5 – 10% CO2 in the incubation chambers; media that contain bicarbonate become alkaline very rapidly due to loss of CO2 when removed from the incubator. • The low pKa of bicarbonate (6.1) results in suboptimal buffering throughout the physiological pH range. • NaHCO3 buffer requires appropriate CO2 concentrations in the gas phase. The reactions are: CO2 dissolves in aqueous solutions. The CO2 concentration in liquid is described by Henry’s Law. H: Henry’s law constant CO2 in an aqueous solution forms a bicarbonate ion. The equilibrium is described as: From the definition of pH and pKa

The pH of the solution is affected by PCO2 and HCO3-.

From the equation, one can plot the relationship among HCO3-, PCO2 and pH.

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How do bicarbonate and CO2 work together as a pH buffer? The equilibrium equation, with pKa = 6.36 can be used to plot the relationship between pH and bicarbonate concentration. The relationship is also dependent on CO2(g). Two lines are shown for two different CO2(g) concentrations. Each point on the line represents the corresponding pH and carbonate concentration. To buffer the medium at a given pH one has to select a combination of bicarbonate and CO2(g) concentrations.

Figure 4.2: Relationship between sodium bicarbonate concentration, atmospheric carbon dioxide and pH

Alternative Buffer • Sodium beta-glycero-phosphate (20 mM) also functions as a detoxifier of ferric chloride hydroxo compounds (i.e., Fe3+ chelator) • Zwitterionic buffers: HEPES (N-2hydroxyethylpiperazine-N-2-ethane) is used between 10 – 50mM. • Alternative buffers can be growth inhibitory at high concentrations (>25 mM) • Requires balance of osmolality (by adjusting bulk salt levels, e.g. if 2 mM is used and 40 mM of NaHCO3 is eliminated, the osmolality must be balanced with NaCl / KCl. Need to maintain Na+ / K+ ratio (~30:1).

Table 7. Cell Culture Tested Biological Buffers Description

pKa value at 37° C

Anhydrous Mol. Wt.

Working Concentration (mM)

Glycine 1.0M

9.53

75.0

50 ‑ 200

Glycylglycine

7.95

132.1

10 ‑ 20

HEPES

7.31

238.3

10 ‑ 28

MOPS

7.01

209.3

10 ‑ 20

Sodium Bicarbonate

6.28

84.0

2 ‑ 26

TRICINE

7.80

179.2

1 to 100mg / l in 5 - 10 days (~ 0.1-1 pg / cell / day)

Stable Cell Line Development

transcription and transduce large quantities of starting material. However, if too successful, the overexpression of some genes can be toxic to cells.

When transiently expressed, the exogenous vector does not get integrated into the genome. After transfection, they exist as extra-chromosomal elements. Most plasmid vectors, except for some episomal vectors, do not self-replicate and are gradually lost in about a week’s time. Even some viral vectors, such as adenoviral vectors, do not persist over a long time and are largely lost in a month. This is fitting for the overproduction of protein, where the aim is to generate a short and intense, rather than a long and sustained, burst of protein production. Thus, in transient situations, the percentage of cells actually taking up and expressing exogenous DNA (i.e., the transfection efficiency) can significantly affect the overall expression level.

HEK-293 (293; human) and COS (African green monkey) cells are frequently used for the transient expression of proteins, but product quality parameters, such as glycoform profile, may differ for materials derived from different species (e.g., human versus Chinese hamster). However, CHO cells are unique. Transient material produced in CHO cells is more representative of the material produced from stable CHO cell lines, so there has been an increasing use of CHO cells for stable expression. Unfortunately, the transient transfection productivity in CHO is relatively low compared to that of 293 host cells.

What Makes a Hyper-Producing Cell Line? Two types of cells are most commonly used as host cells for the generation of stable production cell lines: CHO cells and myeloma cells. Virtually all therapeutic proteins made with these cell lines are secreted out of the cell and can be harvested from their culture fluid. CHO cells, which do not secrete any protein in an appreciable amount in their native state, must be made to develop the capability of protein secretion while becoming producers. In contrast, myeloma cells have well-

METHODS AND STRATEGIES IN CELL LINE DEVELOPMENT | 129

developed protein secretion machinery, left over from their original purpose of secreting antibodies.

Host Cell

Protein Secretion

CHO DG44

serum dependent, adherent, DHFR-/-

nil

CHO DXB11

serum dependent, adherent, DHFR

+/-

nil

CHO K1

serum dependent, adherent, proline dependent

nil

SP2/0

serum dependent, suspension

plasma cell

Myeloma NS0

serum dependent, suspension, cholesterol-dependent

plasma cell

Typical Characteristics of Producing Lines • Serum independent • Suspension growth • Highly secretory, associate physiological change • Energetic • Secretory pathway • ROS-redox balance • Sustained growth in stationary phase • Ready switch to lactate consumption • Glycosylation capacity

Many scientists have studied the changes that occur during the maturation of B-cell to plasma cell, both at the proteome and transcriptome levels. The cellular alterations appear to include elevated energy metabolism, higher protein secretion and glycosylation capacity, as well as an increased redox balance, to counter the effects of reactive oxygen species.

B-cells and plasma cells have only one functional immunoglobulin gene in their genome. In the developmental maturation process, one of the two gene copies in the diploid alleles is inactivated, thus preventing the production of two immunoglobulin molecules in a single cell (i.e. “allele exclusion”). Even a single copy of a transgene is sufficient for a cell to become a super secretor, like the plasma cell. Therefore, to transform a myeloma cell into a high producer of protein, one needs only to introduce a single copy of the product gene into the appropriate locus of the genome. The pre-existing cellular machinery is already tuned to secrete a high level of protein.

CHO cells, contrary to myeloma cells, must have modifications to enhance their protein secretion machinery and to acquire characteristics of highproducing cells. In addition to bolstering the protein secretion machinery, a high-performing cell line should also have superior growth and metabolic characteristics. Manufacturing conditions differ profoundly from laboratory cultivation. For example, the prolonged stationary phase in a fedbatch culture, in the presence of lactic acid, allows for a longer period of protein production at a stage when the cell concentration is high, thus leading to a maximal product titer. The important question, in this case, is how to identify candidate cell lines that harbor those desirable traits. In the past decade, numerous transcriptome and proteome studies have been conducted to examine the traits leading to hyper-productivity. Biotechnologists now realize that there is

METHODS AND STRATEGIES IN CELL LINE DEVELOPMENT | 130

‘Best’ Producer Cumulative effect of combination of different changes may not be the same, some may not be additive

Potentially a large combination may give rise to similar productivity.

Different changes may lead to the same incremental improvement of cell characteristics necessary for high productivity

Energy Metabolism

Secretion

Redox

Growth/Death Control

Fig. 5.1: Hyperproductivity of recombinant protein in producing cells contributed by multiple cellular functions. Many alternative combinations of superior traits may lead to high-productivity.

probably no single master regulator that can be “turned on” to make a CHO or NS0 cell a hyperproducer. Hyper-productivity is the culmination of multiple changes in multiple cellular pathways, such as metabolism, secretion, redox balance, and growth/death control. The acquisition of hyperproductivity is more likely to involve diminutive gene expression changes on a vast scale, rather than larger alterations in only a few master switches.

Basic Steps for Generating a HighProducing Cell Line

Steps in Cell Line Creation • Transfection • Selection • Amplification • Single cell cloning • Screening • Adaption

A few basic steps are generally followed to make a host cell become a high producer of the desired product. First, a transgene coding for the product protein is typically introduced to the host cell using a plasmid. In addition to the transgene, the plasmid carries a gene that confers a selectable trait, such as antibiotic resistance, so that after transfection, a selective pressure can be applied to enrich for those cells which have internalized the plasmid. The plasmid does not replicate in mammalian cells and would otherwise be gradually lost as cells multiply. By applying selective pressure over an extended period, all of the cells that are selected for would have the plasmid integrated into the chromosome.

After a stable cell population is obtained, preliminary in vitro screening is then performed to screen for clones with the highest production levels. This is often fulfilled by assaying the product concentration in the supernatant of a 96-well plate. Another common practice is to grow cell clones as small colonies on soft agar and then perform immunoassays in situ to identify those with a larger immunoprecipitation zone around the colony. The next step, the amplification of transgene copy number, is practiced in some instances (such as when CHO cells are used), but not all (such as when myeloma cells are used). To do this, the stable cells are subjected to a high concentration of an inhibitor

METHODS AND STRATEGIES IN CELL LINE DEVELOPMENT | 131

to the amplification marker, which the cells require for survival. This high concentration of inhibitor kills the vast majority of cells, except those that have multiple copies of the marker and resistance genes.

As the amplification marker multiplies, the adjacent integrated transgene(s) are also co-replicated. The number of copies may increase dramatically in different regions of the genome, thereby giving rise to high levels of transcription and translation. Through this process, in some high-producing cells, the transcript level of recombinant IgG heavy chain becomes the most highly expressed transcript in the cell. An excessive expression of protein can overwhelm cell’s protein folding capacity and lead to an unfolded protein response in the endoplasmic reticulum and, thereby, induce apoptosis. Consequently, cells which have not developed appropriate machinery to handle the increased production may not survive.

Fig. 5.2: Typical steps in introducing a transgene for generating high producing cell lines for manufacturing.

After amplification, the selected cells not only have multiple copies of the transgene and a high level expression of resistance and product genes, but they also have developed the secretory capacity to allow for enhanced protein secretion. These clones have a high propensity to become high producers, but not all of them do. Hyper-productivity also requires many other traits, such as the capability to quickly grow to a high cell density and the capability to sustain a high viability over a long duration in the stationary phase.

Subsequently, single-cell cloning is performed on those surviving cells, typically by sorting single cells into culture wells by flow cytometry. Cloning can also be performed by dispensing cells (approximately 0.2 cell per well) into multi-welled culture plates, so that the probability of having more than one cell in each well is very low. Thus, all cells that arise from a given well all originated from the same cell. Single cell cloning is necessary because a pool of stable cells would have vastly different genetic backgrounds, perhaps in the loci of exogenous gene integration, and potentially have different mutations caused by those integration and other unknown aberrations. Such a mixture of cells of genetic heterogeneity is called a “cell pool”.

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After single cell cloning, the productivity of each clone is then assessed and those with high productivity are isolated. The selected clones are further expanded for stock preparation, growth characterization and product quality assessment. In some cases, the producing cells are then adapted to growth conditions more amenable to manufacturing conditions.

Methods of Gene Transfer

DNA-Calcium Phosphate Co-Precipitation DNA and calcium chloride is added dropwise into a HEPES buffer with sodium phosphate (1 mM). A fine precipitate forms in 5-30 min, and is added directly to the cells (~240μg/106).

Electroporation Expose cells to a high-voltage electropulse in the presence of DNA solution. This introduces pores in the plasma membrane, allowing entry of DNA. Duration of pulse and strength of electric field varies with cell type.

Lipofection/Lipid Mediated Gene Transfer A mixture of DNA with amphipathic compound (DOTMA, DOPE, etc), that simultaneously interacts with DNA and hydrophobic portions of the membrane, allowing passage of DNA into the cell.

Single cell cloning is considered critical for establishing a production cell line, as the cells arising from a single cell are genetically homogenous. It is practiced regardless of whether there is an amplification step or not. Although the host cells used for establishing production lines are all aneuploid and can be prone to further genome reorganization and epigenetic reprograming, a culture of cloned cells are much more homogenous than cell pools. From a single cell at the beginning of single cell clone to the end of a production run, the production cell may have gone through more than 60 doublings. If that is extended to the entire production lifetime, the number of cell doublings may be greater than 80 doublings. It is important to minimize the outgrowth of mutated cells in the original pool, during the course of cell expansion, by single cell cloning during the generation of production cell lines. A number of different methods are commonly used to introduce expression vectors into host cells. The choice of method is dependent on cell type, the available quantity of cells and plasmids, and the experience of the lab practicing it. Although different methods depend on different mechanisms of plasmid uptake by cells, all methods require the cytoplasmic membrane to first become permeable to plasmids. Plasmid delivery by calcium phosphate precipitation, cationic polymers, and liposomes relies on direct interactions of the particles, or lipid vesicles containing plasmid DNA, with the cellular membrane through an endocytosislike of mechanism. In electroporation and microinjection, physical force is used to introduce openings in the cell membrane for DNA entry. The “Methods of Gene Transfer” table lists

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DNA-Calcium Phosphate Co-Precipitation

commonly used methods of DNA introduction. All DNA and calcium chloride is added dropwise into a HEPES methods use a very high plasmid-to-cell ratio, but buffer with sodium phosphate (1 mM). A fine precipitate only a moderate DNA concentration. While up to forms in 5 - 30 min, and is added directly to the cells (~2- a thousand copies of the plasmid can enter cells, 40μg/106). only a small proportion actually translocate to the nucleus and are transcribed to allow for the Electroporation expression of selectable marker resistance gene. Expose cells to a high-voltage electropulse in the presence of DNA solution. This introduces pores in the plasma membrane, allowing entry of DNA. Duration of pulse and strength of electric field varies with cell type.

Table 2. Estimates of Number of DNA Molecules per Cell for Commonly Used Non-Viral Gene Transfer Methods. Method

DNA concentration (μg/mL)/pM

Cell concentration (106 cells/mL)

Number of DNA molecules per cell

Calcium phosphate

50 / 15

5

1.8*106

DEAE dextran

10 / 3

5

3.6*105

Lipofection

40 / 12

5

1.5*106

Electroporation

40 / 12

10

7.3*105

Basic Elements on a DNA Plasmid

Fig. 5.3: A typical vector for introducing transgene into host cells for recombinant protein production.

Plasmids facilitate the introduction of specific genes into mammalian cells. They also contain elements that enhance the transcription and translation of the product gene. Both viral and bacterial plasmid vectors are commonly used for introducing transgenes into mammalian cells for research. However, the prevailing vehicle used for introducing product gene for recombinant protein production is a plasmid vector rather than a viral vector. Although viral vectors are used often in gene therapy, they are rarely employed for establishing production cell lines for therapeutic proteins. The expression of the product gene may be driven by a constitutive, inducible, or conditional promoter. Conditional promoters, driven by specific endogenous factors or events, are frequently used in the research of differentiation and development. It allows a reporter gene or selectable gene to be expressed after a particular differentiation event. It enables the selection or sorting of differentiated cells

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Elements in a Vector

• Promoter • Coding sequence (often with introns) of gene of interest • poly A signal • Selectable marker • Other elements of plasmid (cloning site, origin of replication)

Target Gene • Positional cloning: screen library for chromosomal markers known to flank the gene of interest, and then “walk”, testing individual genes identified in the region. • PCR amplify

Promoter

• Constitutive • Conditional • Inducible • Native dynamic regulated

in a population. For example, the albumin promoter is expressed and activated specifically in liver cells. When driving the expression of green fluorescent protein (GFP), cells of the liver lineage become green.

For protein production, the vast majority of vectors employ a constitutive, and very strong, promoter. Traditional viral promoters, such as SV40 late promoter or the CMV promoter, are frequently used. In the past few years, constitutive promoters, such as the promoters of elongation factor 1 (EF-1) and glyceraldehyde dehydrogenase (GAPDH), from CHO, have been isolated and used in product gene expression. In addition to a strong promoter, enhancer elements can also be included in the intron of the transgene construct, to ensure a high level of transcription. It is rather common to see that at least one intron is included in the product gene construct. Furthermore, the DNA sequence of the product gene is often “codon optimized” to match the efficiency of translation machinery of the host cell. Through codon optimization, one can replace the codon for a rare tRNA with a more abundant tRNA, to avoid the translation rate being limited by the supply of the rare tRNA. In addition to the promoter, enhancer, and the product gene, the vector must also has a selectable marker, and if amplification is to be involved, the amplification marker.

In the course of introducing DNA into host cells, only a fraction of them actually express the plasmid. Although the number of plasmids entering each cell is likely to be very high (probably in the order of hundreds to thousands of plasmids), the probability of their entry into the nucleus and subsequent integration onto the genome is very low. These plasmids are not capable of selfreplication; they only replicate after integrating into the host chromosome. Free plasmids in the cell are, thus, gradually degraded or otherwise lost. After transfection a very large fraction cell population are untransfected. To identify and select

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those that have the transgene, a selectable marker is included in the plasmid. Cells that have at least one copy of plasmid integrated into the chromosome and express the selectable marker gene will survive in the presence of selectable marker.

Recessive Selection

• DHFR (dihydrofolate reductase) • On DHFR deficient background • TK (thymidine kinase) • On TK deficient background

Dominant Selection

• Antibiotic resistance • Neomycin • Hygromycin • MDR (multi-drug resistance) • DHFR

Reporter Gene • • • •

gfp family β-galactosidase luciferase secreted alkaline phosphatase

Selectable markers are classified into two categories: dominant and recessive. A recessive marker resides in cells with a particular genetic background and causes a growth deficiency. Then, the introduction of a compensatory gene leads to overcoming the deficiency. For example, a cell line without a functional dihydrofolate reductase (DHFR) gene requires the additional supplementation of thymidine and glycine in the culture medium for cell growth. The introduction of a functional DHFR enables it to grow without the supplements. Therefore, after the transfection of plasmid containing DHFR, only the transfectants will grow in the absence of thymidine/glycine. Similarly thymidine kinase (TK)-defective mutants require thymidine to be included to the culture medium. The introduction of a functional TK gene allows for cell growth in the absence of thymidine.

Conversely, the presence of a dominant selective agent is lethal to the cell. By introducing the selectable marker gene to the cell, the cell is endowed with a resistance to the selective agent. The most frequently used selectable markers, and their mode of action in mammalian cells, is listed in the adjacent table. Each resistance gene encodes an enzyme, which modifies the selective chemical agent to destroy its activity. The phosphorylation and acetylation reactions employed for inactivating antibiotics require intracellular reactants (ATP, acetyl group donor). Those enzymes are, thus, only effective in destroying the selective agents intra-cellularly. The hydrolysis enzyme, on the other hand, may be active even when released into medium after cell lysis. In any case, in the selection process, the concentration of the selective chemical agent decreases with time and the rate of decrease is dependent on the concentration of transfected cells. Thus, the optimal concentration for selection for each agent is not only dependent

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on cell line but also on its concentration. Clonal selection and population selection may have rather different optimal concentration of selective agent. Another class of selective agents interferes with the uptake of a toxic selective agent. The multidrug resistance gene (MDR) confers cells with resistance by increasing their ability to pump toxic substances, such as colchicine, out of cells. Its overexpression allows for selection from a background of cells not expressing MDR. Table 2. Commonly Used Drugs for Selection of Stably Transfected Mammalian Cells Antibiotic

Family

Mode of Action

Resistance gene

Mode of resistance

Gene Drug size (bp) concentration range (μg / mL)

Geneticin (G418)

Aminoglycoside

Block protein synthesis Neomycin by inhibiting elongation phosphotransferase (npt)

Phosphorylation of Geneticin

795

100 - 800

Hygromycin B

Aminocyclitol

Inhibit protein synthesis by disrupting translocation and promoting mistranslation

Hygromycin phosphotransferase (hpt)

Phosphorylation of Hygromycin B

1011

10 - 400

Puromycin

Aminonucleoside

Block protein synthesis by causing pre-mature chain termination

Puromycin N-acetyltransferase (pac)

Acetylation of Puromycin

603

0.5 - 10

Blasticidin S

Peptidylnucleoside

Inhibit protein synthesis by interfering with peptide bond formation

Blasticidin S deaminase (bsr)

Deamination of Blasticidin S

396

1 - 10

Zeocin

Bleomycin (Glucopeoptide)

Intercalate into and cleave DNA

Bleomycin resistance protein (ble)

Bind stoichiometrically and prevent Zeocin from binding DNA

375

0.1 - 50

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Amplification

• The strategy is to use a mutated form of the enzyme that has a lower catalytic activity or to use an enzyme inhibitor • DHFR with methotrexate (MTX) • DHFR: dihydrofolate+NADPH → tetrahydrofolate+NADP • GS with methionine sulphoximine (MSX) • The metabolic enzyme (amplifiable marker) needs to be amplified in order to supply sufficient reaction rate for cell survival • Glutamine Synthetase: Glutamate + ATP + NH3 → Glutamine + ADP + Pἱ

Two systems are commonly used for gene amplification in mammalian cells: DHFR and glutamine synthetase (GS). The most commonly used gene amplification system is based on the DHFR gene, whose chemical antagonist, methotrexate (MTX) can be used to drive gene amplification. DHFR is an enzyme which catalyzes the conversion of folate to tetrahydrofolate, a compound required for the biosynthesis of glycine, thymidine monophosphate and purine. Methotrexate, a folate analogue, binds and inhibits DHFR, thereby leading to cell death in the absence of thymidine and purine in the medium.

When cells are selected for growth in methotrexate, the surviving population contains increased levels of DHFR which results from an amplification of the DHFR gene. This is also accompanied by amplification of 10 – 10,000 kilobases of DNA surrounding the site of integration. Therefore, by introducing a gene of interest (i.e. protein product gene) alongside the DHFR gene, coamplification of the product gene can be achieved.

The amplification process can be perform as a single step of MTX exposure over one to two weeks, or in multiple step-wise increases in methotrexate concentration. As MTX concentration is increased, surviving cells with higher degrees of DHFR gene amplification are obtained. Highly methotrexate resistant cells may contain several thousand copies of the DHFR gene.

DHFR based amplification is more efficient in a DHFR defective genetic background. Otherwise, endogenous DHFR may get amplified without concurrent amplification of the product gene. Chinese hamster ovary cells deficient in DHFR were isolated after ethyl methanesulfonate- and UV irradiation-induced mutagenesis. These DHFRdeficient cells require the addition of thymidine, glycine, and hypoxanthine to the media. These cells do not grow in the absence of added nucleosides unless they acquire a functional DHFR gene. The glutamine synthetase (GS) selection system is based on the biosynthetic pathway of glutamine

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from the substrates glutamate and ammonia. Most mammalian cell lines require glutamine supplementation in their culture media to grow since their endogenous GS activity is low. The promoter region of GS in CHO cells is rich in CpG and evidences indicate that GS in CHO is silenced possibly by methylation of cytidine.

DHFR Amplification • More efficient in DHFR- background • CHO DXB11; one DHFR is deleted, the other has missense mutation • CHO DG44: both DHFR deleted • With sufficiently high levels of MTX, amplification can be carried out in DHFR+ background

The GS expression vector contains a glutamine synthetase gene along with the gene of interest, allowing for selection by growth in glutaminefree cell culture media. The GS gene is usually driven by a weaker promoter, typically the SV40 promoter. With a high concentration of the glutamine synthetase inhibitor, methionine sulphoximine (MSX), it is possible to select for transfectants with gene amplification.

Several variations on the systems described above have been developed for increasing achievable expression levels. DHFR is used in conjunction with an impaired neomycin resistance gene. After transgene induction and under G418 selection only cells with the vector integrated in a transcriptionally active region will express neomycin resistant transcripts at high enough level to survive. Since the locus of integration is transcriptionally active, the high expressing clones isolated after amplification have only a few integrated gene copies.

After amplification, lasting for a week to two weeks typically, the concentration of antagonist selective chemical agent is reduced. With a lower level of selective pressure the number of copies of transgenes may also decrease and resulting in a decrease in its transcript level and productivity. The propensity for losing transgenes is probably affected by the loci of integration on the chromosome, with those near the distal end of the chromosome arm being more prone to dislodge from genome. Usually a clone becomes more stable after the initial drop of copy number and product titer upon the reduction of selection pressure. In most cases the remaining transgenes are stable in subsequent cell cultivation. Nevertheless a low level of selection pressure is often maintained to suppress any possible deleterious mutants which may have a lower copy number and faster growth rate.

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Classical DHFR Amplifiable Vector

There have been a number of variations of this method. Some methods use another resistance marker, such hygromycin or neomycin resistance, for the first selection of cells co-transfected with DHFR and the gene of interest, followed by amplification by selection of amplified cells in the presence of high concentration of MTX.

Fig. 5.4: A vector for DHFR based amplification of transgene.

Classical DHFR transfections employ a plasmid in which DHFR is tethered downstream to the gene of interest driven by a promoter (e.g., SV40 enhancer/ promoter). The plasmid is used to transfect CHO cells (such DXB11) deficient in DHFR. The transfected cells are selected in nucleotide-free medium, in the presence of methotrexate (MTX). Subsequently, MTX concentrations are increased to enrich for cells that have multiple copies of DHFR and, concomitantly, the gene of interest.

The DHFR/MTX system has been widely used for the generation of antibody-producing cells. The example shown uses a two-plasmid system. DHFR resides on the plasmid containing the gene encoding the light chain. Note that instead of cDNA, the gene of interest is interspersed between two intron sequences. The heavy chain gene, on another plasmid, has Neo as the drug resistance marker. The two plasmids are co-transfected into CHO cells, often at a stoichiometric ratio that slightly favors the heavy chain plasmid. The transfected cells (which co-express DHFR/light chain plasmid and the heavy chain plasmid) are then enriched using MTX treatment.

Fig. 5.5: A gene construct for introducing heavy chain and light chain molecules of immunoglobulin gene.

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Glutamine Synthetase (GS) as Selectable Amplifiable Marker Glutamine synthetase (GS) is selectable marker in most mammalian cells, as they require glutamine for growth in culture. It is used frequently in myeloma and CHO cells. The transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be achieved by using the inhibitor of GS, methionine sulphoximine (MSX).

Fig. 5.6: A vector with GS selectable marker.

Directing Integration to a Transcriptionally Active Region The locus effect on are more

of transgene integration influences the expression level, due to a position gene expression. There are strategies to select clones whose transgenes likely to have integrated into transcriptionally active region (hot spots).

An impaired neomycin phosphotransferase, which confers neomycin G418 resistance, has been used successfully for hot spot integration. In the impaired enzyme, the translation initiation site has been mutated to reduce its translation initiation efficiency; thus more transcripts are needed to synthesize the same levels of proteins that confer G418 resistance. Moreover, an artificial intron is introduced to decrease the level functionally active Neo transcripts, by increasing the frequency of unsuccessful splicing. As a result, only those clones which have the Neo inserted into a transcriptionally active region of the chromosome will have a high enough expression level to overcome drug selection. Most of the selected resistance clones have only one copy of the transgene. The selected clones can be further amplified using the traditional DHFR system. With the increased probability of transcription, fewer clones will need to be screened to obtain high producers, and most obtained clones have low levels of DHFR amplification. Furthermore enhancement is obtained using a single promoter (CMV) for heterologous protein and selectable (amplifiable) marker, while having an IRES between the heterologous gene and selectable marker gene.

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Cell Adaptation

• From transfection to working bank takes about 4 - 6 months.

Host cell Target gene

• adaptation to suspension growth • anti-apoptotic gene • glycosylation modulation

Transfection

Cell pool, selection, cell clone

Selection of higher and stable producer

Initial drug testing (chemistry, biological, and animal)

Adaptation to suspension growth

Initial process development

Adaptation to serum-free/ Animal-component-free growths

Working cell banking

Process development & Scale-up • feed batch/perfusion • metabolic shift

Fig. 5.7: Typical steps in cell line development.

In a typical cell line development process, a large number of product-secreting clones are selected and subjected to further screening of their growth characteristics, product titer, and product quality, glycosylation patterns, and other post-translational modifications. In some cases, the cells do not grow well in the culture environment used in production (e.g., high agitation rates or altered medium composition). They often have to be “adapted” to new culture conditions by long-term cultivation with a gradual change of environmental factors. Over time, the cells gradually develop the ability to grow under the new chemical and physical environment. The presence or absence of a physical surface for cell attachment is probably the most drastic difference in cultivation conditions. Most normal diploid cells used for virus production are strictly anchorage dependent. Some cells, like myelomas and hybridomas, are suspension cells that can be readily grown in a mixing vessel. Many cell lines commonly used for recombinant protein production, including CHO, BHK, and HEK293 cells, are derived from adherent cells. Although, not being strictly anchorage dependent, they often prefer adherent growth given a compatible surface.

When cultivated in suspension, the unadapted cells either fail to grow or form large aggregates with extensive cell-cell contacts and intercellular adhesion. They can be adapted to grow in suspension by being cultured in shaker flasks or spinner flasks. The initial growth rate is slow, and dispersing agents like heparin sulfate, reduced serum (if any), or calcium incorporation may be necessary to prevent adhesion to the wall of the culture vessel in the early stage of adaptation. Gradually, the growth rate is increased and the cells eventually adapt and appear indistinguishable from regular suspension cells. The adaptation of cells to a new nutrient environment is also commonly practiced in cell line development. For example, the requirement of complex lipid additives and growth factors may be reduced or even eliminated through adaptation, although sometimes these adapted cells exhibit lower productivity.

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Fig. 5.8: A timeline for generating an industrial producing cell line.

Stability of Selected Clones

• From thawing (2x108 cell) to production (20m3) needs at least 16 doublings. • Stability is tested over 40 doublings

• Normal diploid cells used in vaccine production are “stable” within the accepted population doublings • Continuous cell lines have variable karyotypes in culture

Mutations and epigenetic alterations occur in cultured cells at relatively high frequencies. Some of these events are affected by culture conditions. For example, many types of stem cells are prone to differentiation, even by changes in cell density, oxygen tension, growth factor concentration, and cell aggregation state, depending on the particular cell type. Cells used for human vaccine production are mostly diploid and are considered to be stable under established cultured conditions. These cells do not exhibit any visible phenotypic alterations or macroscopic chromosomal abnormalities within the accepted range of doubling, or until they reach senescence. The stability of these cells is not a general concern in bioprocessing.

In contrast, the extensively selected, hyper-producing recombinant cell harboring transfected, and often amplified, transgenes have a higher propensity to lose their high productivity. To begin with, the aneuploid host cells used to generate those hyper-producing cells are less stable than their diploid counterparts. In addition to having abnormal chromosomal counts, many of the chromosomes also have macroscopic structural aberrations. Such chromosomal alterations accumulate over time, generating cells of divergent karyotypes from the same parental line. Divergent karyotype and chromosomal abnormality

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Cell Line Stability Issues • Productivity decrease over time • Product quality changes over time • Karyotype changes over time • Microscopic chromosomal aberration occurs over time • The last two issues may not be critical for protein biologics, but are of concern for cell therapy applications

Possible Causes of Instability in Recombinant Protein Productivity • Mutation, especially in intergenic region in pivotal product gene loci • a producing cell may contain many copies of product genes, but maybe only a dominating few contribute to most product transcript • Epigenetic silencing of pivotal genes, at DNA level or at histone reprogramming level • Loss of copies of product gene due to deletion or chromosomal rearrangement

is, thus, an inherent nature of producing cells derived from continuous cell lines. Even though production lines may not be stable (in relation to ploidy), they must maintain their production characteristics related to growth, productivity, and product quality over the number of population doublings required for creating sufficient cell banks. They must also be hardy throughout the thawing process, as well. Assuming that a product life time of 10,000 runs at 10,000 liter scale, with a final cell concentration of 1010 cells/L, the selected cell clone will have to double nearly 80 times. This number of doublings greatly exceeds that required of a fertilized egg to grow into a hamster, a mouse, or even a human adult. In that long duration of time, the occurrence of mutations, epigenetic changes, and chromosomal rearrangement in some cells of the population is unavoidable and probably cannot be eliminated with today’s technology. In discussing cell line stability, we decided to focus on property changes that affect productivity and product quality. The critical component for sustaining productivity over time is to prevent any cell with a lower productivity from overtaking the population, or to prevent a very high rate of productivity loss in the majority population.

A gross and rapid change of productivity in a large fraction of cells may occur upon the removal or reduction of selective pressure after transgene amplification. This problem is alleviated by employing a lower degree of amplification and by establishing the clone only after the copy number of transgene has stabilized. For long-term stability, one could carry out serial cultures for 40 to 60 doublings and examine the productivity, as well as the transgene copy number. From a thawed liquid-nitrogen frozen cell bag of 1010 cells to a production reactor, cells may undergo 15 doublings, so a 40-doubling test of stability will certainly give sufficient margin.

The stability of product quality is more difficult to assess. Mutations in genes affecting product quality, such glycosyl-transferases, may occur and lead to a subpopulation of cells that produce inferior

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• Production cell lines are tested for their ability to retain the product gene in the genome and produces the product • The focus is production stability, not cell/genome stability

product. Mutations causing protein sequence alteration may occur in one or more copies of product genes in all, or a subpopulation of, cells. Since not all copies of product genes in the cells are transcribed and translated at equal efficiency, not all mutations of product genes in a production cell population will be manifested to the same degree. With deep sequencing technology, one may be able to detect such mutations, even at minute level, in the consequent producing cell population.

Automation and High Throughput Technology

PerkenElmer - Basic Model

Tecan -- more automated with multi-plate capabilities and computer interface

Fig. 5.9: Examples of high throughput cell clone screening system.

Developing a cell line for production purpose is a very labor-intensive process. To increase the probability of obtaining a very high producer, a large number of cells need to be isolated at every step, which involves productivity variation from successive rounds of selection and amplification. In the past decades, lab automation and highthroughput technology have become an integral part of bioprocess development. Liquid and cell handling, both cell pool and cell clones, quantification of product titer, data acquisition, data processing and analysis, and archiving have all becoming automated.

Many of the automated systems are based on culture plates, or wells, and resemble other liquid handling systems for high throughput chemical screening. The difference is that a incubation system, with temperature and atmospheric control for gas mixture and humidity, is necessary. Robotic arms are often used to move plates onto the working “stage” and allow multiple manipulations to be performed on multiple plates without human interference. In many cases, the system is installed inside a clean room or clean hood to minimize microbial contamination.

The culture handling system is usually integrated with an assay system to assess product titer and cell growth. Multi-step assays and screening protocols can be performed by transferring culture fluid into automated assay systems. The results can be directly integrated into culture handling systems to further expand wells or plates selected for

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• Basic liquid handling manipulations • Distribution of liquid to 96 - 384 well plates • Sampling/removal of liquid • Transfer from plate to plate • Cherry picking – transfer from well to well

More complex models are capable of: • Cherry picking • Multiple plate handing (i.e. movement of plates from a “hotel” to the pipetting stage) • Multiple “steps” performed sequentially (e.g. DNA preparation protocols) • Other “add-ons” like PCR machines, incubators, spectrophotometers, etc

further investigation. An integrated microscopic imaging system can provide the added capability of determining anything from clonal colony growth to colony morphology to ensuring that cells picked from each well are single-celled clones.

Another type of automated system integrates cell cloning with product titer assessment by performing cell screening on agar plates. In this case, the secreted product molecules (mostly antibodies) are entrapped in agar that contains antibodies against the product. A halo ring of immunoprecipitation zone is formed around the colony. The size of the halo ring reflects the amount of product secreted. Image analysis is then used to extract the data for selecting high producing clones to pick.

Concluding Remarks Under best culture conditions, a hyperproducing industrial cell line derived from CHO or myeloma cells can secrete 50-100 pg protein/cell-day, a level which rivals professional secretors in vivo. Such remarkable cell lines are created through the combination of optimized genetic constructs, selection, amplification, cell clone screening, and the insight of picking the “right” clones. Although the specific productivity of the producing cells has increased by about one order of magnitude, the methodology of generating high producing cells has remained largely the same in the past three decades. The entire process is still empirical and very labor intensive. High-throughput cell handling and screening systems allow for the screening of a large number of potential high producing clones in the early stages of cell line development. However, subsequent steps of adaptation, growth characterization, and testing

of stability is still labor intensive. The use of host cell lines, which have been adapted or modified to harbor all desirable growth characteristics, have greatly reduced the need of adaptation. There is an increasing movement toward miniaturizing cell culture while still simulating large-scale reactors, although this progress is still limited. The advance in genomics has brought about a fundamental change in the way we can study the process of cell line development and brightened the prospects that we can gain mechanistic insight into hyper-productivity. This knowledge may allow us to quickly select the “right” clone by examining the transcriptome or genome of the candidate cells. With more tools for genome engineering becoming available, it may also become feasible to impart on the cells favorable genome-wide modifications.

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Stoichiometry and Kinetics of Cell Cultivation Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Mass and Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Mass and Size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material Balance on Cell Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Variation in Cell Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino Acid Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Growth of Mammalian Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative Description of Cell Growth & Product Formation . . . . . . . . . . . . . . . . . . Stoichiometric Ratio and Yield Coefficient. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integral Cell Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetic Model of Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Model Describing Growth and Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . Monod Model and its Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environment, Kinetics and Stoichiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147 148 148 149 150 152 153 154 156 158 159 162 162 164 165 166

Introduction Cultured cells take up nutrients to generate energy and to make more cell mass and products. For manufacturing, it is important to supply a sufficient quantity of nutrients. These nutrients allow cells to grow and produce product while minimizing the formation of waste product. To make the manufacturing process efficient one must also produce the targeted product quantity within a given period. Therefore, one must not only know how much nutrients to supply, but often also how fast to deliver in order to sustain the production environment. We use stoichiometric principles to determine how to supply the correct quantity of nutrients; and use kinetic principles to guide the process along a desired path. Awareness of these concepts is key to an overall understanding of how to culture cells. This chapter discusses stoichiometry,

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kinetics of cell growth, and also gives the typical values of stoichiometric and kinetic parameters commonly encountered in cell culture processes.

Cell Mass and Composition Cell Mass and Size Table 1. Typical Dry Weight of Cells Bacterial

10-12 g / cell

Yeast

10-11 g / cell

Average Animal Cell

3-6 x10-10 g / cell

Table 2. Average Composition of an Animal Cell Pg/Cell

Range

Percentage % of Dry Biomass

Wet weight

3500

3000 - 8000

Dry weight

600

300 - 1200

Protein

250

200 - 300

10 - 15

~50 - 70

Carbohydrate

150

40 - 200

~1 - 5

~5

Lipid

120

100 - 200

~1 - 2

~5

DNA

10

8 - 17

~0.3

~2

RNA

25

20 - 40

~0.7

~4

Water Volume

55 - 80 4x10-9cm3

Both microbial and mammalian cells vary widely in size. In general, the dry biomass of bacteria, yeast, and animal cells is in the order of 10-12, 10-11, and 5 x 10-10 g per cell, respectively. The most abundant chemical species in a cell is water, accounting for 90% of the volume of plant cells, 80 – 85% of animal cells, and 70% of bacterial cells. Since cell cultivation is carried out in aqueous environments and the amount of water taken up by cells during growth is extremely difficult to assess, the material balance on cell culture is typically performed only on “dry” matter, excluding water. Our discussion on stoichiometry will be largely based on dry biomass of cell number, as commonly practiced in cell culture.

Macroscopically, cells are made of a few classes of macromolecules (protein, DNA, and RNA) or macromolecular assemblies (primarily lipid bilayer membranes). These organic matters constitute the vast majority of the dry mass in a cell. Protein molecules constitute the largest portion among them, providing the machinery for DNA, RNA, and protein synthesis. Protein molecules also serve as the structural components of the cell and execute all of the catalytic, transport, and communication functions. The lipid content of an animal cell is greater than in a bacterium. The abundance of organelles contributes to their higher content and their lipid bilayer membrane in an animal cell.

Intracellular carbohydrates exist as oligosaccharides on many proteins and lipids. Carbohydrate also exists as ribose in DNA, RNA, and nucleotides (e.g., ATP, GTP, etc). Only a small fraction exists in a free (or phosphorylated) form. The cellular content of carbohydrate is harder to estimate, because it usually exists as a part of other molecules.

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Material Balance on Cell Growth

Growth of Biomass Involves: • Consumption of nutrients • Production of new biomass • Excretion of products

Glucose Amino acids Other macronutrients (lipid, nucleotides, etc.) Micro-organic nutrients (vitamins) Bulk salts Trace minerals Oxygen

→  CELLS →

Biomass Product Carbon dioxide Water Lactate, NH3 Excreted amino acids

Example of the “equation” for hybridoma growth: C6H12O6 (glucose) + 0.15C6.14H12.36N1.50O2.08 (weighted average of amino acids) + 0.34C5H10N2O2 (glutamine) + 1.39O2 →2.37CH1.97N0.26O0.49 (cell mass) + 0.0058CH1.83N0.14O2.06 (antibody) + 1.53CO2 + 1.28H2O + 1.44C3H6O3 (lactate) + 0.16NH3 + 0.13C4H7NO2 (alanine) Such a formula is used when one performs metabolic flux analysis.

The principle of material balance holds true in cell culture. The total mass of inputs and outputs, and the amount accumulated in a system, is always in balance. The two most common, and most abundant, nutrients in cell culture are glucose and glutamine (although some cultures do not require glutamine). They serve both as constituents of cell mass and as sources of energy. Other common inputs to cell culture processes include lipids, lipid precursors, vitamins, and salts; these will be discussed in the Medium Design chapter, as these nutrients supply key cellular constituents, but contribute less to generating energy.

To generate energy, glucose is converted to lactate, CO2, and H2O through glycolysis, the TCA cycle, and the pentose phosphate pathway. Glutamine is deaminated, releasing NH3, before its carbon skeleton is used for energy metabolism. Consequently, the accumulation of metabolites, including lactate, NH3, CO2, and H2O, is commonly seen in cell culture. In many cases, the amino group from the metabolized glutamine and other amino acids are exported as nonessential amino acids (such as alanine, asparagine, and proline), in addition to being excreted as NH3.

Energy metabolism satisfies the energetic needs of making biomass, through the biosynthesis of DNA, RNA, protein, and organelles. Other important energy-intensive aspects of cellular events are the uptake of nutrients and the sustained balance of cellular osmosis and membrane potential. As will be discussed later, the cell’s cytoplasmic and mitochondrial membranes have a negative electric potential that must be maintained, at the expense of energy, to sustain cell viability The process of growing cells and producing a product can be formulated into an “overall biomass synthesis” equation. This equation can be viewed as, essentially, the “apparent” composite of all reactions involved in generating energy and synthesizing biomass. At the center of the reaction will be biomass, so a formula for the cell mass

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can then be established, based on its elemental composition. Usually, we use a formula that neglects all elements except for carbon (C), hydrogen (H), nitrogen (N), and oxygen (O). This formula is only useful for describing the ratio of those elements. One can arbitrarily assign the stoichiometric numbers to give them different “formula weights”. In the example shown, the stoichiometric number of carbon is chosen to be 1. Others may prefer to assign the formula mass to be 100. The inputs in a cell growth process include all nutrients consumed by cells to proliferate. Since only C, H, N, and O are considered in the stoichiometric equation, we consider only glucose, glutamine, and other amino acids. Other minute media components containing C and N (such as vitamins or nucleotides) are neglected. Instead of writing down all amino acids separately, one may also use a weighted average, according to the stoichiometric ratio of their consumption.

The outputs include “new” cell mass that has been generated as the result of nutrient consumption, as well as the metabolites and product that have been excreted. One can write a molecular formula describing the formation of the protein product, by knowing its composition. Typically, the metabolites excreted also include lactate and ammonia, as well as some non-essential amino acids.

Variation in Cell Volume Table 3. Size of Animal Cells Cell Type

Volume (μm3)

Hybridomas Endothelial Cells Trypsinized and reattached before spreading occurs

12 - 20 1400 - 2500

17

2000

Chinese Hamster Ovary cells (suspension)

1200 - 1800

Chinese Hamster Ovary Cells (anchored)

1300 - 1800

Human foreskin fibroblasts (FS-4)

Diameter (μm)

7000

~14

The volume of a typical animal cell is a few pico liters (about 1,000 times larger than bacteria). The average cellular diameter ranges from 10 to 20 µm. Even for the same cell line, one can detect great size variations over a range, since cells immediately, before and after mitosis are about two times different in their size. At a given time in a growing culture, cells are in different stages of the cell cycle and their size distribution is somewhat larger than two fold. For aneuploid cells, the distribution of size is typically greater than normal diploid cells. The distribution of cell size changes with the growth stage. Rapidly growing and quiescent cells may have different sizes. Furthermore, in

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• Cell Volume exhibits a distribution at any point in time

a culture, cells that lose viability often become visibly smaller, as measured by flow cytometry.

Cell size varies amongst different cell types. Many types of stem cells are fairly small. Their nucleus spans more than 70% of the cell diameter and their cytoplasm is relatively small. Liver cells and antibody-secreting plasma cells are at the other end of the cell size spectrum, and contain a significant amount of cytoplasm for protein secretion.

• Dead cells are often smaller • Varying with culture stage

It is instructive to remember that the volume of a sphere (which is a reasonable approximation of a cell) is proportional to its diameter raised to the third power. Therefore, cells that are twice as large in diameter are eight times larger in cell volume. Although cell number is traditionally used for the quantification of cell concentration, it may not sufficiently capture the difference when comparing different processes, in which cell sizes are very different.

Fig. 6.1: Cell size change during a batch culture

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Amino Acid Composition Table 4. Amino Acid Composition of Cells and IgG Cell Composition Standard IgG Mean deviation composition ALA

9.03

0.32

5.31

ARG

4.74

0.32

2.43

10.08

0.59

0.26

0.04

12.62

0.63

GLY

9.14

0.57

6.98

HIS

2.22

0.07

1.67

ILE

5.73

0.35

2.43

LEU

9.00

0.68

6.83

LYS

6.85

0.49

6.98

MET

2.27

0.14

1.37

PHE

3.73

0.31

3.49

PRO

5.51

0.58

7.13

SER

6.19

0.16

12.90

THR

5.42

0.22

7.74

TYR

2.73

0.14

4.10

VAL

6.54

0.27

9.10

ASN ASP CYS GLN GLU

3.49 3.95 2.43 5.01 5.16

Microbial and plant cells often grow on simple carbon sources supplemented with an inorganic nitrogen source, such as ammonium or urea. The cells convert inorganic nitrogen to all 20 natural amino acids that are used to make proteins. Animal cells lack the capability to make 11 to 12 of those 20 natural amino acids. These essential amino acids must be supplied for animal cell culture, to enable them to grow and make products. Thus, knowing the amino acid composition of cells, and of the protein product, is important.

The protein content and composition of cells change under different growth conditions; however, they are seldom measured. Nevertheless, literature values of some cells are available, as well as the amino acid composition product, IgG. Given target levels of biomass and product to be produced, a stoichiometric amount of all essential amino acids must be supplied. In addition to essential amino acids, which must be provided, non-essential amino acids are usually also supplied. However, these can be derived by metabolic transformation from other amino acids and can be considered “substitutable”.

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Intracellular Fluid Table 5. Intracellular Concentrations of Amino Acids mM

mM

Ala

0.2 - 2.0

Lys

0.1 - 0.6

Ang

medium concentration • At high cell concentration, cell consumption can cause depletion in medium • Need to supplement about 1x in feed medium

for the design of the next feed medium. Using these methods, a very effective medium is typically achieved with fewer than three rounds of optimization.

Some medium components are consumed in very small quantities and their concentrations remain virtually unchanged in culture. This includes many inorganic ions, such as sodium, calcium, and sulfate. From a stoichiometric point of view, the feed medium does not need to include these nutrients. As feed is added, however, the volume of the culture increases and causes the medium components to be diluted. Thus, these components are included at low levels in feed medium (typically 1X concentration or less). In some cases, inorganic salts, such as NaCl, are completely eliminated from the feed medium to reduce any changes in culture osmolarity.

Some ions, such as magnesium (complexed with ATP), phosphate (as free phosphate or in nucleotides), and potassium, are present at a much higher concentrations inside the cell than in the medium. At high cell concentrations, the amount of these ions taken up by the cells may become significant. Therefore, it is often necessary to compensate their consumption by supplying them in the feed medium. In addition to basal medium components, protein hydrolysates, serum, insulin, transferrin, vitamins, and lipid additives are also used in culture. These additives supply minute nutrients, which are consumed or become inactive with time. However, the concentrations of these medium components are not usually measured and their consumption rate is mostly unknown. In the absence of a reliable measurement consumption rate for those additives, one has to rely on an order-of-magnitude estimate of the upper and lower limits of their consumption rate. The feeding rate of those additives is then chosen to maintain their concentrations above a minimum threshold, while below a maximum tolerable limit which is experimentally determined.

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Control Strategies for Fedbatch Cultures

Developing Fedbatch Strategy Define objective of feeding. Possibilities include: • Sustain nutrient level • Manipulate growth rate in a range Define what criteria to manipulate • e.g., what level of nutrient to control? Mode of feeding • Continuous vs. intermittent How to deliver the feed medium • By direct measurement of nutrient or infer from indirect measurement

Control Objective and Criteria: Productivity and Product Quality • Control objective extends beyond acheiving high productivity to high product quality and consistency • Require an understanding of relationship between manipulated variables and control objective

How the feed medium is delivered to a culture may affect the performance of a fedbatch process. Online measurements may be employed to determine the consumption of the reference nutrient in real time, then used to drive a fully-automated system to feed nutrients continuously. At the other extreme, one can use off-line monitoring of nutrient level and manually feed the nutrient periodically. Three elements should be considered in developing a feeding scheme: 1) the control objective and the control criterion (level of different nutrients to be maintained), 2) the mode of feeding (continuous or intermittent feeding), and 3) a control strategy for determining the timing and amount of feed medium to be delivered.

The simplest strategy is to allow nutrient levels to vary, within a wide range, by adding large quantities of feed media at widely-separated time intervals (e.g., once or twice per day) based on off-line measurements or historical data. Such a periodic feeding scheme is very simple and is usually sufficient to avoid depletion or overfeeding of nutrients. For more specialized cases, especially those aiming to manipulate cell metabolism, a more frequent measurement of parameters, along with well-controlled feeding schemes, are necessary. A common objective of nutrient feeding is to increase product titer by increasing cell concentration. In recent years, the objective has been gradually extended to assuring product quality and consistency. Culture conditions may affect glycoform or protein folding. The depletion of a particular amino acid may cause an amino acid to be mis-incorporated into the protein product. Culture conditions may also affect the frequency of deamination or glycation of the protein product after it is secreted into the medium. Furthermore, extensive cell death may cause product degradation or desialylation. The control objective of feeding may be to reduce glycation by setting a control criterion to keep

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Possible Control Objective and Control Criteria Control Objective • Reducing desialylation by reducing cell death and sialydase release • Reducing glycation Control Criteria • Control osmolarity below set point • Control glucose level below set point

glucose concentrations below a certain level, or to minimize amino acid mis-incorporation by maintaining amino acids above a certain level. Another method of control is to minimize protein quality deterioration by maintaining a long, slow growth phase and avoiding a death phase.

After defining the control objective, the next step is to identify the process variables that cause the process to deviate from the control objective. Furthermore, a relationship between the key process variable and the control objective, such as inducing lactate consumption, should be available so that the process variable can be controlled within an optimal range. For mammalian cell culture, although the control objectives can be defined, the factors that affect the path to the objective are not easily identified. Lacking knowledge of the relationships between manipulated variables and the control objective makes the optimization of control criteria difficult. In the past few years, it has become empirically evident that maintaining a moderately high osmolarity in a late stage of culture can increase the productivity of recombinant proteins. A high osmolality increases productivity, perhaps, by enhancing the expression of stress responsive proteins, which facilitate protein folding, although the exact mechanisms are not known. By keeping the osmolality only at a moderately high level to avoid causing a rapid decline of cell viability the culture can be sustained over a longer period of high specific productivity, leading to high final product concentration. This practice is commonly used in industrial manufacturing. An increasingly common practice is to employ atypical culture conditions, which may not be optimal for long-term maintenance or expansion of cells in the final production reactor. These conditions may include very high glucose concentrations (15 g / L or 83 mM), low pH (6.9 vs. 7.2 for optimal growth), and/or low temperature (33/34 oC vs. 37 oC). These conditions have been found to affect the duration of culture and the overall cell metabolism, resulting in an increased product titer. A high concentration of glucose

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also increases osmolality. This is compensated by reducing the salt concentrations to keep osmolality around 300 mOsm during the cell growth stage.

Feeding Strategies Feeding by Direct Measurement of Nutrient Consumption

In-Time Measurement for Control • On-line or off-line

Example •

On-line sampling coupled • HPLC for amino acids • Enzyme assays for glucose, glutamine, lactate

Different Way of Coupling Feeding to On-Line Measurement • Single stream with fixed stoichiometric ratio • Multiple streams ratios among different nutrients can be adjusted

Direct measurement of nutrient concentrations is the most straightforward way to determine the amount, rate, and timing of feed to be added. Based on current concentrations of nutrients, one can determine how much medium should be added to sustain the nutrient level for a given period of time.

The concentrations of some nutrients can be determined on-line, although off-line nutrient measurement is more common. Glucose and glutamine are the two nutrients most commonly measured and used as controls because their concentrations can be determined rather rapidly in laboratories. Direct measurement of these compounds on-line can be implemented using an auto-sampling device, in series with commercially-available immobilized enzyme/membrane-based measurement devices.

HPLC can also be implemented as an on-line approach for the measurement of glucose and amino acids. This technique requires a series of processing steps for sample preparation before injection into the HPLC. A significant lag time between sampling and delivering control action is unavoidable. However, since the doubling time of mammalian cells in culture in generally exceeding fifteen hours, even an hour lag time in HPLC measurement of amino acids is acceptable. Industrially HPLC on-line analysis has been implemented at pilot plant scales.

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Proportional Feeding With Base Addition Rationale • In lactate production state L / G is relatively constant • Base added to neutralize pH at 1 mole base / mole lactate • Use stoichiometric ratios to lactate to feed • Easy to implement, but its sensitivity is limited by buffer in medium

A widely-used control strategy in bioprocessing is to proportionally add medium according to the amount of base added to the culture for controlling pH. This provides the option of continuous on-line feeding without an on-line nutrient measurement device. In most cultures, a large fraction of the glucose consumed is converted to lactic acid. To maintain culture pH, one mole of base is added to neutralize each mole of lactic acid produced. If the stoichiometric ratios between lactic acid production, glucose consumption, and other nutrients are relatively constant, the rate of lactic acid production can be used to estimate the consumption rates of glucose and other nutrients.

Barring the effects of pH buffers (such as sodium bicarbonate or HEPES), the base addition rate is indicative of lactic acid production. This method is simple and easy to implement. However, it is highly sensitive to CO2 level in the gas phase and sodium bicarbonate concentration in the medium.

Proportional Feeding With Turbidity • Turbidity is a good indicator of total cell density (but not viability) • Feed nutrients proportional to cell density • Reliable during growth stage • Capacitance probe can measure viability

Proportional feeding according to base addition is not well-suited for processes requiring a highly accurate control of feed rate. Medium buffer capacity can cause delays in base addition and decrease the overall sensitivity of the method.

The use of an online laser turbidity probe can provide accurate estimations of cell concentration in culture. Simple proportional feeding with turbidity works well during the exponential growth phase, when viability is high and the growth rate is relatively constant. However, near the end of the exponential growth phase, when viability drops, an assessment of viability or metabolic activity must be used to adjust feed rates to avoid over-feeding. An alternative measurement to turbidity is measurement of capacitance, which reflects viable cell volume, i.e., viable cell concentrations and cell sizes. This may be used for better control of nutrient feeding rate.

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Proportional Feeding With Oxygen Uptake Rate (OUR)

• Most sensitive among all on-line measurement of metabolic activity • Require developing a computer algorithm • Need to develop stoichiometric ratios to oxygen

Among different measurements of nutrient consumption, the oxygen uptake rate (OUR) is most accurate for assessing cellular metabolic activities. OUR measurements, unlike pH, are not masked by buffers in the medium. A small amount of oxygen consumed in culture, even in the range of 10 µM, can be accurately determined using a dissolved oxygen sensor. For other nutrients which are often present at millimolal or hundreds of micromollal levels, such sensitivity of measurement cannot be easily achieved especially for on-line measurement.

With its high sensitivity, small changes of OUR can be confidently detected on-line and in real time, thereby providing an immediate indication of changes in metabolic rate. On-line oxygen consumption data can then be used to determine nutrient demand, using established stoichiometric ratios and control continuous feeding.

Delivery of Feed Medium Parameters in Feeding • Initial culture volume vs. feeding volume • Feed concentration • High concentration desired, but stability/ precipatation concern • High salt content from solubilization • Single feed vs. multiple feed solution • Need adjustable stoichiometric ratio • Continuous vs. step-wise feeding • Step-wise feeding • Frequency • Equal volume each time or proportional to cell density • Operational simplicity vs. controllability

After determining the amount of feed media to be added, one needs to decide on the mode of medium delivery (e.g., the frequency and amount of feed to be delivered at each feeding). The method of feed media delivery is constrained by equipment and also by the composition of the feed medium. The feed medium usually consists of a solution of concentrated amino acids and other organic nutrients, which, when kept over a long time, tend to precipitate.

The main consideration in determining the proper feeding frequency is the acceptable range of nutrient concentration. The concentration of nutrients will fluctuate between a high level at the time of feeding and the low point immediately before the next feeding. More frequent feeding reduces the deviation from the set point. On-line feeding, by coupling to base addition, turbidity, or to OUR measurement, is easily implemented by computer control and is almost continuous. When feeding is coupled to less frequent off-line measurements, medium is typically added manually, a few times a day.

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Processes employing continuous feeding to control nutrient levels in a small range will require substantially more effort than intermittent feeding strategies. While allowing more control over environmental conditions, the superiority of continuous feeding in terms of extending culture lifespan and increasing productivity has not been clearly documented, except in the case where a metabolic shift is the desired outcome. In fact, with simple off-line monitoring, robust, intermittent feeding strategies is standard industrial practice.

Online Estimation of Stoichiometric Feeding Detecting Stoichiometric Ratio Change During the Culture • Monitor glucose, lactate, glutamine, OUR • Monitor ∆L, ∆G, ∆G in, ∆OUR and check their ratios over time

A challenging issue of stoichiometric feeding is the adjustment of the feeding rate, or feed composition, in response to metabolic changes throughout a culture [12]. This is particularly critical when fedbatch cultures are used to elicit a metabolic shift. Changes in metabolism over the course of a culture are commonly seen, as evidenced by the nonlinear relationships between specific nutrient consumption rates. Many such changes bear little consequence on cell growth or productivity, but in some cases the effect is profound.

For simplicity, major changes in feed composition or feed rate are only made when profound changes in metabolism are observed. For example, in a late stage of growth, cells may cease to produce lactate and consume glucose at a slower rate. Excessive glucose feeding may, then, reverse the metabolism in the favor of lactate production.

Major changes in the metabolic rates of glucose, lactate, and glutamine can be detected by monitoring their stoichiometric ratios throughout the course of a culture. On-line measurement of nutrient levels would allow for timely detection of changes in stoichiometric ratios; however, its widespread implementation in industrial processes will require further development of reliable, automated sampling methods. Without on-line, direct measurements of glucose and lactate to detect changes in stoichiometric

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ratios, one may resort to indirect estimation. For example, changes in OUR and the base addition rate may be an indication of a changing metabolic state, as the ratio of OUR to lactate consumption changes significantly when cells switch from a lactate production state to a consumption state.

Concluding Remarks Fedbatch culture is the prevailing mode of cell culture in the final production of manufacturing recombinant proteins. It is also commonly used for extending the period of cell expansion for pre-production culture. In the latter, the culture conditions and feeding strategy are designed for optimal cell growth, whereas in the former, the conditions are often designed to elicit a high

productivity, high cell concentration, and high product accumulation. The design of feed medium and the selection of the feeding control strategy are critical to the successful implementation of fedbatch culture. By applying stoichiometric principles to feed medium design and by using a well-designed feeding strategy, an optimal fedbatch culture process can be implemented with relative ease.

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Cell Retention and Perfusion With contributions from Sadettin Ozturk, Chun Zhang, and Weichang Zhou Practice of Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of Perfusion Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material Balance on Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Recycling Factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of Cell Retention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Incline Settling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acoustic Resonance Enhanced Settling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spin Filter Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microfiltration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternating Tangential Filtration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

249 251 251 253 254 254 255 256 257 258 259 260 261

Practice of Perfusion Culture Marketed cell culture products using perfusion bioreactors Product

Company

• RecombinantTM, Antihemophilic Factor (recombinant), (Factor VIII) • Kogenate-FS (Factor VIII) • Aldurazyme • Naglazyme • ReoPro (IgG Fab Fragment) • Remicade (IgG1 • Simponi (IgG1) • Xigris (Protein C) • Cerezyme • Fabrazyme • Myozyme/Lumizyme • Gonal-F (follicle stimulating hormone) • Vpriv (velaglucerase alfa) • Replagal (agalsidase alfa) • ReFacto (Factor VIII)

Baxter Bayer BioMarin Centocor (J&J) Eli Lilly Genzyme (Sanofi) Serono (EMD) Shire Wyeth (Pfizer)

Many sectors of the chemical process industry underwent major transformation during the second half of the 20th century. One of the most significant changes is the switch from batch processes to continuous processes. By minimizing equipment turnover time and process start-up time, a continuous process can be sustained at a high rate of productivity over a long time and achieve a higher throughput than a batch process.

Increasing adoption of the continuous process method also sparked much research into bioprocessing. Many enzymatic biocatalyst processes have long been operated continuously, similar to most waste treatment or biodegradation processes. Contrary to the practices of the chemical process industry, however, continuous process only became more common in the later years of microbial and cell culture bioprocessing. A vast majority of biochemical processes involving microbial or animal cell cultivation are batch

CELL RETENTION AND PERFUSION | 249

Advantages Better Product Quality • Better controlled culture environment (nutrients & byproducts) • Pseudo steady state operation (ease of control) • Shorter residence time • Higher cell viabilities & lower concentration of impurities • Critical for unstable molecules More Economical • Higher cell concentrations & higher productivities • Smaller bioreactor size • More flexible • Faster start up in process development Disadvantages Longer Cycle Time • Longer process development & validation time • Higher contamination risk • Higher equipment failure risk • Potential regulatory/licensing issues

processes for many reasons. First, unlike catalysts and reactants do not change their behavior over time in chemical reactors (although catalysts may gradually lose their activity), microbes and cells may mutate, evolve, and change the make-up of their population and production capacity. Second, the risks of microbial contamination and equipment failure make long operations undesirable, especially in manufacturing, for which process robustness is of paramount concern. This is especially true when producing high value pharmaceuticals. Finally, the current product capture and purification operations are all designed for batch mode. Even if the production is operated in a continuous mode, the process is not designed to realize all of the advantages of a continuous process.

A continuous culture is constrained by the maximal flow rate at which it can operate. One cannot operate at a flow rate that is faster than cells’ growth rate; otherwise cells are washed out and incapable of replenishing the culture content. This is particularly daunting for processes that must be performed at a flow rate higher than the cell growth rate. In some cases, cells produce growth inhibitors that must be continuously removed by media replenishment. In other cases, cells must be grown at a low growth rate to achieve a high productivity, resulting in the media being removed at a faster rate than the growth rate.

To overcome this shortcoming, a cell recycling system can be added to continuous culture. By recovering cells from the effluent flow and returning them to the reactor, one can operate a continuous culture beyond its natural limitation of the dilution rate (flow rate divided by the bioreactor volume). With a higher cell concentration in the reactor, the overall throughput of the reactor is then also higher.

When cell culture was first adopted as the production vehicle for biopharmaceuticals, continuous operation was explored as an industrial process. Researchers realized the growth of mammalian cells in batch culture is impeded by lactate and ammonium accumulation. A continuous process, thus, alleviates growth inhibition by removing metabolites through

CELL RETENTION AND PERFUSION | 250

a continuous flow of medium. However, the high cost of medium, especially serum, and the low product concentration made continuous culture impractical.

To enable continuous operation, cells are retained in the reactor while the media flow flushes out metabolites. This can be accomplished by immobilizing cells on some solid particles to prevent them from being flushed out by media flow, or by separating cells out from the effluent stream and recycling them back to the reactor. Thus, the general idea is continuous culture with cell retention. Theese processes are often called perfusion, which is reminiscent of the procedure of flowing fluid through an organ or tissue.

Analysis of Perfusion Culture Material Balance on Perfusion Culture

Fig. 12.1: Schematic of a continuous culture with cell recycle

A number of biotherapeutic proteins are produced by perfusion processes. Some recombinant antibodies could easily be produced with a fedbatch process instead. It should be noted that the selection of process mode is often based on the availability of in-house expertise and many other factors. However, in the cases of labile products that may be degraded or otherwise inactivated over time, a perfusion culture alleviates the loss of productivity that cannot be easily overcome in a batch culture. For products that accumulate only at very low concentrations, a perfusion process may also present a competitive advantage over a batch or fedbatch

One can analyze a perfusion culture system by performing material balances on the reactor system. The flow rates (F) of fresh medium entering and exiting the system are the same, to keep the volume in the reactor constant. The fresh medium stream is free of cells. We also assume that the reactor is well mixed, so that the nutrient (substrate) concentration in the reactor is the same that it is in the effluent stream. A cell separator is used to process the reactor effluent stream to return a concentrated cell stream with a given cell concentration, cx, back to the reactor. Note that the effluent stream from the reactor has a higher flow rate than the fresh feed, with a flow rate of (1+α)F instead of F, to balance the recycle stream

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For the balance equation on biomass for the bioreactor is 

dx V = α Fcx − (1 + α ) Fx + μ xV dt

Balance on the cell recycle system gives: 

(1 + α ) Fx = αFcx + Fx2 (1 + α − α c) Fx = Fx2 x2 =1 + α − α c x

The balance on the substrate on the bioreactor   V

μx ds = Fs0 − V − F (1 + α ) s + α Fs dt Y

Defining F/V=D. Applying steady state conditions:

= 0 α Dcx − (1 + α ) Dx + μ x μ= D(1 + α − α c) 0= Fs0 −

μx Yx s

0= D( s0 − s) −

D = Dilution rate practically c>1, so D>μ

V − Fs

μx Yx s

Fig. 12.2: Comparison of cell concentration profile at different dilution rates with and without cell recycle

Cell recycle (or retention) can accomplish: • A higher cell density in reactors • A higher dilution rate than maximum specific growth rate

at the αF flow rate. The symbols used are the same as those in the stoichiometry and kinetics chapter.

Material balance can be performed on both the reactor and the cell recovery/recycling device for both cells and substrate. An important parameter affecting the performance of the system is the ratio of cell concentration in the purge stream, x2, to that in the reactor, x. This ratio is affected by the recycling factor, α, and the cell concentration factor, c by x2/x = 1+α-αc. Note the ratio should be always smaller than 1, so that there is a concentration effect by the cell recovery device.

An important conclusion from the steady state analysis is that with cell recycling, the dilution rate, which is defined as the flow rate divided by the reactor volume, is larger than the specific growth rate; whereas without cell recycling (i.e., α=0), the dilution rate is always the same as the specific growth rate. As seen in the figure about cell recycling, the cell concentration in the reactor is higher than without cell recycling and the reactor can be operated at a dilution rate higher than the growth rate. Therefore, for cells with a doubling time of one day, the maximum flow rate that can be used without cell recycling would be one volume a day. With a perfusion culture, even a few volumes a day may be operated depending on the cell retention factor (c). A higher retention factor permits a higher cell concentration. Depending on the retention device used, the efficiency of retention may vary; for instance, it may decrease rapidly at the high dilution rate, causing the cell concentration to decrease rapidly.

The analysis described above is for a bioreactor with an external cell recovery device. The same principle applies to a system with an internal device. The consequence of employing an internal device is the same: the dilution rate can be higher than otherwise. It allows a fast nutrient flow rate to be used to reduce metabolite concentrations, while keeping cells in the reactor and allowing cell concentrations to become maximal.

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Effect of Recycling Factor

In a perfusion system operated at steady state, the amount of cells purged (Fx2) from the cell recovery device equals the amount of cells produced through reproduction (µxV). To keep a high viability and steady state, a small amount of cells are purged,. The system is operated at a dilution rate, D.

For a given dilution rate, one may employ a different recovery device with a different cell retention efficiency. Using the equation as an illustration, given the same dilution rate and specific growth rate, one may choose different combinations of α and c. If one chooses a highly efficient cell concentrator with a large c, then α that is used would be smaller, and vice versa.

Fig. 12.3: A high cell concentration factor allows for a low recycle ratio while achieving the same enhancing effect of cell cycle

This concept illustrates that at a given dilution rate, when using a highly efficient cell separator (such as a centrifuge that creates a concentrated recycle stream), a low recycling rate (αF) can be employed. Conversely, when using an inefficient cell separator that gives a low degree of cell concentration, then a large recycling rate needs to be used; in other words, cells will need to be pumped out of the reactor and pass through the cell separator to recycle more frequently. In large-scale operations, the cell stream could potentially stay outside the reactor for a long time, so oxygen starvation is a concern. Consequently, the fluid stream out of the reactor is often chilled, first, to reduce oxygen consumption. With a device that gives a low c value, cells will need to be subjected to the extra environmental perturbation of being chilled and pumped more often. This factor should be considered in selecting a cell recovery method.

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Methods of Cell Retention Selecting the method of cell separation very much depends upon the way cells grow. The larger the particles are, the easier they are separated. Many processes employ microcarriers with particle diameters ranging from 0.2 mm to 2 mm. These cellladen microcarriers can be easily separated from media stream by sedimentation. In some cases, cells are grown as large clumps, or aggregates, of 1 – 2 mm in size, also making sedimentation readily applicable. Other considerations in selecting a cell separator are the required throughput purge rate, the concentration factor (c), and the scale of operation.

Because of the limit of the concentration factor (c) of a cell separator, even though the purge rate (F) may not be large, a large recycling factor (α) may be necessary. Note that the capacity of the separator required is not only based on the dilution rate (D, or F/V), but also on the flow rate out of the reactor ((1+α)F). If a device with a small concentration factor, c, is used, the capacity of the separator will have to be substantially larger than what is needed to process the purge stream alone.

Sedimentation Conical Settler • Selective removal of dead cells • Low separation efficiency • Cell settling velocity ~ 2-10 cm / h • Good for larger cells • Long residence time outside of the bioreactor

Fig. 12.4: A settling cone for cell retention

The simplest cell separator is perhaps a settling cyclone. The fed stream from the bioreactor enters into the settler in its midsection, where the stream splits into flows in two directions. The upward stream, drawn by a pump for a purge stream, encounters a large cross sectional area and, thus, the vertical velocity is much smaller than the cell’s setting velocity, so the cells move downward. The downward stream, on the other hand, faces a decreasing cross sectional area and, thus, increases its vertical velocity as it carries cells downward. A transient zone separates the two well-developed upward and downward flow regions. In this transition zone, cells are separated and carried downward into the reactor. Such a settler is a convenient device for laboratory operations. As the reactor scale increases, the flow rate also needs to increase proportionally, but the cross sectional area of the settler increases only

CELL RETENTION AND PERFUSION | 254

with 2/3 power of the settler volume. The efficiency of cell separation decreases rapidly as the scale increases, making it ill suited for larger operations.

Incline Settling

In a simple settling tank, the direction of fluid flow and cell settling are along the same axis (both vertical). A sufficiently long transient zone is necessary to separate the cell and cell-free streams.

Operating parameters • Cell size and concentration • Perfusion rate • Settling area • Length of the plates Inclined Settling • While a particle is moving upward with flow, it also settles toward the bottom plate • It is “collected” upon hitting the bottom • Eventually, the particle rich zone has a higher fluid density and begins to move downward • The particle-rich stream is recycled to the bioreactor

purge stream

Particle Recovery

co n

ce t

rat ed

ce ll

str ea

m

Particle Setting

feed from reactor

recycle to reactor

Fig. 12.5: Cell separation in an inclined settler for cell retention

To enhance the separation efficiency, the settler is often inclined so that an angle exists between the fluid flow direction and the particle settling direction. A particle is considered “collected” once it settles on a settler’s surface, since the fluid velocity on the surface is zero.

In an inclined settler, the feed cell stream enters at the bottom and moves upward. Inside the settler, cells begin to “settle” down vertically due to gravity. If a cell particle hits the surface at the lower plate, it is “collected” and does not get carried out by the effluent stream. Eventually, the cells settled on the bottom plate form a layer of fluid with a higher density than the stream above. This heavy stream then moves downward, carrying the cells along. At the steady state, there are three streams in the system: the feed stream, the effluent stream (carrying unsettled cells), and the concentrated cell stream at the bottom. In industrial design, multiple inclined plates are used in a single settler. In such designs, the feed stream and the returning cell stream do not cross each other by partitioning their flow path in different zones. In some cases, mechanical vibration is applied to the plates to prevent settled cells from sticking to the surface and being lysed.

The residence time in the settler has to be at least as long as the particle settling time. With a high cell concentration in the stream, oxygen starvation is a major concern, as it may induce apoptosis and cell lysis. Therefore, the stream passing through the settler is often chilled to reduce the cell’s metabolic rate.

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Centrifugation Centrifugation Technology • Excellent separation efficiency • High perfusion capacity • Little clogging • Easy scale-up • Vulnerable to mechanical failure in long term continuous operation Three different designs: • Westfalia Disc type • Continuous cell recycle back to fermenter • Centritech Lab • Disposable separation unit

Fig. 12.6: The cell loading and cell ejecting regions of a disc centrifuge for cell recycle

Centrifugation is a standard unit operation in many downstream recovery processes. In the early days of perfusion process development, it was among the first to be exploited for cell retention. As early as the mid 1980s, the Japanese company Teijin had employed centrifuges for perfusion culture of hybridoma cells used to produce antibodies for cancer imaging.

However, most centrifuges are not designed for long term and aseptic operations. Early use of the centrifuge for perfusion was only intermittent and was for batch harvest and cell biomass recycling, as well as for the replenishment of fresh medium. A number of autoclavable or stem-sterilizable centrifuges are now available. These and the disposable bagbased centrifuge are all capable of processing up to hundreds of liters of medium a day, and are suitable for continuous use in perfusion culture.

The disc-type centrifuge is analogous to a multiple parallel plate settler, except that the parallel plates are rotating and generate a centrifugal field for cell settling. The disposable bag system employs three tubes: a feed tube, an outflow tube for the heavy (cell-rich) stream, and an outflow tube for the light stream. The unique design of an inverted question mark allows the three tubes to rotate, along with the centrifuge, without being twisted.

CELL RETENTION AND PERFUSION | 256

concentrated cell stream

cell

dilute stream feed

cell movement

Fig. 12.7: A disposable bag based centrifuge (Centritech) for cell recycle

Acoustic Resonance Enhanced Settling This device uses acoustic energy to enhance Mechanical/Acoustic Trapping

• Enhanced sedimentation (Cell aggregation) • Slightly favorable for viable cell retention • Heat generation may create temperature gradient • Low separation efficiency • Moderate capacity (200L/day per unit) Operating parameters • Cell density, perfusion rate, power input

cell agglomeration. As the cell stream from the reactor passes through the acoustic chamber, cells are induced to agglomerate. This gives rise to a faster settling velocity. With increased settling velocity, sedimentation is easily accomplished without resorting to multiple plates.

As cell concentration or operating conditions (i.e., flow rate and temperature) change, the energy and residence needed for agglomeration may also

CELL RETENTION AND PERFUSION | 257

“light” single cell stream moves upward

“heavy” aggregate stream moves downward

change. Sensors for detecting cell agglomeration will help stabilize the operation of the device.

transducer

reflector

agglomeration zone

feed

recycle stream cell aggregates

Fig. 12.8: An acoustic cell agglomeration device for cell retention

Spin Filter Separation

Fig. 12.10: A spinning filter (or rotating cage) for cell retention

Rotational Cage Internal and External • Remove dead cells/debris Operating Parameters Affecting Performance • Screen pore size (1-120 μm) • Perfusion rate • Rotation speed • Screen surface area • Draft tube • Screen materials: • Stainless steel, DNA & RNA deposit, • Teflon, Polyamide 66, polyethylene, better

Fig. 12.9. Picture of an acoustic cell retention apparatus

The term “spin filter” is used to refer to two different designs that may have rather different mechanisms of operation. Both employ high porosity filter with relatively large openings (~20 - 100 µm) installed on the wall of a rotating cage. Both are submerged in culture fluid. A pump draws medium from inside the cage as the purge stream. The culture fluid passes through the filter to enter the cage, then is withdrawn by the pump to become the effluent stream exiting the reactor. The flow into the cage has a lower cell concentration than the bulk culture fluid, thus achieving the overall retention of cells in the reactor. A rotating cage rotates along the center shaft of the impeller agitator at a low speed. The centrifugal field is typically insufficient to push away cells along the outside wall of the cage. Yet, a boundary layer of liquid around the cage probably exists, in which the cell concentration is lower than in the bulk. As a result, the fluid drawn across the filter is lower than in the bulk. Furthermore, there is little filter cake formation. The screen on the cage is, thus, not exactly a filter. Nevertheless, the system has been employed in scales of up to hundreds of liters.

CELL RETENTION AND PERFUSION | 258

Centrifugal Cage dilute purge stream

recycle stream

Using such a device, the operation is less prone to variation, due to changes in fluid dynamics. It gives the freedom of operating in different regions in the reactor. In some variations, it is installed outside of the reactor and used as an external cell retention device. However, the device

spinning

stationary

differs from the traditional centrifugal filter used in the recovery process in chemical industry in an important way in that no filter cake is formed. In fact, if cell cake is formed on the screen wall of the cage, cell death is likely to occur in the cake and leads to process failure

Fig. 12.11: A Centrifugal filter for cell retention

Microfiltration

External Loop for Cell/Harvest Separation • High shear, tangential flow • At high transmembrane pressure, cell deformation occurs • Fouling caused by high molecular weight DNA, protein, lipids, anti-foam occurs after days of operation • Difficult to scale-down • The degree of concentration in a single pass is relatively small

Cross Filtration Model

feed

The rotating cage is notoriously difficult to scale up, as its operating mechanism is not well understood. Later modifications of spin filters increased its rotation rate up to hundreds of rpm, allowing it to operate like a centrifugal filter. The centrifugal force is sufficient to push the cells away from the surface of the filter, thus drawing liquid through with a lower cell concentration than in the bulk.

Microfiltration uses membranes of different configurations, including parallel plates and hollow fiber devices, and has a pore size of around 4 µm. Microfiltration was among the first techniques used for cell retention. Its widespread use has been impeded by membrane fouling, which is especially severe when a high concentration of proteins or complex medium is used in the culture. With the use of low protein medium in the past decade, the problem of protein fouling has lessened, but clogging by dead cells remains problematic.

return to reactor cell-free permeate

cell membrane very high flux may cause cell damage

Fig. 12.12: Microfiltration membrane for cell retention

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Alternating Tangential Filtration

Different configurations of microfiltration devices all use tangential flow, meaning the feed stream flows in a direction parallel to the membrane, while the filtrate flows across the membrane. The more recent use of a pulsatile flow system, in which a diaphragm is used to rapidly reverse the flow direction, has reduced fouling.

from reactor

return to reactor

product harvest stream

product harvest stream

diaphragm in closed position

diaphragm in open position air

air Rapid pulsatile flow in reverse directions minimizes fouling.

Fig. 12.13: An alternating tangential flow hollow fiber device for cell retention

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Concluding Remarks Perfusion culture operation was explored very early on in cell culture development for obvious reasons: (1) the low throughput from batch operation can be enhanced by switching to continuous operation, (2) a simple continuous culture would have too low of a cell concentration to make the process economical, and (3) a dilution rate faster than the cell growth rate is needed to purge the metabolites accumulated in culture. Its widespread adoption was inhibited, however, by concerns about regulatory requirements and the lack of a clear definition of BATCH for product manufacturing. Researchers were also concerned about the lack of a scalable cell recovery device for long operations. These hindering factors have largely been overcome. Although cell line stability for sustained production of a product is still a concern, evidences have shown that, with proper control of cell age and cell stock, stability may not be an overriding concern.

volumes of inoculum that are used to scale up. The carrying over of a large amount of spent medium from seed culture is undesirable; evidence seems to suggest that a high metabolite concentration at inoculation can negatively affect a cell’s metabolic characteristics in the main culture. Thus, using the cell recovery devices for preparing inoculum, especially to increase the initial cell density, could potentially enhance process performance.

Another area that may change in the near future is the increased use of fortified medium in perfusion culture, as we have seen in fedbatch cultures. Using fortified medium (instead of medium with a standard composition) can reduce the flow rate and increase cell and product concentrations.

Given the intrinsic advantages of continuous operations and the advances in cell retention technology, we may begin to see more widespread application of perfusion culture in the coming As cell retention technologies become mature, one years. It has considerable potential to increase may also see this application go beyond perfusion the capacity of high throughput processes, reduce culture. The same devices can be used to concentrate reactor sizes, and possibly minimize product quality cells and remove metabolites, as well as to quickly fluctuations through steady state operations. replace the fluid phase for cell freezing operations. Current inoculation operations are limited by the

CELL RETENTION AND PERFUSION | 261

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Scaling Up and Scaling Down for Cell Culture Bioreactors Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanical Agitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Agitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Power Consumption and Mixing Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Power Consumption of Agitated Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Dimensionless Numbers for Stirred Tank Reactors . . . . . . . . . . . . . . . . . Effect of Scale on Physical Behavior of Bioreactors . . . . . . . . . . . . . . . . . . . . . . . Mixing Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nutrient Starvation Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixing Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nutrient Enrichment Zone, Mixing Time vs. Starvation Time . . . . . . . . . . . . . Mixing Time Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Scaling Up and Mechanical Forces on Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Scaling Up and Oxygen Transfer and Carbon Dioxide Removal. . . . . . . . . . . . . . . . . . Material Balance on Oxygen in Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aeration Rate and Superficial Gas Velocity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gas Flow Rate in Scaling Up: A Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Scaling Up on CO2 Removal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263 265 265 266 266 268 269 271 271 272 272 273 274 275 276 278 280 281 284

Introduction Translation of the process scale is one of the most difficult issues in bioprocessing, and it is probably one of the least systematically studied subjects in the field. Few engineers are involved in designing large-scale equipment using small-scale experimental data, but many will be developing processes in laboratories and at pilot plant scales for eventual implementation in a production scale. Others may be involved in troubleshooting investigations for production plants using laboratory equipment. Therefore, an understanding of the factors affected by scaletranslation is important in carrying out those studies.

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Translation of Scale

Objective • Prediction of process performance • Specify operating conditions from one scale to another Major Effect of Scale • Oxygen (Gas) transfer • Heat transfer • Shear force • Compression force • CO2 removal

Fig. 13.1: Schematic of a large scale cell culture fermenter



A reactor may be scaled up geometrically similarly or non-similarly. Geometrical similarity refers to maintaining the ratio of the main geometrical lengths, such as height over diameter, as well as the relative size of the internal parts (e.g., impeller, flow diverter, etc). In this chapter, our discussion will focus on geometrically similar cases, which are conceptually easier to grasp, although this is not always the best approach in scaling. In scaling up geometrically similarly, all length dimensions of the rector are scaled proportionally. Consequently, the surface area will increase with the length dimension to the second power, while the volume will increase to the third power of the length. As a result of scaling up, the scale-related surface area per unit volume of equipment will decrease. In microbial fermentation, the decrease in surface area to volume ratio causes the impediment for removal of heat generated from metabolism and mechanical agitation. In mammalian cell processing, the metabolic heat generated is less a concern. However, the process may still be sensitive to other variables related to scale change. As the scale of an equipment changes, the physical and mechanical parameters may not be maintained constant. Frequently, it will not be possible to keep all key operating parameters constant between different scales. This may lead to changes in the chemical environment and, ultimately, cell physiology and productivity. The objective of scaling-up and scaling-down is, therefore, not to strive to keep scale-related parameters at constant, but to define the operating range of scale-sensitive physical and mechanical parameters so that the cellular physiological state and productivity can be maintained within an acceptable range. At times, this may require an adjustment of the chemical environment at different scales.

SCALING SCALING UP UP AND AND SCALING SCALING DOWN DOWN ON ON CELL CELL CULTURE CULTURE BIOREACTORS BIOREACTORS | 264

Mechanical Agitation Purpose of Agitation • Gas-liquid mass transfer • The higher shear field near the impeller tip produces small bubbles, thereby increasing gas-liquid interfacial area (provided that bubble coalescence is not correspondingly increased) • Suspension of solid (e.g. microcarriers, soymeal) or dispersion of liquid • Liquid-liquid, liquid-solid mass transfer (e.g. hydrocarbon culture, quick mixing of pH neutralizing base) • Minimization of pellets or aggregates • Pellets are cell aggregates or mycelial microorganisms (streptomyces, molds) • Mixing, especially for viscous fluid (e.g. xanthan gum) • Fermentation, broth of mold culture

Mechanical agitation in a stirred bioreactor keeps cells in suspension, provides mixing to create a more homogeneous chemical environment, and creates a flow pattern that increases the retention time of gas bubbles in the culture fluid to enhances oxygen transfer. In the cases that cells are grown as aggregates, agitation also helps reduce the formation of oversized particles.

In microbial fermentation, oxygen demand is rather high (often exceeding 150 mmole / L-hr). To increase the efficiency of the oxygen supply, extensive agitation is used to break up air bubbles. In many fermentations of mycelial mold or actinomycete, extensive agitation is used to overcome the high viscosity of culture fluid and to reduce the mycelial pellet size to enhance oxygen transfer.

In cell culture processes, the oxygen demand is nearly two orders of magnitude lower than that in microbial fermentation. Adequate oxygen supply can usually be accomplished by much less intensive agitation, which is also sufficient for providing sufficient mixing and suspension of cells.

Mechanism of Agitation

Table 1. Characteristics of Impellers Characteristics

Propeller

Disk Turbine

Flow direction

Axial

Radial

Gassing

Less suitable

Highly suitable

Dispersing

Less suitable

Highly suitable

Suspending

Highly suitable

Less suitable

Blending

Highly suitable

Suitable

The flow patterns generated by different impellers in a stirred tank are generally classified as one of two types: axial flow or radial flow. An axial flow pattern refers to primarily upward or downward flow due to the pumping action of the impeller. In a radial flow pattern, the liquid moves primarily outward toward the wall of the vessel. In cell culture processing, impellers generating axial flow are used because the shear fields generated by axial flow patterns are lower than those generated with radial flow patterns. The Rushton disk turbines, as are often used with multiple installations in large reactors, are the predominant type used in microbial fermentation. In this design, the sparger is placed directly underneath the disk turbine. Gas bubbles from the sparger rise to hit the disk and are directed outward.

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The blades, rotating at a fast speed, then break the bubbles up. In the immediate surrounding region of the blade, a very high-energy dissipation is predicted by computer simulations, which would contribute to bubble break up, but also create a high shear zone which can potentially cause damage to cells.

Fig. 13.2: Fluid flow patterns in a stirred tank reactor: axial flow type vs. radial flow type

Flat Blade (Rushton Turbin)

The propeller or pitch blade type impellers, on the other hand, focus on moving the liquid to create the lift for mixing and suspending solids. The impeller diameter to tank diameter ratio should be higher for microcarrier culture. While the “propeller three blades” is used extensively in microbial fermentation to enhance oxygen transfer, the “axial flow three blades” provides less shear stress and a more uniform velocity in the entire discharged area than the “propeller three blades.”

Axial Flow Blade

Propeller Three Blade

Fig. 13.3: Three types of impellers commonly seen in stirred tank reactors

Power Consumption and Mixing Characteristics Power Consumption of Agitated Bioreactors N

PO

H V

Di DT

Fig. 13.4: Notation of an impeller based mixing reactor. H: liquid height, V: liquid volume, N: impeller rotation rate, Po: agitation power, Di: impeller diameter, DT: tank diameter.

In designing equipment and analyzing physical systems, of which the scale spans over a wide range, one often needs to develop a correlation between different design variables. When data collected from different scales and under different conditions are plotted together, they inevitably give rise to different correlations. Each of those correlations is good for that particular scale or a range of scales. They are, thus, of limited value.

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In order to find a correlation that is applicable to different scales over a wide range of operating variables, the experimental data are often plotted in “dimensionless variables” to develop correlations among the experimental data. These dimensionless variables are a combination of experimental variables. In such combinations, the units of dimensions from individual variables cancel each other out with the idea being that a correlation between dimensionless variables is not sensitive to scale. The dimensionless correlations obtained from experiments performed on different scales should hold on any scale, including those that have not been investigated.

Fig. 13.5: Relationship between impeller Power number and impeller Reynolds number for different types of impellers

Various relationships expressed in dimensionless numbers are fundamental to fluid mechanics, mass transfer, heat transfer, etc. The correlation between friction factor (f) and Reynolds number (Re) is used universally in the design of fluid flow in pipes. The plot of friction factor and Re show two regions: at low Re, f decreases linearly until Re = ~2000, where there is a short break, then it continues at a relatively constant value at a high Re region. The first region is recognized as the laminar (or viscous) flow region and the constant tail is the turbulent flow region.

A similar plot has been generated for power n consumption in a stirred tank reactor. The Reynolds number is now denoted as ReI (Impeller Reynolds Power Number (Np): 3Po 5 number) to indicate that it is based on the length N Di t (diameter) of the impeller. The dimensionless number for power consumption by impeller is the Po = Impeller power (ungassed power, Po indicated indicated ungassed) power number, Np. Correlations between Np and ReI N = Impeller speed have been generated for various types of impellers. They all exhibit a similar behavior to the f vs. Re Di = Impeller diameter plot for fluid flow in a pipe. In all these Np vs. ReI ρ = Fluid specific gravity plots, a rapid decrease with increasing impeller μ = Fluid viscosity Reynolds number is followed by a constant value • For all practical purposes a bioreactor is always region; the decreasing region and the constant operated in the turbulent region region represent the two correlations for viscous In turbulent regions: (Np) is constant, independent of flow and turbulent flow regimes respectively. In (ReI) the turbulent regime, the power number is constant (Eq. 1) over a wide range of impeller Renolds number, but Po = K 3 5 tN Di the value changes with different types of impellers. 2 Impeller Reynolds Number (ReI): NDi t

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Other Dimensionless Numbers for Stirred Tank Reactors Three other dimensionless numbers, in addition to power number, are used: • Dimensionless velocity, v / ND • Dimensionless pumping number, Qp / ND3 • Dimensionless blending (mixing time), ϴN v: tip velocity Qp: liquid pumping θ : mixing time

• At turbulent regions, all those numbers are relatively constant.

In bioreactor operations the flow is always in the turbulent regime. Viscous flow is encountered in a stirred tank only when a very viscous fluid, like glycerol, is used. Therefore the power number for impellers is constant for the same type of impeller. In other words, the impeller power divided by N3Di5 (N is agitation rate, Di is impeller diameter) is constant. Three other dimensionless numbers are frequently used to predict the performance of a stirred tank when the scale changes. They deal with three important aspects of bioreactor operations: velocity, volumetric flow rate, and mixing time.

The maximum velocity in a mixing tank occurs at the tip of the impeller. This velocity can be represented by the multiplicative product of the rotation speed of the impeller times its diameter, NDi. (Note: in this chapter, we will ignore π in the discussion of perimeter, area of circle, etc. The constant value π is cancelled out when comparing different scales.)

The amount of fluid that the impeller can move (called “pumping”) is directly dependent on its rotating velocity and the area of the impeller blades. Because we are considering scale translation under geometrically similar conditions, we can use the length of impeller, instead of the impeller blade, to represent the length scale. The pumping, then, is the projected area (D2) of the impeller multiplied by the velocity of its rotation (ND), which gives ND3.

For mixing time, the representative time scale (called “characteristic time”) in a mixing tank is the inverse of rotation speed (1/N).

The dimensionless numbers for the three properties can by obtained by taking the representative velocity (v), liquid volumetric flow rate (Qp), and mixing time (Ө), and divide by their respective characteristic counterpart (e.g., ND, ND3, and 1/N). The plots of dimensionless velocity, pumping, and mixing time against ReI all show profiles similar to the power number plot, with two distinct flow regimes: laminar (viscous) flow and turbulent flow. The values at high ReI turbulent regimes are relatively constant.

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Effect of Scale on Physical Behavior of Bioreactors Scale Translation Approaches • Constant K a L • Constant impeller tip speed (ND ) i • Constant power per unit volume (Po/V) • Constant mixing time

Table 2. Examples of Scaling Up by Keeping Different Parameters Constant. The reactor is scaled up 15.6 times by volume while keeping geometric similarity. Property

Pilot Scale 160 l

Plant scale, 2500 l

P

1.0

15.6

98.0

6.2

P/volume

1.0

1.0

6.2

0.4

N

1.0

0.54

1.0

0.4

D

1.0

2.5

2.5

2.5

Qp (pumping)

1.0

8.5

15.6

6.2

Qp/volume

1.0

0.54

1.0

0.4

NDi (tip speed)

1.0

1.35

2.5

1.0

Using the correlations based on the dimensionless numbers, one can explore the effect of a changing scale on different variables. We assume that the equipment in different scales will remain geometrically similar. In that case, the effect of different reactor sizes can be compared using characteristic length D (the tank diameter). If the tank diameter increases by 10 fold, all the other reactor parts (tank height, impeller diameter, etc.) will increase by the same proportion of 10 fold.

In scaling up different processes, one needs to keep the most important variable(s) constant or within an acceptable range. The commonly used criteria for scaling up are (1) a constant KLa, so that mass transfer can be maintained, (2) a constant impeller tip speed, to sustain a critical value of high shear velocity to break up agglomerating particles or pellets of mycelial cells, (3) a constant power input per volume (usually for less power intensive processes such as crystallization, blending), and (4) a constant mixing time.

Consider the case of scaling up by maintaining power input per reactor volume constant. Recall that the power number is constant in a turbulent region and the power input (PO) is proportional to N3D5. The reactor volume is described by πHD2. Because of geometrical similarity, we can represent H by D and ignore the constant π that does not contribute to scale comparison. The reactor volume (V) is thus represented by D3. In keeping PO/V constant, N3D2 is also constant in different scales. In scaling up as D increases, the rotation speed must decrease by 1/D2/3. It is inevitable that larger reactors will need to be operated at lower rotation speeds.

By similar algebraic manipulation one can also see that scaling up by a constant power per volume (PO/ N3D2) constant will lead to increasing the amount f total pumping (ND3) with the scale. However, pumping per volume will decrease as the scale increases. For mixing time, the trend is an increase with scale. The table compares the effects of scaling up

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by setting different parameters at a constant value. Cases considered include constant power per volume, constant agitation rate, constant pumping rate, and constant tip speed. Often, one chooses not to keep a single value constant, and not to scale entirely geometrically similarly.

Scaling Up Geometrically Similarly By Keeping Power Per Unit Volume Constant

PO/N3DI5ρ is constant in turbulent region. Density of water, ρ, is constant. Thus, (Eq. 1) PO = KN3DI5 The volume of reactor can be expressed as characteristic length raised to the third power, V = πHDt2 = cDi3 (Eq. 2) The power per unit volume is described as Po/V = K N3DI5/ cDI3 = K’N3DI2 = constant This leads to the conclusion that when power per unit volume is kept constant, N3DI2 is also constant. N3DI2 = constant (Eq. 3) Effect on Agitation Rate Comparing scale 1 and scale 2 N13DI12 = N23DI22 N1/N2 = (DI2/ DI1 )2/3 (Eq. 4) The agitation rate N decreases with increasing scale. When the diameter increases eight times, the agitation rate is ¼ in the larger scale. Effect on Impeller Tip Speed Tip speed is described by N multiplied by Di. from Eq. 3

N13DI1 3/ DI1 = N23DI23/ DI2

N13DI13/ N23DI2 3 = DI1/ DI2 N1DI1 / N2DI2 = (DI1/ DI2)1/3 (Eq. 5) Tip speed increases with increasing scale, but only at 1/3 power of the length of scale.

Effect on Mixing Time The decreased pumping per volume also causes an increasing in mixing time when scale increases. The dimensionless mixing time is ΘN. Its value is relatively constant in the high Re number turbulent region.

The capacity of liquid pumping can be described by the impeller tip speed, NDi, by the area that it moves against the liquid, Di2. Under the condition of constant power per volume, N13Di12 = N23Di22. Multiply both sides by diameter to the seventh power. (Eq. 7)

N13Di19 / Di17 = N23Di29 / Di27 N1Di13 / N2Di23 = (Di1 / Di2)7/3 Liquid pumping capacity increases with scale. By dividing both sides by characteristic length raised to the third power, we can obtain the pumping capacity on a per volume basis NDi3 / Di3 = pumping per volume = Qp/V (Eq. 8) (Qp1/V1) / (Qp2/V2) = (Di1 / Di2)-2/3 = (Di2 / Di1)2/3 The pumping capacity per volume actually decreases with increasing scale.

Θ1N1 = Θ2N2

From Eq. 4

Effect on Liquid Pumping

(Eq. 6)

Θ1 / Θ2 = N2 / N1 = (Di1 / Di2)2/3

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Mixing Time The nutrients that are added at the beginning of the culture will eventually become well mixed in the culture fluid. (Note: This may not be true for some microbial fermentation in which some nutrients are supplied in a solid form and dissolve gradually). Mixing problems may arise for those components that are added continuously or intermittently during the cultivation. If the nutrient is added in a fixed position(s), it may not get carried to other locations in the reactor fast enough to meet the cells’ metabolic needs. In some cases, the additive needs to be supplemented at a high enough concentration that has an adverse effect to the culture, and must be dispersed quickly. In these cases, adequate mixing must be provided.

Nutrient Starvation Time Because of its solubility, oxygen is the first nutrient species to be completely consumed

Table 3. Comparison of Oxygen and Glucose Saturation Time in a Typical Culture (For 1010) Oxygen 0.1 mM (50% saturation with air space)

C in Culture

Glucose 1 g / L (55 mM)

Specific consumption rate

1 x 10-10 mmole / cell-hr

Volumetric consumption

1 mmole / L-hr

0.15 – 1 mmole / L-hr

Time to depletion

0.1 hr (6 min)

12 hr

0.15 – 1.0 X 10-10 mmole / cell-hour

In cell culture processes, oxygen is almost always the first nutrient to be depleted. Due to its low solubility in medium, it must be continuously supplied. In comparison, the concentration of glucose maintained in the medium is usually a couple orders of magnitude higher than oxygen. Their ratio of molar specific consumption rates ranges from about 1.0 (when most glucose is converted to lactate) to close to 6.0 (when most glucose is converted to CO2). For oxygen, at a high cell concentration, the depletion time can be as short as a few minutes, whereas the depletion time for glucose is orders of magnitudes longer. Therefore, in reactor scaling, special attention is always paid to oxygen.

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Mixing Time Mixing Time Measurement • Measurement • At t = 0 add tracer • Measure terminal mixing time, defined as the point when an arbitrary chosen uniformity, 90%, is reached

To measure the mixing time, one can inject a dye into the reactor under operating conditions and then use a sensor placed in a fixed location to record the dye concentration over time. The concentration will fluctuate over a large range initially, but will eventually reach a steady value. The time needed for the concentration to reach a range of steady value (such as 90% of its final homogenous concentration) is considered the “mixing time.” If one plots the concentration deviation from its final steady value, ΔC, the time profile can be approximated by first order kinetics.

Fig. 13.6: Measurement of averaged mixing time in a stirred tank

Nutrient Enrichment Zone, Mixing Time vs. Starvation Time

In a stirred tank reactor, a nutrient is added at a fixed position. Consider a fluid element carrying cells. When it passes by this position, it acquires the nutrient and then moves away to circulate around the reactor. On average, the same fluid would return to this feeding zone after a duration of one characteristic mixing time. It is important that the amount of nutrient that the fluid acquires at the nutrient enrichment zone is sufficient to sustain the metabolic needs of the cells before it returns to the zone. Therefore, the mixing time needs to be shorter than the starvation time. The starvation time is dependent on cell concentration and the consumption rate. If the mixing time is longer than the starvation time, a discernible concentration difference of the nutrient would appear in different locations in the reactor. Pockets of low concentrations would emerge and their locations may change over time, as the fluid flow pattern and cell concentrations are not constant. A sensor that is at a fixed position may, thus, not reveal the presence of such pockets.

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Mixing Time Distribution

Fig. 13.7: Measurement of circulation time distribution in a stirred tank

Mixing Time Distribution Measurement • Add radio emitter to the reactor i, a sensor picks up the signal when the emitter circulates around and passes by • Measure circulation time for each encounter and plot the frequency distribution of the circulation time • Determine the mean and median circulation time and the standard deviation σ • One can plot distribution of circulation time as a population density function. The portion of circulation whose time of circulation lies between t and t + Δt is the area under the curbe between t and t + Δt.

If we follow a particular fluid element in a reactor, it will come to the nutrient enrichment zone, go away, and come back repetitively. The time interval between its return to the nutrient enrichment zone, however, will not be uniform. The mixing time described above is an average mixing time. But the mixing time that is physiologically important (e.g., the return time to the nutrient enrichment zone) is not a single value of the average mixing time, but is distributed over a range.

Imagine that we use a ball that emits a radio signal. The ball has the same density as the fluid and is carried around by the fluid motion. A sensor at a fixed position in the reactor would pick up the signal when the ball is close and record the time interval between consecutive returns. This time interval of return will distribute over a range as the ball sometimes returns shortly after it moves away, while at other times it roams around the reactor for a while before returning to the sensor. The histogram of the time-interval distribution can be converted to a mixing time distribution function. In general, the distribution follows a logarithmic normal distribution. The mean or median of mixing time is a descriptor of mixing characteristics, but it does not present the entire picture of mixing. Given the same median or mean mixing time, two reactors may still have a very different mixing time distribution.

Imagine that the location of the sensor is also the position of nutrient feeding. Those cells circulating with the particular fluid element receive nutrient only when the fluid returns to that position. If the circulation time is longer that a critical time, then the nutrient level seen by those cells may fall below the critical value. A wide distribution of mixing time can be a concern. Even though the frequency of exceedingly long circulation time is low, nutrient starvation may occur in those rare occasions and trigger apoptosis or cause other irreparable damage to cells. In reactor operation, the fraction of mixing

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Population Density Distribution f(t)

time that is longer than the critical time should be minimized by either improving mixing or by setting a limit on the cell concentration.

Probability of Starvation

τ 0

1 Circulation Time

2

Critical Circulation Time

Fig. 13.8: Mixing time distribution and critical mixing time for nutrient depletion

Mixing Time Distribution • The mixing time in a tank is not uniform. If the average mixing time is 6 minutes, many fluid elements will have a mixing time shorter than 6 minutes, and others will have longer times. • If the critical mixing time is 6 minutes, the average mixing time should be shorter than that.

Scaling Up and Mechanical Forces on Cells In a turbulent flow, the fluid’s kinetic energy is transferred by swirling pockets of fluid, called “eddies”. Turbulent regimes are comprised of eddies of different sizes, characterized by velocity fluctuations.

The hydrodynamic forces experienced by the cells may arise from fluid-cell and cell-mechanical parts interactions. Cells, being neutrally buoyant particles, follow the motion of the relatively larger eddies. In general, the direct impact of cells on the impeller is minimal and cells generally flow by impeller blades without suffering much direct mechanical impact. However, these large eddies cause the formation of cascades of smaller eddies, which may impart damage to cells by dissipating all of their energy on the cell surface. The size of the eddy relative to the size of the cell is thought to be an important factor that damages cells. Smaller eddies in the size range of cell surface motif, i.e., much smaller than cell diameter, are considered to be more damaging than eddies that are larger than cells. Studies examining cell death

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• Microeddies cause cell damage

Fig. 13.9: Eddies surrounding a cell suspending in a stirred tank

• Eddy size increases with scale if the power per unit volume decreases with scale

caused by turbulent flow often assume that the cell death rate is proportional to the Kolmogorov eddy concentration, and cell damage occurs when the eddy size is smaller than a critical eddy size. As the scale increases, the eddy length increases. Thus, strictly from the viewpoint of the average eddy size, the effect of turbulence on cell damage will not become more severe in scaling up. One should be cautioned that the eddy size is not uniform, but distributed over a range. As the scale increases, the distribution function of the eddy size also varies. Further, the discussion here does not take into account the effect of gas mixing. The phenomenon of mechanical stress caused by combined agitation and aeration is rather complex in scaling up.

Scaling Up and Oxygen Transfer and Carbon Dioxide Removal When scaling up a process, we aim to produce cells and product in quantities proportional to the scale. To meet that goal, we normally provide all nutrients in a proportional amount, to meet the increased metabolic needs of cells. For liquid nutrients, increasing the nutrient provision in proportional to cellular needs on a large scale is easily met. However, for oxygen and CO2, which are supplied and removed through gas aeration, scaling up presents a challenge because of the constraints of physical factors.

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Material Balance on Oxygen in Bioreactor

Supplying oxygen to the growing cells in the bioreactor involves blowing air through the culture fluid, and transferring oxygen from the gas bubbles into the culture fluid. The amount of oxygen carried out from the reactor is less than that supplied into the reactor. This difference is the amount transferred into the liquid phase. Material balances on oxygen can be performed on the gas entering and leaving the reactor (the gas phase balance), as well as on oxygen being transferred from gas bubbles to liquid (the liquid phase balance). The oxygen transfer rates calculated from the gas phase balance and from the liquid phase balance are equal.

• The dynamics of the dissolved oxygen concentration are described by the balance between OTR and OUR at a quasi-steady state.

At steady state, OTR=OUR

(Eq. 9)

(Eq. 10)

Oxygen Balance on Gas Phase

• On the gas side, the oxygen transferred from the gas side to liquid side is reflected in the difference of oxygen concentration between gas inlet and gas outlet.

(Eq. 11)

Gas phase balance is performed by taking the difference between the oxygen input rate at the inlet and the oxygen output rate at the outlet. That difference is the amount of oxygen that has been transferred into the liquid. While oxygen is transferred into the liquid phase, CO2 (produced by cells in the medium) and water vapor are stripped out of the culture broth, thus the volume flow rate in the outlet may differ from the inlet. Assuming ideal gas behavior (PV = nRT), the molar flow rate of the oxygen at the inlet and the outlet is the total air flow rate (PQ/RT) multiplied by the molar fractions of oxygen at the inlet and the outlet YO and YO respectively. The oxygen transferred into the liquid is simply the difference between the molar flow rates of oxygen in the inlet and the outlet. 2,in

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2,out

Oxygen Balance on Liquid Phase

On the liquid side OTR and OUR is described by Eq. 11 since the rate of change of dissolved oxygen is small. In cell culture process, the air flow rate can be considered to be the same at the inlet and outlet. Overall, the relationship is described as:

(Eq. 12) (Note: P/RT converts volume flow rate Q to molar flow rate using ideal gas law. P is the pressure of gas phase)

On the liquid side, the oxygen transfer rate (OTR) is described by the overall mass transfer coefficient (KLa) and the driving force (C*-C). For small reactors, we assume that liquid is well mixed. This assumes that the concentration measured at the outlet is the same as the concentration in the reactor. C* at the air outlet should be used for the driving force calculation. For a large reactor, one assumes that the gas phase behaves like a tubular reactor (plug flow), and a logarithmic mean of the driving force is used. We assume that a quasisteady state, i.e., the change in dissolved oxygen (dC/dt), is very slow compared to the oxygen consumption and rate of transfer. Thus, the oxygen uptake rate (OUR) can be approximated by the OTR.

C* is the dissolved oxygen concentratoin in equilibrium with the gas, which may differ in different parts of the bioreactor. For small scale bioreactors, one can assume both liquid phase and gas phase are well mixed. The gas phase in the reactor is thus the same as that in the exit gas stream. Thus, C* is related to the oxygen concentration at the exhaust gas by Henry’s law constant:

(Eq. 13)

For large scale bioreactors, the inlet and outlet oxygen concentrations may be very different. One uses the logrithmic mean driving force described below:

(Eq. 14)

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Aeration Rate and Superficial Gas Velocity

D = tank diameter Q = aeration rate vl = culture volume in reactor Vs = gas superficial velocity A = cross-sectional area of the tank H = heights of culture volume

Superficial gas velocity = gas flow rate/reactor cross sectional area vs = Q/A = Q/(πDt2) (Eq. 15) The reactor volume increases with length scale raised to the third power, while the cross sectional area increases only with the second power. V1 / V2 = D13/ D23 A1 / A2 = D12/ D22

Now we examine the gas phase balance. In scaling up, if we keep Q/V constant, we will be able to maintain the same oxygen level at the inlet and outlet (Yin, Yout) and sustain the same oxygen transfer rate (OTR). However, when scaling up, Q/V is likely to decrease. If OUR is to be sustained, then Yout has to be smaller to maintain the material balance.

Then, consider the liquid phase balance. OTR is KLa multiplied by (C*-C). Because oxygen level at outlet, Yout, is lower, C* is also lower. How can OTR be kept at the same level as in the small scale? One possibility is to increase KLa while keeping C at the same level as in the small scale (thus allowing (C*-C) to be smaller). The will require an increase in agitation power. The other possibility is to increase (C*-C) to the same level as in the small scale by allowing C to be maintained at a lower level, if the reduced C has no adverse effect on culture performance. Alternatively one can use oxygen enriched air to increase C* to maintain (C*-C) at the same level as in the small scale. However, this will lead to increased level of CO2 accumulation as will be discussed in the next section. Therefore, overall oxygen transfer becomes a challenge in scaling up because the aeration rate cannot be increased proportionally with the scale.

When scaling up one may choose to increase the air flow rate proportional to the increasing reactor volume, Q1 /Q2 = V1 / V2 One can see that vs1 / vs2 = D1/ D2

(Eq. 16)

Superficial gas velocity will increase linearly with increasing scale.

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Aeration Rate and Oxygen Transfer Driving Force

Now we examine the gas phase balance. In scaling up, if we keep Q/V constant and keep KLa constant in the liquid phase, we will be able to maintain the same oxygen level at the inlet and outlet (Yin, Yout) and sustain the same oxygen transfer rate (OTR). However, when scaling up, Q/V will actually decrease so if OUR is to be sustained, then Yout has to be smaller to maintain the material balance.

Then, consider the liquid phase balance. We have kept KLa constant in this analysis. To keep OTR also constant, the driving force must be sustained. However, the driving force is the logarithmic mean of the oxygen concentration at the inlet and the outlet. With a lower level of Yout, the driving force for oxygen transfer will actually be lower. Therefore, overall oxygen transfer becomes a challenge in scaling up because the aeration rate cannot be increased proportionally with the scale.

When scaling up, we aim to maintain OUR and OTR at the same level. Considering mass balance in the gas phase: From Eq. 12:

Given that Q2/V2 is smaller, and Yin (oxygen concentration in the inlet air) is the same, Yout,2 in the large scale will be smaller From Eq. 13 and Eq. 14, since Yout,2 is smaller, so is C*. Considering the liquid phase, OUR and OTR will be maintain at the same level in the two scales: (KLa)1(C*1 – C1) = (KLa)2(C*2 – C2) This can be accomplished by • Allowing C*2 to be lower while maintain C2 = C1. KLa2 in the large scale must increase. Keep KLa2 at the same level as in small scale, then the concentration driving force of oxygen must be increased to the level of the small scale by • Allowing C2 to decrease • Increasing C*2 by using enriched oxygen

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Gas Flow Rate in Scaling Up: A Summary

The physical constraint of a reduced cross-sectional area to a reactor volume ratio with increasing scale poses a challenge in oxygen transfer. In scaling up, the airflow rate may not be increased proportionally to the culture volume because an overly high superficial air velocity is problematic.

Table 4. Effect of Scale on Oxygen Transfer Reference Scale

Constant Air Flow

Scale (volume)

1

1,000

Constant Superficial velocity 1,000

Cross Sectional Area

1

100

100

Air Flow Rate

1

1,000

100

Superficial Air Velocity

1

10

1

O2 Consumption/ CO2 Production

1

1,000

1,000

Q(yin –yout)

1

Comments

1

May reach flooding

(yin –yout) has to be very large, i.e. yout is small Need to increase KLa or power input

If air supply increases proportionally with scale, foaming can become serious and flooding may occur.

In microbial fermentation, a high airflow rate eventually causes “flooding” (i.e., the impeller is swamped by gas bubbles) and loses its capacity for pumping liquid. For cell culture, the aeration rate used is substantially lower than the flooding aeration rate. However, potentially a different problem may arise. Antifoam agents are not used as extensively in cell culture as in microbial fermentation because of potential damage to cells. At a high superficial velocity control of foaming may become problematic.

When scaling up, aeration rate is not increased proportionally to the culture volume. Because less air is given to the same volume of culture, more oxygen has to be taken out from the gas phase to meet the oxygen demand. This causes the oxygen level in the gas phase to be lower. As a result the driving force for oxygen transfer is also lower in the large scale.

In scaling up it is a common practice to take a middle ground. The airflow rate per reactor volume is decreased somewhat, but the superficial velocity is allowed to increase (albeit less than proportionally to the culture volume) to minimize the loss of the oxygen transfer driving force. In some cases, the air is enriched with oxygen, while in other cases, the agitation rate is increased when oxygen falls below a set point during the cultivation.

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Effect of Scaling Up on CO2 Removal • The mass transfer coefficients for oxygen and carbon dioxide are about the same. • R.Q. (moles of CO2 produced/ moles O2 consumed) for mammalian cells is very close to 1.0. So, oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) are about equal. • The toxic level of CO2 is around 15–20% (110 mm Hg – 150 mm Hg). • The carbon dioxide removed from the reactor (at steady state) is the difference between its concentrations in the inlet and outlet gas:

(Eq. 17) • CER and CCO2, in are the same in reactors of different scales • If cell concentration and metabolic activity remain constant. • Q / V is smaller in large scale. • Inevitably CCO2, out will have to be higher on a large scale. • The driving force for CO2 removal decreases with increasing scale.

The respiratory quotient for most cells, using glucose and glutamine as the main source of energy, is very close to 1.0. Thus, every mole of oxygen consumed by the cells generates about one mole of CO2. A very active culture with a high cell concentration can produce more than 100 mmole/L of CO2 per day. In comparison, a cell culture medium has about 20 - 40 mM of sodium bicarbonate. The amount of CO2 produced by cells, thus, far exceeds that which is added as buffer to the media. Many cells, such as hepatocytes, are rather tolerant to CO2 but others are more sensitive. The growth of most cells may begin to be affected at a CO2 concentration of 15% (114 mm Hg) so continuous removal of CO2 from the culture is important.

When scaling up, the airflow rate per reactor volume may not increase proportionally. As a consequence, less air is used to strip CO2 from an equal volume of culture media. If the metabolic activity of the culture remains the same, the same amount of CO2 produced by the cells is now being removed using less air. The CO2 level will then be higher in the air exiting from the large-scale reactor.

The rate of CO2 stripping is dependent on the concentration difference of CO2 in the liquid and the gas phase. A higher concentration in the gas phase diminishes the stripping efficiency. The increased CO2 concentration in the exit air, thus, causes further accumulation of CO2 in the liquid phase. To compensate for the reduced O2 driving force from scaling up, one can use O2-enriched air. However, such a measure cannot compensate for the diminished stripping efficiency of CO2. Therefore CO2 accumulation patterns in large scale and small scale bioreactors can be rather different.

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• To achieve the same molar transfer rates for oxygen and carbon dioxide (although in opposite directions), the driving force for carbon dioxide has to be around 100 mm Hg, a level similar to that for oxygen transfer. • The upper bound of CL for CO2 should be below the inhibitory level of 110-150 mm Hg. If the driving force is 100 mm Hg, then C* will have to be 10-50 mm Hg. That is 1.5-6.5% CO2 in air. • To strip off carbon dioxide a sufficiently fast air flow rate must be used to ensure the CO2 concentration in the gas phase is low enough to provide a large driving force.

(Eq. 18)

The overall mass transfer coefficient for CO2 is slightly lower than that of oxygen because of its larger molecular weight. However, the difference, approximately the square root of their molar weight ratio, is very small. One can consider the KLa to be about the same.

Unlike O2, the solubility of CO2 in aqueous solution is very high. At the gas bubble interface, O2 in the liquid phase can be assumed to be in equilibrium with the gas phase, so (C*-C) is a good estimate of the driving force. The same assumption is not always valid for CO2.

CO2 in the medium exists as CO2, HCO3, and CO3-2. At the interface, CO2 crosses the film and is transferred out of solution, but HCO3- does not. So, HCO3-must dissociate to CO2 before being transferred to the gas phase. The kinetics of HCO3-, the dominant form of CO2 in aqueous solution, to dissociate to CO2 is slow. Because of the slow kinetics, the actual driving force is smaller than what can be estimated from the total CO2 (g) concentration.

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Chemical Environment in Scale Translation

In scaling up or scaling down, the chemical environment may also be affected. For cell culture processes, many of those changes are caused by different CO2 accumulations at different scales. CO2is a major contributor of the pH buffer in a cellular environment. CO2 produced by the cells is excreted to maintain a physiological range of intracellular pH.

Carbon dioxide is transported through the plasma membranes as CO2 and HCO3-. CO2 can diffuse through cellular membranes, while HCO3 transport is mediated by transporters. A symporter cotransports HCO3- and H+, while another antiporter co-transports Cl- and HCO3- in the opposite direction.

Fig. 13.12: Schematic of the removal of carbon dioxide produced by cells

As CO2 builds up in the culture fluid, a higher Clgradient is needed to “drive” HCO3- out, otherwise the intracellular HCO3- level will be higher. Because the airflow rate per reaction volume is not kept constant in scaling up, the CO2 level in the culture will be higher on a larger scale. This will affect the intracellular CO2/HCO3- levels. However, experimental evaluation of the effect of scale on cellular level of CO2 and intracellular pH is still lacking.

• CO2 produced by cells can diffuse through the cell membrane • Most CO2 becomes HCO3- and is excreted through transporters • Accumulation of CO2 in medium may affect intracellular pH

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Concluding Remarks In scale translation, the relationship of geometryrelated physical parameters are not kept constant, because the volume and the surface area of the reactor change in different proportions relative to length. Therefore, one has to define the critical range of various scale-sensitive variables and choose scaling up criteria to ensure the operation is within the optimal region. In most cases, one chooses not to scale up completely geometrically similarly. Most large-scale reactors have a larger height to diameter ratio than the smaller scale ones. Nevertheless, the physical constraints on scaling up are the same regardless of whether one scales up geometrical similarly or not. In scaling up, the gas flow rate is also likely to change in its proportion to the reactor volume. This causes the mass transfer characteristics to be different for different scales. While the dissolved oxygen can be controlled at the same level, the CO2 concentrations profiles are likely different for reactors of different scales. Differences in CO2,concentration in the reactor will cause pH control actions, including base and CO2,addition and

CO2 stripping, to vary at different scales. Difference in pH control actions may further change the chemical environment of the culture. Given that the physical and chemical parameters related to scaling up cannot easily be manipulated or controlled, one may resort to selecting cells which are less sensitive to those parameters. Understanding scale-sensitive parameters and a sound knowledge of estimating the range of those critical parameters will greatly facilitate the scale translation of cell culture processes. In process development involving scale translation, one should aim to reproduce or to predict the conditions of physical constraints, as well as the resulting chemical environment. It may not be possible to replicate all physical and chemical parameters on drastically different scales. Ultimately, one should identify and aim to control critical physical parameters, to minimize variations in the chemical environment.

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Cell Culture Genomics Gene and Genome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What is a Gene?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organization of Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Packaging DNA into Chromatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Mechanisms that Mediate Epigenetic Regulation . . . . . . . . . . . . . . . Genome Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteome Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequencing Technologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sanger Method and Sequencing Technology Evolution . . . . . . . . . . . . . . . . . . . High Throughput NextGen Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Expression Exploration in Cell Culture Processing . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285 285 287 290 291 291 293 293 299 302 302 304 306 308

Gene and Genome What is a Gene?

Mouse Genome Encodes • ~30,000 genes coding for proteins • ~1,600 genes coding for RNA • 800 tRNA genes • 350 tRNA genes • ~450 other ncRNA (noncoding RNA genes)

A gene is a sequence of DNA that encodes for an RNA and protein product. Up until a quarter century ago the prevailing notion was that one linear sequence of DNA directly encodes for one gene. After the discovery of alternative splicing, however, our understanding quickly changed. Recent findings have highlighted a relatively large number of alternatively splicing genes in the mouse genome. A large number of these genes are translated into different protein sequences. Our knowledge of the relationship between a gene and its expression product is evolving. For instance, we now know that two genes may reside on opposite strands of the same segment of DNA (and will be transcribed in opposite directions), or they may reside in the same strand of DNA and are overlapping.. A gene may encode for two types of end products: 1)

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Table 1. Distribution of Mouse Genes Protein coding genes

~30,000

Pseudogenes

~4,000

RNA coding genes

~1,000

rRNA

~800

tRNA

~350

snRNA

~150

miRNA

~300

rcRNA

~450

Gene Structure in Eukaryotes • Exons make up the mRNA; intervening sequences called introns are also present • Splicing of pre-mRNA entails removal of intronic regions, addition of polyA tail and 5’ cap • Alternative splicing is common

a protein, or 2) a non-protein-coding RNA (ncRNA). In its entirety, the mouse genome encodes for a total of about 37,000 genes. Among them, just over 30,000 genes code for proteins, 4,000 code for socalled pseudogenes (genes whose identity can be traced to an ancestor but are no longer translated into a functional protein), and the remaining 1,600 code for RNAs (including 800 rRNAs, 350 tRNAs, and other non-coding RNA, or ncRNA). The average mammalian gene spans a region encompassing approximately 23 to 28 thousand base pairs (kbp) in length. Typically, the promoter region is located upstream of the transcribed region (about 1 kbp or longer, sometimes as long as 10 kbp) and the transcribed region is used to generate a primary transcript, or pre-mRNA. The primary transcript starts with a 5’ end untranslated region (5’ UTR) and ends with a 3’ UTR. It also contains a number of protein coding exons and introns. After the introns are spliced out, the mRNA, or mature transcript, remains and includes: 1) the 5’UTR, 2) the exons, 3) the 3’UTR, and 4) a newlyadded polyA tail. The mRNA is ultimately exported out of the nucleus for translation in the cytoplasm. Many primary transcripts undergo alternative splicing. In these cases, portions of exons are stitched together under different conditions (e.g., in different tissues, at different times, or occurring at different frequencies) to give different mature mRNA species. The stitching point of two consecutive exons may not be the same under alternative splicing. In this case, splicing may result in a shift of the reading frame, which would affect the exons downstream of the splice junction and perhaps give rise to completely different protein sequences.

The Functional Annotation of the Mammalian genome (FANTOM) consortium has generated the most complete mouse gene sequence database to date. It has uncovered a large number of protein coding genes with alternative splicing. These events have been found to produce very different protein sequences, a large number of polymerase II transcribed ncRNAs (with polyA tail), and

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antisense RNAs that may have regulatory roles. The prevalence of so many classes of variants poses ambiguity in the definition of a “gene”. For instance, if one were to count each independent gene product as a different “gene”, the total number of genes could be estimated to be upwards of 50,000.

Organization of Eukaryotic Genes 23-28kbp

~1 kbp AUG intergenic region

Promotor

UGA Exon1

Intron1

Exon2

Intron2

Exons

Transcription

5’ UTR

intergenic region

3’ UTR UGA

AUG primary transcript

Alternative splicing

Splicing polyadenylation

5’ UTR AUG AUG

UGA UGA

3’ UTR AAAAAAAAAAAA

AUG

UGA

AAAAAAAAAAAA

start

stop

AUG

UGA

start

stop

AAAAAAAAA

Export to cytosol

Translation

protein 1

protein 2

Fig. 14.1: Expression of an eukaryotic gene. Alternative splicing into two different proteins are shown.

Organization of Genome Composition of a Typical Mammalian Genome • • • •

Coding Sequences Intronic Sequences Repetitive Sequences Other intergenic sequences

~ 1-2% ~ 20-25% ~45-55%

The number of genes in each organism varies greatly, from 700 genes in a simple parasitic mycoplasma to over 30,000 in mammals. Those studying the “minimum set of genes” that are required for life have derived a gene set ranging from 270 to 350 genes.

As the number of genes increases with increasing complexity of organism, the additional genes and gene products acquired tend to affect a cell’s interaction with its environment (e.g., transporters, signaling molecules, and their receptors). In other words, data suggest that the increased complexity of higher organisms requires the formation of a more sophisticated communication, both at the cellular and organism level.

With an increasing number of genes, one also sees an increasing size of the genome. By convention, the size of a genome is quantified using the number of bases in a haploid genome. A bacterium’s genome

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Eukaryotic Genome is Organized into Chromosomes • Human: 22 pairs; x,y • Mouse: 19 pairs; x,y • Rat: 20; x,y • Chinese hamster: 10 pairs; x, y • Mouse chromosome size: 61 Mbp (Chr 19)to 197 Mbp (Chr. 1)

Repetitive Sequence • Short RNA-derived interspersed elements (SINES, 90-300 bp) • Long interspersed nucleotide element (LINES,>500 bp) • Retrovirus like elements with long terminal repeats (LTR) • DNA transposons • Widely distributed simple sequence repeats: • Direct repetitions of short k-mers such as (A)n, (CA)n or (CGG)n • Segmental duplications, consisting of blocks of 10–300bp of the genome • Blocks of tandem repeated sequences, such as at centromeres, telomeres, the short arms of acrocentric chromosomes and ribosomal gene clusters, these also include satellites and microsatellites.

is about 3 Mbp to 8 Mbp, while a fungus’s genome is a bit less than one order of magnitude larger than that of bacteria, from 12Mbp to 30Mbp.

In contrast, mammals have about a genome size of 2-3 Gbp, nearly 1,000 fold higher than bacteria. While the mammalian genome is nearly 1,000-fold larger, it contains only 10 times more genes. A typical protein-coding gene in bacteria is about 1 kbp, or 330 amino acids, while an equivalent sequence in mammals is about 1.3 kbp. Including introns and UTRs, a mammalian gene in total is 23-28 kbp, which is substantially larger than a bacterial gene.

The gene-encoding regions, including introns, account for only about 25% of the mammalian genome. The rest of the genome consists of other intergenic sequences, including promoters, regulatory elements, and regions not yet explored by scientific inquiries. Additionally, nearly 50% of a mammalian genome consists of repetitive sequences. That number is even higher in some plants, giving them a genome size even larger than mammals. The repetitive sequences reside not only in intergenic regions, but also in introns, UTRs and upstream or regions adjacent to genes. Repetitive sequences fall into different categories; some are short, while others are long, up to 500 bp. Some are the result of transposons or the remnants of retrovirus infection throughout evolution.

The presence of repetitive sequences presents a barrier to the quick and accurate assembly of DNA sequencing reads. In DNA sequencing, the DNA molecule is first fragmented and then the independent fragments are sequenced. The assembly algorithm searches for the overlapping regions and attempts to stitch them together into longer contiguous sequences (or contigs). Therefore, a fragment that has a repetitive end can often be assigned to multiple loci as it is not a unique sequence. One solution to this is to sequence very long fragments of DNA, over the stretches of repetitive sequences.

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Table 2. Genome Size of Representative Organisms Common Name

Taxonomy

Species

Genome Size

Estimated Number of Genes

Mycoplasma

TENERICUTES

Mycoplasma pneumoniae FH

8.11E+05

670

Bacteria

PROTEOBACTERIA

Escherichia coli DH10B

4.69E+06

4,271

Bacteria

FIRMICUTES

Bacillus subtillis subtillis 168

4.21E+06

4,354

Yeast

FUNGI-ASCOMYCOTA

Saccharomyces cerevisiae S288C

1.21E+07

6,273

Slime Mold

PROTISTS-MYCETOZOA

Dictyostelium discoideum AX4

3.40E+07

13,362

Roundworm

NEMATODES

Caenorhabditis elegans

1.00E+08

20,935

Fruit Fly

ARTHROPODA

Drosophila melanogaster

1.37E+08

21,116

Chicken

CHORDATA-BIRDS

Gallus gallus

1.00E+09

17,935

Frog

CHORDATA-AMPHIBIA

Xenopus tropicalis

1.70E+09

20,500

Human

CHORDATA-PRIMATES

Homo sapiens

3.17E+09

53,894

Mouse

CHORDATA-MAMMALS

Mus musculus C57BL/6J

2.72E+09

37,261

Rat

CHORDATA-MAMMALS

Rattus norvegicus BN/SsNHsdMCW

2.70E+09

35,427

Dog

CHORDATA-MAMMALS

Canis lupus familiaris

2.40E+09

24,661

Chinese Hamster

CHORDATA-MAMMALS

Cricetulus griseus

2.70E+09

32,476

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Packaging DNA into Chromatin

DNA molecule

packed with histone proteins to form chromatin chromatin forms fiber-like structure

may condense tightly or packed loosely in different regions of chromosomes

Fig. 14.2: Packing of a segment of DNA into highly condensed region and relatively open region

• Chromatin: complex of DNA and protein in which genetic material is packaged within the cell

In bacteria, the genome is typically arranged into a single, large chromosome. Most chromosomes are circular, although linear chromosomes are also seen. Conversely, in eukaryotes, the genome is segmented into different chromosomes. Chromosomes are not merely DNA molecules wrapped loosely together. A typical E. coli cell (2 µm x 1 µm in size) has a genome of about 1.5 mm in length. If all 46 diploid chromosomes of the human genome were strung together, they would form a ~2 m x 2 nM string. The chromosome is, thus, not merely a string of DNA packed haphazardly into the nucleus. It requires extensive manipulation and the work of specialized machinery to allow it to be packed into a dense volume, while still remaining accessible for transcription.

Each chromosome is a molecule of double-stranded DNA. Packaging of DNA occurs at multiple levels. At the local level, small regions of a DNA molecule form a complex with DNA binding proteins, chiefly histones, to form a “beads-on-a-string”like structure. That form is further condensed into packed beads, called nucleosomes. In further condensation of the structure, some regions are more open and accessible to transcription (this is called euchromatin), while other regions are more densely packed, and are called heterochromatin.

• Histones: principal protein components of chromatin • Nucleosome: fundamental sub-unit of chromosome which consists of 165 bp of DNA wrapped around an octamer of core histones

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Epigenome The term ‘epigenetics’ was introduced in the 1940’s to describe “the interactions of genes with their environment, which bring the phenotype into being.” • ‘Heritable changes in gene expression not encoded in the DNA’ • Essential for genome packaging and fundamental to development • Epigenetic alterations influenced by the environment; for example, identical twins can be susceptible to different diseases • Epigenomics: Representing the totality of epigenetic marks in given cell type

Molecular Mechanisms that Mediate Epigenetic Regulation

Although the genome of each cell of a higher mammal may encode more than 30,000 genes, at a given time it may actively transcribe only a fraction of these. A typical CHO cell in culture, for instance, expresses only about 16,000 genes. Some genes are ubiquitously expressed in all tissues; others are tissue- or timedependent. At the transcriptional level, a large array of transcription factors are responsible for regulating the expression of genes according to the tissue type, timing by developmental stage, or by event (such as stress or exposure to some signaling molecule). At a higher level, transcription is also regulated by the accessibility of the gene loci, which is further controlled through epigenetic events. Epigenetic regulation does not result in a mutation, as no change occurs to the underlying DNA sequence. It does, however, cause a heritable change in the cellular phenotype. Unlike a mutation, which originates in a single genomic locus of a single cell, epigenetic changes can occur and affect gene expression on a global level. For instance, when stem cells differentiate, or when fibroblasts are transformed into iPS (induced pluripotent stem) cells, global epigenetic changes occur on the chromosomes to affect the reprogramming of genetic circuits.

In cell culture, cells are often “adapted” to new culture conditions, such as differing growth factors or adjusting to growth in suspension. In such processes, the entire population of cells shift their phenotype. Such processes are less likely to be mutation events and are more likely to involve epigenetic regulation. In the generation of producing cells, the host cell transforms from a non-secretor to a professional secretor in a short time, accompanied by a vast change of cellular properties. Although it is possible that mutations may be responsible for some of the changes, it is very likely that epigenetic events are the major

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• Chromatin modification • Covalent modifications of histones • Histone variants • Nucleosome remodeling • DNA Methylation • Non-coding RNAs

Table 3. Types of Histone Modifications Chromatin Modifications

Residues Modified

Functions Regulated

Acetylation

Lysine

Transcription, Repair, Replication, Condensation

Methylation

Lysine

Transcription, Repair

Methylation

Arginine

Transcription

Phosphorylation

Serine, Threonine

Transcription, Repair, Condensation

Ubiquitination

Lysine

Transcription

drivers to in the dramatic shift to high productivity.

The major mechanisms of epigenetic modification involve DNA modifications and histone modifications. Of the DNA modifications, the methylation of Carbon 5 of cytosine is one of the most common. Once methylation occurs, the mark is highly stable and can be passed on to daughter cells. Note that this change does not constitute a change or mutation in the DNA sequence. Much of the DNA methylation occurs on cytosines that reside in CpG dinucleotides (its complementary strand, 3’GpC -5’, is also methylated). Regions of the genome with a high GC content, where the CpG sequence is very frequent, are often called “CpG islands”. Such regions, when upstream of a promoter, can play key roles in the regulation of gene expression. Methylation of a CpG islands primarily leads to gene silencing, as has been shown in the cholesterol dependence of NS0 cells. For instance, Hsd17b7, a key gene in cholesterol synthesis, is silenced by CpG methylation in NS0 cells. Accordingly, demethylation treatment of NS0 cells led to the rapid emergence of cholesterol-independent cells. Methylation is also likely to be involved in the glutamine dependence of CHO cells, due to methylation of a CpG island upstream of the glutamine synthetase gene. Of note, methylation does not only occur in CpG islands; nearly a quarter of methylation seen in embryonic stem cells is not in a CG context. This non-CG methylation, however, is more transient and mostly disappears after differentiation.

At the histone level, acetylation, methylation, phosphorylation, and ubiquitination may occur on different amino acid residues of histone proteins. Each histone protein has multiple sites that may be modified, resulting in a large combination of possible histone modifications, each affecting the packing of DNA and the accessibility of genes in the region. Both histone and DNA chemical modifications require specific enzyme-mediated reactions. Their maintenance and removal also requires specific enzymes.

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Knowing the mechanisms of epigenetic regulation, it is possible to intervene using chemical inhibitors of the necessary enzymatic reactions. For example, 5’-Azacytidine is used to facilitate demethylation. TrichostabinA and sodium butyrate are also used to interfere with histone modifications. The alternation of the epigenetic status can also be induced by the introduction of exogenous genes, as in the process of reprogramming induced in the derivation of iPS cells. reprogramming induced in the derivation of iPS cells.

Genome Scale Analysis Transcriptome Mouse Genome Encodes • ~15,000 genes are expressed in a given cell • Highly abundant genes generally don’t change transcript level over a wide range • Rare genes can be very dynamic • 1,000 fold change in transcript level in 30 min is common in bacteria. A similar change in differentiating stem cells usually takes days.

Table 4. mRNA in a Typical Somatic Human Cell

Superprevalent (Abundant) Intermediate Complex (rare)

Number of Species

% of mRNA by mass

10 - 15

10 - 20

1,000 - 2,000

40 - 50

15,000 - 20,000

40 - 45

At a given time a typical mammalian cell transcribes about 15,000 genes into RNA. The vast majority of those transcripts are present at only very low levels, with some even as low as a few copies in each cell, with another small fraction expressed at intermediate levels. Only a very small number of genes are expressed at extremely high levels. Genes in the last class, the so-called “abundant genes”, encode proteins such as ribosomes, GAPDH (3-phosphoglyceraldehyde dehydrogenase), and actin.

In some recombinant cells, the product gene, which is highly amplified, also falls into this category. Because the sheer number of transcripts for each abundant gene is very high, the total mass of those RNAs can constitute up to ~10% of all mRNA. Such genes usually do not undergo a very large degree of change in their expression level. For instance, one rarely sees even two-fold changes in the transcript level of most abundant genes under different culture conditions. However, keep in mind that, because they are so abundant, even a 10% change in the level of a gene in this category is much greater than even a 10-fold change in a rare gene. The genes that most frequently undergo very large changes in expression are the rare genes. These rare genes often encode for products that are gene regulators or other products that are powerful even at minute levels. For this reason, they are kept at very low expression levels and are not expressed when not needed.

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Expressed Sequence Tags (ESTs) • Transcripts from cells of tissues are isolated, reverse transcribed to cDNA and cloned into E. Coli to construct an EST library • Clones of E. Coli are sequenced to give rise to fragment or the entire length of transcript • Sequences are assembled and annotated • The data gives transcriptome profiles of (abundance level) of transcripts of different genes under different conditions • The sequence data are used to design microarray and assemble the genome

The transcript levels of many genes are relatively stable at different times and under different conditions, while some are relatively dynamic. Overall, the rate of change of gene expression in mammalian cells is rather slow compared to bacteria. We see over three orders of magnitude decrease in transcript levels within half an hour in bacteria; however, even under stem cell differentiation conditions, a similar level of change in mammalian cells usually occurs over days. To explore the dynamics of gene expression in different tissues and in different diseases or differentiation stages, transcripts were isolated from those tissues and directly sequences. Those transcripts are typically called expressed sequence tags (ESTs). The collection of those ESTs form the core of the database of various genes in different species.

Capturing the dynamics of transcripts at a global level, i.e., on a genome scale, has become readily available in the past decade through the use of DNA microarrays and, more recently, through deep sequencing. Transcriptome profiling through arrays and sequencing remains the cellular analytical tools that are truly global and capable of genome-wide survey.

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DNA Microarrays

Fig. 14.3: Classical DNA microarray prepared from cDNA clones of an EST library and the use of it as a two-dye microarray

Table 5. Available Microarray Technologies Affymetrix

Agilent

Nimblegen

Manufacturing technology

Photolithographic manufacturing

Noncontact inkjet printing

Maskless synthesis using digital micromirro device

Probe Length Feature size Multiplexing

25 - mer 5 - 18 μm No

60 - mer 65 μm 2-, 4-, and 8-plex

60 - mer 16 μm 2- and 4 - plex

Two prevailing forms of microarrays are currently utilized: cDNA microarrays (commonly used for complex mammalian genomes) and oligonucleotidebased microarrays. The difference between these methods lies in their type of probes. In the case of DNA microarrays for microbial species the primers are designed to specifically amplify gene fragments from genomic DNA that will then serve as probes. These probes are designed specifically for unique segments of gene sequences. The probes spotted on a cDNA microarray for mammals, on the other hand, are amplified from cloned cDNAs using universal vector primers. Usually designing probes based on specific gene sequences is too costly with the large number of genes involved. They usually lack the specificity to differentiate many isoforms or alternatively-spliced variants. Oligonucleotide-based microarrays, in contrast, utilize much shorter (20 - 80 bp), specifically designed, then chemically-synthesized probes. Multiple probes covering different regions of each gene are often used to increase the specificity.

With the decreasing cost of oligoDNA microarrays and direct sequencing, cDNA microarrays are being phased out. cDNA arrays rely on using two fluorescence channels for relative measurement renders them inconvenient for comparison of a large number of samples.

Long oligo microarrays are typically comprised of 50 - 70 bp probes synthesized onto a glass slide. Affymetrix arrays are made by the direct synthesis of eleven sets of short 25-mer probes onto the chip through photolithography-based technology. Typically, multiple probes are employed for a given gene or contig over a region of a few hundred base pairs of each target transcript. The photolithographic in situ synthesis technique requires the construction of masks for each layer of nucleotides added to the probes. The process is extremely costly. In contrast, using a digital micromirror, NimbleGen technology directs light to tiny spots to allow chemical reactions to occur only in the lighted spots without using masks, thus drastically

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reducing the manufacturing cost of making the array. light source

mask (each layer four masks for A, T, G, C)

array “layer by layer” synthesis (total ~25 layer for 25-mers of DNA) Fig. 14.4: Mask-based layer-by-layer in-situ photosynthesis of oligonucleotide on array surface (Affymetrix system).

digital mirror light source

Both Agilent and NimbleGen allow multiplexing (i.e., multiple independent samples can be hybridized to separate arrays on a single slide). Both of these array formats also support a dual mode system that provides the option of using the routine twocolor experimental design (Cy3 / Cy5 based) or one-color (Cy3 only) on a single platform.

When the DNA array is used for two-channel comparison, usually a common reference is used. Such common references are usually acquired by mixing mRNA samples from different tissues under different culture conditions to ensure that the vast majority of transcripts are present. When the DNA array is used for two-channel comparison, usually a common reference is used. Such common references are usually acquired by mixing mRNA samples from different tissues under different culture conditions to ensure that the vast majority of transcripts are present.

“layer by layer” synthesis ~60-mers of DNA

no mask needed

Fig. 14.5: Digital mirror-based photosynthesis of oligonucleotides on array surface (NimbleGen system).

RNA-seq • Direct counting of abundance level of reads for each gene, normalized to gene length • Very wide dynamic range of detection • Quantification not affected by low sensitivity or saturation as in fluorescence detection • Does not require sequence information for

With the ability to generate tens of gigabases in a single run, high throughput sequencing technologies are becoming an affordable and powerful tool for transcriptome profiling.

A first step in transcript profiling is the removal of rRNA by oligo(dT) capture of mRNAs, which targets their polyA. Subsequent reverse transcription for cDNA synthesis is performed either by polyAbased priming or by using random primers. RNA-seq methods take mRNA samples, shears them

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into fragments, and reverse transcribes them into cDNAs, which are then sequenced using one of the high throughput sequencing technologies. The resulting output is a large number of sequences on the order of 50 - 150 bp. Each string of sequence is called a “read”. Depending on the depth of sequencing and the abundance level of the nucleic acid fragment, a segment of nucleic acid may be sequenced only a couple times or up to million times.

RNA-seq is not 3’ end biased; the abundance level of a transcript is represented by the number of times that segments of the transcript have been sequenced. The more abundant and the longer the transcript is, the more frequently its sequence reads will appear.

Using this method, one can sequence as deeply as is necessary to detect almost all transcripts in the cells, even those that are rare. In microarray analysis rare transcripts usually do not yield enough signal to give confidence to results. Furthermore, very high abundant genes are usually detected in the non-linear, near-saturation range of signal, thus lacking good sensitivity for quantification. Such problems are not present when RNA-seq is used, as it gives a much wider dynamic range.

Another major advantage of RNA-seq is that it requires no prior EST database or genome sequences of the species to be probed, whereas for DNA array probe design, at least the sequences of the genes to be probed must be available. For the species whose genome sequence or EST database is available, the reads from sequencing are mapped to the exon sequences for enumeration of hit reads and for normalization to sequence length. Even if no genome sequence or EST database is available, the reads can be still assembled into contigs. In most such cases, the contig annotation can be obtained from a related species, and the reads can be mapped back to each contig and then counted to give an estimate of transcript abundance.

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Table 6. Commonly Used DNA Microarray Formats Microarray Type cDNA

oligoDNA

Probe sequence generation

Common Uses

Sample Numbers

Hybridization method

Quantification Method

Universal primer amplified EST probes

Commonly used for mammalian EST library based array

Sequence specific primer amplified probes

Generally used for microbial species whose genome has been sequenced

Normally two channel comparisons (can go up to four)

Typically mRNAs are isolated and reverse transcribed to cDNA which is the labeled with a fluorescent dye

Synthetic specific 50-60mer, can have multiple probes per gene

Cross hybridization among different closely related sequences can be minimized

Measure the ratio of intensity of the same transcript from different samples. Comparison of multiple specimens from different samples must use ratio of ratios unless a common reference for all samples is used. Can use a pooled RNA as a common reference to facilitate the comparison of multiple samples. Direct comparison of levels of different transcript in the same specimen is difficult.

Two Channel

Single Channel

RNA-Seq

Photolithographically synthesized 25mers, multiple probe set per sequence

Interrogate multiple segments of about 500 bp region using both “perfect match” and “mismatch” probes to compute the signal

Single Channel

mRNA is reverse transcribed, then transcribed into cRNA, which is then fragmented and biotin labeled before hybridizing to the probes on the array.

The intensity gives an estimate of the abundance of transcript for each gene. Data can be used for both intraarray comparison (different genes in the same specimen) as well as inter-array comparison (ratio of expression level as in cDNA array).

Direct sequencing. Coverage depends on sequencing depth. For CHO, 20GB gives good coverage of most genes

Use for reaching depths sufficient to detect rare genes. Discerning heterogeneity in transcript or genome. Also for transcriptome profiling of unsequenced genome

Single sample per channel, or multiplexing barcoded sample

Short reads are sufficient for sequenced genome. If assembly is required, long reads are preferred.

Direct counting of sequence reads per gene, normalized to gene length.

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Proteome Profiling

Proteome profiling is another means of surveying global changes in gene expression. It is a powerful complement to microarray transcriptome profiling, serving to examine gene function directly at the protein level. Proteomic analysis allows for the investigation of the primary amino acid sequence, protein-protein interactions, and post-translational modifications on a large scale. In proteomics, mass spectrometry has established itself as an indispensable tool.

2D Gel Electrophoresis

2D-gel electrophoresis allows for the simultaneous analysis of a many protein molecules. In this method, a complex protein sample is resolved in two dimensions according to charge (isoelectric point) and mass on a polyacrylamide gel, followed by staining. The stained protein spots are then characterized using sophisticated image analysis tools (such as PDQuest). The difference in staining intensity of observed spots allows for relative quantification between protein samples.

Figure 14.6: Typical flow of 2-D electrophoresis-based protemics.

• 2D gel electrophoresis • First Dimension: Isoelectric focusing (IEF), separation by charge. IPG (immobilized pH gradient) strips; usually pH 4 to 7 • Second Dimension: SDS-PAGE, separation by molecular weight • Staining types: Coomassie Blue (sensitivity in the ug range); silver staining (sensitivity in the ng range); SYPRO RUBY(sensitivity in the pg range; fluorescent dye; most suitable for quantification) • Image Analysis: identification of differentially expressed spots by eye or with the aid of specific software packages (PDQuest)

This method, however, is not suitable for some proteins. For instance, proteins present at lower levels are not easily detected. Also, some proteins co-migrate and cannot be resolved. Finally, proteins with charges outside of the isoelectric range of the gel or highly hydrophobic proteins are also unable to be resolved in the gel.

Once a protein spot of interest is identified, the spot is excised and purified for further analysis, such as direct sequencing or mass spectrometry for protein identification. Electrospray ionization (ESI) and matrix-assisted laser desorption/ ionization (MALDI) are two techniques commonly used to volatize and ionize the proteins or peptides for mass spectrometric analysis.

• MALDI-TOF and ESI • Differentially expressed spots are extracted from 2D gel, and subjected to proteolytic digest, and peptide finger print analysis using MALDI-TOF

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2D LC

Shotgun LC Based Methods SILAC • Use non-radioactive isotope for labeling cultured samples • Mix samples for isolation or enrichment of some cellular fractions • Sample can be analyzed by PAGE or 2D LC • The fractions can be analyzed in mass-spec for identification iTRAQ • Use isobaric tag for different samples 2D LC • Protein mixture is subjected to proteolytic digestion peptides are amenable to LC separation to reduce complexity • Column types: First dimension often exchange, second dimension reverse phase • Electrospray injection into mass spectrometer • For identification of molecules, not quantification

Fig. 14.7: iTRAQ labeling of proteolytic peptides for 2-D liquid chromatography.

In applying two-dimensional liquid chromatography (LC) for analysis of protein mixtures, a proteolysis of the protein (typically using trypsin) is first performed. This process reduces proteins to oligopeptides of mostly 15 - 30 amino acids long, which facilitates separation by liquid chromatography. In the first dimension, the peptides are separated into fractions. Each of the selected fractions is then injected into the second LC for further separation before injection into the mass spectrometer (MS) for detection. LC-LC/MS methodologies have the advantage of being capable of analyzing complex protein mixtures; however there was previously no way to quantify different expression levels among samples until recently, due to the application of stable-isotope labeling to LC-LC/MS. This method makes use of the fact that pairs of chemically-identical analytes with different isotope compositions can be differentiated in a mass spectrometer by their difference in mass-tocharge ratio. The ratio of signal intensities for the pair accurately indicates an abundance ratio for the two analytes. Two commonly used labeling techniques are SILAC (Stable Isotope Labeling by Amino Acid in Cell Culture), and iTRAQ (Isobaric Tagging for Relative and Absolute Protein Quantitation).

Although all proteins in a sample are, technically, included in the analysis using non-gel-based techniques, these methods are still not a true global surveying tool. In actuality, only a few hundred to a few thousand proteins are identified in the analysis of a sample due to limitations in the resolving power of liquid chromatography. The capture of a peak from a sample is stochastic, and highly abundant proteins are detected over those present at lower levels. The isolation and identification of low abundance proteins can be enhanced by repetitively analyzing the same fractions in the mass spectrometer and by excluding peptides that have already been identified in the previous analysis. This is very expensive and tedious so exhaustive surveys of the proteome space are not commonly practiced.

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Chemical isotope labeling of proteins in vitro (after protein isolation) using isobaric Tags for Relative and Absolute Quantification (iTRAQ) is a highly versatile and widely-used method. iTRAQ tags have a reporter group, a balancer group, and a peptide reactive group that tags the N-terminus of every peptide. One of the unique features of iTRAQ is that up to eight samples can be labeled with different tags and analyzed simultaneously, while other methods can only label two samples.

ITRAQ

The combined balancer and reporter groups have an identical mass of 145 for all four labeling pairs of balancers (mass 28 to 31) and reporters (mass 114117). The MS spectra for each of the four labeled samples look identical. The reporter-balancer fragment stays intact, giving rise to the same m/z ratio. This allows for protein identification using the combined signal of the four samples. Upon MS/MS fragmentation, the bond between the reporter and balancer group is broken. The reporter groups then appear as peaks in the low mass region, and quantification of the peak area for each reporter group gives the relative abundance for a peptide between the labeled samples.

Fig. 14.8: Isotopic labeling of peptides.

SILAC

Fig. 14.9: Stable isotope labeling of amino acids in cell culture (SILAC) for proteomics.

In vivo labeling methods, such as Stable Isotope Labeling with Amino acids in Cell culture (SILAC), use deuterated leucine, or other isotope labeled amino acids, to differentially label one of the protein samples by replacing an amino acid in the cell culture medium, thereby allowing the isotope to be incorporated into the cellular proteins. The combined labeled and unlabeled samples are then analyzed by LC-LC/ MS, generating two spectra for each peptide fragment, one shifted precisely by the mass of the deuterated amino acid. Differences in peak height provide the means of quantification between samples. This method allows two samples to be mixed prior to protein isolation, thereby eliminating systematic errors due to protein isolation efficiencies. It is well suited for use when subcellular enrichment protocols are used (as in the case of organelle fractionation) prior to LC-LC/MS analysis. One

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notable limitation of this method is that the labeling time required is relatively long, to allow for sufficient incorporation of the isotope into cellular proteins.

Sequencing Technologies Sanger Method and Sequencing Technology Evolution

Table 7. Summary of Sequencing Methodologies Technology

Read Characteristics

Applications

Sanger Sequencing (ABI)

500-1000 bp 384 reads/run

Gold standard all purpose sequencing

Roche Sequencer FLX/454

400 bp/read 1-7 Gbp/run

Re-sequencing, expression profiling, SNP/methylation

Illumina/Solexa

36-150 bp/read 20 Gbp/run

Re-sequencing, expression profiling, SNP/methylation

SOLiD (ABI)

30-50 bp/read 20 Gbp/run

Re-sequencing, expression profiling, SNP/methylation

Helicos

35 bp/read 20 Gbp/run

Re-sequencing, expression profiling, SNP/methylation

• Target DNA template is used for DNA synthesis • Fluorescently labeled nucleotide analogues (dideoxynucleotides) is added to the synthesis reaction; wherever an analogue is incoporated into the elongating DNA, the reaction terminates • Each of the four dideoxynucleotide chain terminators is labelled with a different florescent dye. • The newly synthesized and labeled DNA fragments are separated by size (with a resolution of just one nucleotide)

DNA sequencing technology has undergone revolutionary changes in the past few years. Traditional Sanger sequencing has dominated the field for nearly three decades, and it is still the prevailing method used for sequencing small regions of DNA. For large-scale, or genome-wide sequencing, a number of high throughput methods have rapidly changed the scope of sequencing. They are now used for genome sequencing, transcriptome profiling, transcription initiation site surveys, transcription factor binding site profiling, and epigenetic alteration studies.

Sanger sequencing gives relatively long reads, while newer methods give shorter reads. Some of the reads are too short for efficient assembly and are used primarily for “resequencing”, i.e., for sequencing the genome of individuals of a species whose genome sequence is already available. Other newer methods, such as 454 and Illumina, produce reads that are at least long enough for assembly.

All of the new methods and the Sanger method share the same “reading” scheme. Each time a nucleotide is incorporated into an elongating DNA molecule a signal is emitted. Two approaches are adopted to detect the emitted signal, (a) amplifying the signal by having many DNA molecules emitting the same signal, (b) using very sensitive detection methods to detect even the signal emitted from a sigle molecule.

In Sanger sequencing, each target DNA molecule is first cloned into E. coli so that a sufficient amount of pure DNA molecules can be obtained by growing the E. coli clone. Those DNA molecules are then used as templates for DNA synthesis. By using altered nucleotide analogues that cannot be used in DNA synthesis, the elongation stops randomly as soon as an analogue (instead of the genuine nucleotide) is incorporated. Given a proper titration of the ratio

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of analogues to normal nucleotides, there will be a DNA molecule terminated at every base of the DNA template. After synthesis, the mixture is elongated and the terminated molecules are separated by chromatographic separation at a single base resolution. Since each analogue is labeled with a fluorescent color, the colorimetric chromatogram is finally used to read off the sequence.

This clone handling is very expensive and time consuming. Furthermore, single-base resolution can be accomplished only up to at most 1.2 kbp, even with capillary electrophoresis. Fig. 14.10: Conventional Sanger sequencing of DNA.

Sanger Sequencing • Enrich target DNA fragments by cloning into a E. coli plasmid

- Specific primers at two ends of target DNA Collect plasmids, use specific primer to start DNA synthesis from one end

• Add nucleotide analog, ddNTPs in addition to dNTPs at low frequency in the reaction mixture • Incorporation of a ddNTP terminates DNA elongation. • Probabilistically a small fraction of elongating DNA is terminated at every base, resulting a collection of synthesized DNA fragments of different length • Separate those DNA molecules using capillary electrophoresis • Each of the four ddNTPs fluoresce at a different wavelength. As each DNA fragments comes out of electrophoresis, the fluorescence is “read” to give the identity of the base

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The most fundamental change from the Sanger

High Throughput NextGen Sequencing method to newer methods is the omission of E. coli

Next Generation Sequencing Technologies • Massively Parallel Local Amplification • ‘Sequencing-by-Synthesis’

clones. Two approaches were taken to bypass cloning. In the first approach, localized PCR amplification is performed: a single DNA fragment is amplified in a droplet or in a locus on a surface. This allows a large number of identical DNA fragments to be used to generate signals for detection as they incorporate nucleotides when used as templates for synthesis. In the second approach, single molecule detection is achieved. As long as each molecule is separated from the others, the signal from nucleotide incorporation into an elongating DNA can be detected. The increased sensitivity eliminates the need for cloning.

The current prevailing methods, sometimes referred to as “next generation sequencing technologies”, fragment the DNA molecules randomly without resorting to cloning. A key feature of these methods is the massive and parallel amplification of each DNA fragment individually, thus creating up to millions of clusters, or colonies, of DNA molecules of the same sequence. With the 454 technology, each fragment is immobilized on a bead contained in an oil / water emulsion droplet, thus forcing all of the PCR products of that particular fragment to also be immobilized on the same bead.

Fig. 14.11: Two strategies of localized PCR amplification of DNA molecules for enhanced signal detection.

On the other hand, the Illumina Solexa sequencing technology immobilizes the fragment on a solid surface and confines the PCR products of the fragment in the locale, thereby forming a cluster of identical sequences on the surface.

These ingenious approaches allow a large number of identical sequences to be isolated so that the light emitted from reactions in these clusters can be detected using a single sensor, such as a CCD camera. This contrasts with the traditional Sanger method, which requires a sensor at the end of each electrophoretic capillary, thus drastically reducing the cost of sequencing. Both methods then employ base-by-base synthesis. The 454 technology works by sequential addition of

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454 Sequencing Technology • Immobilize one DNA fragment per bead • Suspend each bead in an emulsion droplet • PCR amplify DNA in each bead • Place each bead in one well • Perform synchronized synthesis by adding one nucleotide at a time • Each nucleotide incorporation emits a photon • Homo-oligomer emits stronger light intensity (works only for short homo-oligomer sequences) • Can sequence up to 1000 bp in length Illumina Sequencing Technology • Immobilize DNA fragments on surface with adaptor • Localize PCR reaction to create clusters of DNA fragments with complementary strands • Perform synchronized DNA synthesis from one strand, then on the other strand • Upon the extension of one base, the reaction is terminated because the functional group for further extension of the added nucleotide is protected; this prevents incorporation of more than one nucleotide when homo-oligomer sequences are encountered • Can sequence up to over 100 bp; very high throughput

one of the four nucleotides and photons are emitted upon the incorporation of a base. The Illumina Solexa methodology employs fluorescently-labeled nucleotides that are also reversible terminators. One base is incorporated and interrogated at a time, since further elongation of the chain is prevented. When all clusters are scanned at the end of a cycle and the base has been determined for each colony, the fluorophores are cleaved off and terminating bases are activated, thereby allowing another round of nucleotide incorporation.

The drawback of the current generation of these new sequencing technologies is their reliance on the complete synchronization of serial reactions. The reads obtained are relatively short, compared to those from Sanger sequencing. However, this drawback is more than compensated by their improved capability of massively paralleled “reading” of millions of sequences at high speeds and at relatively low costs. Their tremendous parallel sequencing abilities allow for up to a million fragments of a very abundant mRNA species (such as the transcript for the recombinant protein product) to be “read” in a single run, while only single reads can be obtained for thousands of rare genes. Such a dynamic range in sequencing outputs has made them great tools for assessing transcriptome-wide abundance levels.

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Gene Expression Exploration in Cell Culture Processing Transcriptome analysis is a powerful tool for studying cell culture processes, as it is extremely revealing for both subtle and not-so-subtle changes that can occur in many situations (e.g., cell cultivation, the variation of cell behavior over time, and cell line development). It has been used to compare cell performance in different reactor scales, cells of varying productivities, and cells growing under different media composition, and to probe cell changes after long-perfusion culture.

In a striking example, a cell line was subjected to studies under differing culture conditions. The variables included temperature, pH, and reactor size. There was also an uncontrolled variable. Two different lots of the same hydrolysate were used in each run, as is customary (when one lot of material is exhausted, another lot is used). The time-course transcriptome data from all runs were collected and subjected to clustering. All the runs with the same lot of hydrolysate were found to be clustered together, thus overriding the other variables of the experiment.

Fig. 14.12: Comparative transcriptome dynamics between (a) mouse hybridoma cells treated with butyrate and (b) differentiating mouse stem cells (MAPC). MAPC cells were grown on liver lineage differentiation medium for 6 days. Mouse hybridoma cells were treated with 1mM butyrate for 27 h. Both MAPC and hybridoma cell samples were referenced to a corresponding, untreated time sample. The transcript levels were probed with the Affymetrix MOE430A array. For each dataset, average intensities are plotted along the y-axis, and the log2 of the expression ratios are plotted on the x-axis. Each marker represents a gene on the array. The vertical lines mark the bounds of a two-fold expression change. Markers lying outside of these lines are more than two-fold up or down regulated between the two samples compared. In comparing Figures 2a and b, the number of genes that are more than two fold differentially expressed is substantially higher for the stem cell differentiation study than for the butyrate treated hybridoma cells.

Such revelation is possible because of the large number of genes probed. Many subtle changes which, individually, may not be used to draw a confident conclusion, can collectively point to a trend or correlation that is not otherwise easily discernible. There has been some effort in comparing cells of varying levels of productivity. It has now been realized that the “hyperproductivity trait” is a complex phenomenon that is not simply a result of turning on a small number of master genes. Rather, the generation of high producers is likely to involve colossal changes in gene expression that occur in a wide range of genes, but each only to a modest extent. A most important feature of either sequencing- or microarray-based transcriptome analysis is its global coverage of gene expression. This type of analysis sometimes reveals unnoticed cellular processes that may play a key role in some physiological events. For example, it was found that the endocytosis and secretory vesicle retrograde transport pathways

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are upregulated in high recombinant proteinproducing cells. This led to the realization that high producers have an increased capability to recycle components of membrane vesicles that carry proteins destined for secretion from the Golgi apparatus to the cytosolic membrane.

One of the important features of transcript profiling in cell culture processes is the small number of genes differentially expressed and the lower magnitude in the observed foldchanges, as compared to those observed in other biological processes. For microbial cells, it is not unusual to observe more than 20% of the genes as differentially expressed by more than two-fold within ten minutes of changing culture conditions. For cultured mammalian cells, it is rare to see over two-fold expression changes occurring in more than a small percentage of the genes surveyed.

Fig. 14.13: Intracellular processes differentially expressed between high and low producing NS0 cell lines. (a) Each node depicts an intracellular process with a large number of differentially expressed genes. (b) Schematic of the steps involved in vesicle-mediated transport (nodes 4, 5, and 6).

It is important to realize that the cells we deal with are cultivated in artificial conditions, not in their native niche. For an organism the development of a fertilized egg to an adult body incurs many major events and gene expression changes to guide those events. Once the cell is guided to its destined developmental status, the perturbations of other environmental factors are relatively minor in magnitude by comparison. The various events that cells in culture may encounter are only relatively small perturbations compared to developmental or differentiation events that their genome has been evolved to accommodate. It is not surprising that colossal big changes in gene expression are rarely seen in cell culture bioprocess but are frequently seen in stem cell differentiation.

The small change in gene expression, as compared to other organisms and to the in vivo processes, requires one to be more versatile and more skillful in data analysis. However, the power of global transcriptome analysis will fundamentally change our practice in cell culture processing. nor perturbations in comparison to development.

The small degree of gene expression changes, as

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compared to other organisms and to the in vivo processes, requires one to be more versatile and more skillful in data analysis. However, the power of global transcriptome analysis will fundamentally change our practice in cell culture processing.

Concluding Remarks In the past few years we have seen dramatic advances in genome science and technology. The availability and affordability of sequencing technology has changed sequencing effort from species sequencing (i.e., focusing on obtaining a “representative genome sequence”), to individual sequencing (i.e., aimed to acquire genome sequence of an individual human, organism or cell line). The depth of information we are acquiring from genome, transcriptome, proteome and epigenome, is transforming the

way we deal with bioprocess challenges. The application of “-omics” technology in cell culture processing is still largely limited to process analysis. One can foresee that, in a not too distant future, “-omics” technology will be increasingly used for the generation of producing organisms, as well as in the design of biological processes. In some ways, genomics science may also bring about transformative changes in cell culture bioprocessing.

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Index Note: Page references followed by f or t indicate material in figures or tables, respectively. A ABC transporter, 43, 44f, 84 Abraham, Edward Penley, 2 Abundant genes, 293, 293t Acetylation of histones, 292–293, 292t Acetyl CoA in cholesterol synthesis, 87 in lipid metabolism, 86, 86f metabolism of, 81 in tricarboxylic acid cycle, 61, 73 Acetyl CoA shuttle, 86, 86f Acoustic resonance enhanced settling, 257–258, 258f Actin fibers, 37, 38–39, 38f, 46 Active transport, 40–42, 41f Activity parameters, 156, 160f Adaptation, in stable cell lines, 131, 142, 142f, 143f ADCC. See Antibody dependent cellular cytotoxicity Adenosine, in medium, 109, 109t Adenosine triphosphate (ATP) in active transport, 40 consumption in glucose metabolism, 60–61 mitochondrial production of, 29 production in glucose metabolism, 60–64, 66, 70 Adenosine triphosphate synthase (ATP synthase), 61, 63 Adherent cultures, vs. suspension cultures, 202 Adhesion, cellular, 45–46, 53, 142 microcarriers for, 203–204, 204t, 205t vs. suspension, 202 Aeration. See also Oxygen transfer, in bioreactors stripping of carbon dioxide by, 219 surface, for oxygen supply, 220 Aerobic glycolysis, 65–66 Affym etrix microarrays, 295–296, 295t Agarose cell immobilization, 209 Aggregation, 206, 206f, 209, 209f, 212 Agilent microarrays, 295t, 296 Agitation, in bioreactors, 265–266 impellers for, 206, 265–268, 265t, 266f mechanism of, 265–266 purpose of, 265 Airlift bioreactor, 207, 207f, 225 Akt, in regulation of glucose metabolism, 77, 77f Alanine in medium, 108 metabolism of, 81–82 Albumin production of, 13 recombinant, 122 secretion of, 31 serum, in medium, 118, 122

Alginate microcarriers, 205t Allosteric regulation, differential, of glucose metabolism, 75–76, 75f Alternating tangential filtration, 260, 260f Alternative splicing, 285–286 Amino acid(s) alterations, in bioprocessing, 16–17, 16t analysis of, 299–302 in cellular growth, 150, 151, 151t degradation of, 81–82 essential, 81, 108, 108t, 151 in fedbatch culture, 238–240, 239f, 239t, 246–247 in intracellular fluid, 153, 153t in medium, 81, 106–108 metabolism of, 58, 81–82, 81f, 82f, 186 non-essential, 81, 108, 108t, 151 stock solutions of, 108 transport of, 40–41, 43, 81 Aminophospholipid translocases, 84 Ammonia, in cultured cell metabolism, 67, 236 Amphipathic lipids, 22–23 Amplification localized, in DNA sequencing, 304, 304f for stable cell lines, 131–132, 138–141, 140f Amplification maker, 135 Anaerobic glucose metabolism, 58, 65 Aneuploid cells, 54, 133, 143, 150 Animal(s) as production vehicles, 199 transgenic, 12, 14, 14t vaccines for, 8t, 9 Animal component-free serum, 102–103 Animal serum, in medium, 102, 117–119 Anterograde transport, 34, 35, 35f Antibiotic(s) declining price of, 2–3 development of and advances in, 2–4 in medium, 117, 117t as selection markers, 137t Antibody dependent cellular cytotoxicity (ADCC), 90 Antibody products, 5–8, 7t manufacturing of, 11–12 quantity for administration, 11, 11t stoichiometric ratio in, 11 Antibody synthesis, analysis of, 186 Antioxidants, in medium, 116 Antiporters, 41, 42f Apf-1, 52 Apoptosis, 51–53 cell cycle and, 50f death receptor pathway in, 51–52 mitochondrial pathway of, 52–53, 53f morphological changes in, 51 vs. necrosis, 51 Apoptosome, 52 Ascorbic acid (vitamin C), in medium, 109

INDEX | 309

Asparagine in medium, 108 metabolism of, 81 Aspartate, metabolism of, 81–82 ATP. See Adenosine triphosphate ATP-binding cassette (ABC) transporter, 43, 44f, 84 Attenuated vaccines, 4–5 Autocatalytic growth, 156 Automation, for cell line production, 145–146, 145f Axial flow impellers, 206, 265–266, 265t, 266f Azacytidine, 293 B Baby hamster kidney cells. See BHK cells Bacterial genome, 287–288, 289t, 290 Bag-based centrifuge, 256 Bag culture systems, 201–202, 202f Bak protein, 52 Balance equations, 162–165, 175–180. See also Material balance Basal medium, 101 components of, 106–117 optimal concentration of organic nutrients in, 110, 110f Base addition, proportional feeding with, 244 Base-by-base synthesis, 304–305, 304f Batch culture (processes), 233. See also Fedbatch culture in bioreactors, 196–198, 197f vs. continuous processes, 249–251 Bax protein, 52 B cells, transition to plasma cells, 33, 33f, 130 Bcl-2 proteins, 52 Bcl-xL protein, 52 Beef extract, in medium, 124 Beta-carotene, in medium, 116 Beta cells, pancreatic, 31 BHK cells, 8t, 19t adhesion of, 142 aggregates of, 209 as continuous cell line, 49 for transient expression, 129 veterinarian vaccines from, 8t Bicarbonate buffer, 113–114, 115f, 115t Biogen, IDEC ISM facility, 12 Biological fluids, in medium, 118 Biologics, 127. See also specific types Biomass, 147–148, 148t growth of. See also Growth material balance on, 149–154, 149f as objective of medium, 97–99, 125 overall synthesis equation for, 149–150 intracellular fluid in, 153–154 medium to generate, 103–104 metabolic flux analysis of, 184–185, 185f as output of reaction, 176 on substrate, yield of, 158 Bioreactors, 191–212. See also specific types agitation in, 265–266

impellers for, 265–268, 265t, 266f mechanism of, 265–266 purpose of, 265 basic types of, 193–196 batch processes in, 196–198, 197f cell culture, 206–212 cell retention in, 249–261. See also Perfusion culture cell support systems in, 202–206 continuous processes in, 196–197 disposable or single-use systems in, 199–202 fedbatch cultures in, 198, 198f, 212 mixing characteristics of, 193, 266–270 mixing time in, 206, 268–274 operating mode of, 196–198 oxygen balance in, 220, 276–280 oxygen supply for, 220–228 by medium recirculation, 221f by silicon tubing/membrane, 220 by sparging, 220–228 by surface aeration, 220 oxygen transfer in, 206–207, 213–231 driving force for, 214–216, 220, 279–280 enhancing or improving, 216, 220, 221 experimental measurement of, 224–225, 224f, 225f hydrostatic pressure and, 225 in immobilization reactor, 229, 229f mass transfer coefficient in, 216 objective of, 219 in plug-flow reactors, 229–230, 230f rate of, 215–216, 220 scaling up and, 275–280, 280f, 280t surface/interfacial area and, 216 through gas-liquid interface, 212–219, 213f, 214f phases in, 196 power consumption in, 266–270, 266f reactions and reaction kinetics in, 194–196 scale translation for, 263–284 and carbon dioxide removal, 275, 281–283 and chemical environment, 283, 283f constant parameters in, 269–270, 269t dimensionless variables in, 267–270 and driving force, 279–280 effect of scale on physical behavior, 269–270 geometric nonsimilarity in, 264 geometric similarity in, 264 major effects of scale, 264 and mechanical forces on cells, 274–275, 275f mixing time in, 271–274, 271t nutrient starvation time in, 271–274, 271t objective of, 264 and oxygen transfer, 275–280, 280f, 280t physical and mechanical parameters of, 264 Reynolds number in, 267–268, 267f and superficial gas velocity, 278, 278f tracer concentration response in, 193–195, 194f velocity of, 268, 278, 278f

INDEX | 310

volumetric flow rate in, 268–270 Biosimilars, 3, 9–10 approval in Europe, 10, 10t marketed in China, 11t marketed in India, 10t uncertainty about quality of, 10 Biotin, in medium, 109 BiP chaperone protein, 33, 35 Bispecies-transporters, 41–42 Blasticidin S, as selection marker, 137t Blood bags, 201–202 Bok protein, 52 Bristol Myer Squibb, Devens (Massachusetts) facility, 12 Bubble size, in gas sparging, 222–223 Buffer systems, in medium, 113–115, 115f, 115t Bulk ions, in medium, 111–112, 111t C Calcium intracellular and extracellular concentrations of, 100, 100t in medium, 111, 111t Calcium phosphate precipitation, 133–134, 134t Calculated differentiated data, 169 Calculated integrated data, 169 Carbohydrates, as cellular component, 21, 148 Carbon in amino acid metabolism, 81–82 in cellular growth, 149–150 flow or flux of, 67 in glucose metabolism, 59, 61–67 in glutamine metabolism, 59 metabolic flux analysis of, 182f, 183 in pentose phosphate pathway, 64–65 Carbon dioxide in buffer system, 113–114 cellular tolerance of, 281 concentration in medium, 217–218 in glucose metabolism, 60, 61, 66 production of, 219 removal in bioreactors, 219 scaling up and, 275, 281–283 transfer (diffusion) of, 212–219 g-Carboxylation, 12, 14, 16 Carrier-mediated diffusion, 40–42, 41f Carrier proteins in medium, 117, 118, 122 transport by, 122, 122t Caspases, in apoptosis, 51–53 Catabolism of glucose, 60 of lipids, 36 in metabolic flux analysis, 188t Catalase, in medium, 116 Catalytic macromolecular components, of medium, 105–106 CDK4/6-cyclin D complex, 48–49 CDK inhibitors (CDI), 48–49

cDNA microarrays, 295, 295f, 298t Cell(s) chemical environment of, 21–22, 21t, 97–101 composition of, 21–22, 21t, 148, 148t in culture. See Cultured cells death of, 51–53 apoptosis vs. necrosis, 51 cell cycle and, 50f consideration in growth rate, 157 death receptor pathway in, 51–52 from injury (necrosis), 51 mitochondrial pathway of, 52–53, 53f morphological changes in, 51 diameter of, 21 mass of. See Biomass movement of, 46–47 nutritional requirements of, 97–99. See also Medium senescence of, 53–55 size of, 21, 148, 148t sources of, 19–21, 19t volume of, variation in, 150–151, 150t, 151f Cell adhesion, 45–46, 53, 122–123, 142 microcarriers for, 203–204, 204t, 205t vs. suspension, 202 Cell adhesion molecules, in medium, 122–123, 123t Cell aggregates, 206, 206f, 209, 209f, 212 CellCube Module, 201 Cell culture engineering. See also specific processes and materials advances and growth in, 1–4 process robustness in, 14–17 Cell culture products, 4–12. See also specific products alternative technologies for, 12–14 biosimilar or follow-up biologics, 3, 9–10, 10t, 11t in-process structural alterations to, 15–17, 16t manufacturing of, 11–12 from perfusion bioreactors, 249t quality of, 14–17 Cell cycle, 47–49, 48f and apoptosis, 50f checkpoints in, 47–49 cyclins and CDKs in, 48–49 positive and negative cues in, 47–48 Cell expansion. See also Growth; Growth control medium for, 97–99, 125 Cell lines adaptation of cells in, 131, 142, 142f, 143f amplification for, 131–132, 138–141, 140f, 141f automation and high throughput technology for, 145–146, 145f basic steps for generating, 131–133, 132f continuous, 49, 143–144 development of, 127–146 gene expression in, 306–308, 306f, 307f genomics and, 146, 306–308, 306f, 307f host cells for, 127–129

INDEX | 311

hyper-producing, 129–133, 130t, 131f immortalization of, 54 industrial, 8t, 9 screening for, 131 selection of cells for, 131–132 single-cell cloning for, 131–133 sources of, 19–20, 19t stability of clones selected for, 143–145 stability of product quality in, 144–145 stable, 127, 129–146 transfection for, 131–137 veterinary, 8t, 9 vs. cell strains, 53f, 54 Cell membrane, 22–27 composition of, 22–25 dynamic nature of, 23, 26 homeostasis of, 26 lipid bilayer of, 22–27, 83, 87 potentials across, 25–26, 25t protein content of, 25 transport across, 23, 26–27, 39–47 turnover rate of, 26 Cell pool, 132–133 Cell recycling, 250–251 Cell retention methods of, 249–261 in perfusion culture, 249–261 Cell separation methods, 254–261 Cell signaling, 45–46 Cell strain, 53f, 54 Cell support systems, 202–206 Cellulose, for microcarriers, 203–204, 205t Centrifugal cage, 259, 259f Centrifugation, 256, 256f, 257f Centritech Lab centrifuge, 256 Ceruloplasmin, in medium, 116 Channel-mediated diffusion, 40–42 Chaperone proteins, 33, 35, 90 Checkpoints, in cell cycle, 47–49 Chemical environment of bioreactors, scale translation and, 283, 283f of cells, 21–22, 21t, 97–101 Chemically defined medium, 102, 103 Chemical reaction systems cellular system vs., 175–176 material balance for, 175–180 Chick egg, 4, 9, 19 China, biosimilars marketed in, 11t Chinese hamster ovary (CHO) cells, 8t, 9, 19t, 20 adhesion of, 142 aggregates of, 209, 209f as continuous cell line, 49 doubling time of, 154t genome of, 288, 289t on microcarrier, 203f, 204 non-antibody products from, 6t

recombinant proteins from, 8t for stable expression, 129–130, 130t, 146 therapeutic antibody products from, 7t for transient expression, 129 volume of, 150t Chloride ions intracellular and extracellular concentrations of, 100, 100t in medium, 104 transport of, 43–45 Chlortetracycline, in medium, 117 CHO. See Chinese hamster ovary cells Cholesterol biosynthesis of, 86–88, 88f function of, 83, 86–87 in lipid bilayer, 24–25, 24f, 83, 87 structure of, 85, 85f turnover rate of, 26 Choline, as backbone of phospholipid, 22 Chondroitin sulfate, in extracellular matrix, 46 Chromatin DNA packaging into, 290, 290f modifications of, 292–293, 292t Chromium, in medium, 112 Chromosome(s) abnormalities, in cell line, 143–144 in cultured cells, 54–55 in mammalian genome, 288 Cis Golgi, 32, 33 Cisternae maturation model, 33 Citric acid cycle. See Tricarboxylic acid cycle City water, contaminants in, 106 Clonal growth, 110 Clones in Sanger sequencing, 302–303 selected for cell lines, 131–132, 143–145 Cloning, single-cell, 131–133 CMV promoter, 135 cMyc, in regulation of glucose metabolism, 77, 77f Cobalamin, in medium, 109 Cobalt in intracellular fluid, 153 in medium, 112 Codon optimiziation, 135 Collagen in extracellular matrix, 45–46 in medium, 123t for microcarriers, 204, 204f, 205t Combustion, 61–63, 176–178 Compartmentalization, 184 Complex glycans, 92, 92f Complex medium, 102 Complex supplements, in medium, 117–125 Concentration factor, in perfusion culture, 254 Concentration gradient across cell membrane, 21–22, 21t, 40–42, 43–45 across mitochondria, 29–30, 30f

INDEX | 312

Conditional promoters, 134–135 Conical settler, 254–255, 254f Constitutive promoters, 134–135 Contact inhibition, 46, 48, 53, 54f Continuous cell lines, 49 Continuous processes, 249–251 advantages of, 250 in bioreactors, 196–197 with cell retention, 251. See also Perfusion culture disadvantages of, 250 economics of, 250 flow rate vs. growth rate in, 250 Continuous stirred tank reactor (CSTR), 193–196, 193f, 212 reaction and reaction kinetics in, 194–196 tracer concentration response in, 193–195, 194f Copper in intracellular fluid, 153 in medium, 112 COS cells, for transient expression, 129 Co-transporters, 41–42, 42f Cow pox vaccine, 4, 199 CpG islands, 292 CRFK cells, 8t Crisis, 54 Cross filtration model, 259, 259f CSTR. See Continuous stirred tank reactor Cultured cells crisis of, 54–55 epigenetic changes in, 291–293 growth of. See Growth Hayflick’s phenomenon and, 54–55 life span of, 54–55 medium for. See Medium metabolism of. See also Metabolism glucose, 58–70 overview of, 58–59 passages of, 53–55 stoichiometry and kinetics of, 147–166 Cumulative data, 171 Cyclins, 48–49 Cylinders on shakers, 201–202 Cytidine, in medium, 109t Cytochrome C in apoptosis, 52 in electron transfer chain, 63 Cytokine(s) in medium, 97–98 quantity for administration, 11 Cytoplasm, 27–36 Cytoskeleton, 27, 37–39 Cytosol, 27–29 acetyl CoA shuttle in, 86 lipid metabolism in, 83, 85, 86, 87 D Data, types of, 169

Data analysis, 167–174 Data processing, 167–174 cell culture, 169–171 fedbatch culture, 160, 160f mapping data to pathways, 173, 173f. See also Metabolic flux analysis pipeline for, 168–173 spreadsheets for, 168, 170–171 standardized templates for, 168–169 Data visualization, 172, 172f, 174f Death, cellular death of, 51–53 apoptosis vs. necrosis, 51 cell cycle and, 50f consideration in growth rate, 157 death receptor pathway in, 51–52 from injury (necrosis), 51 mitochondrial pathway of, 52–53, 53f morphological changes in, 51 Death phase, of cell growth, 154–155, 154f Death rate, specific, 157 Death receptor pathway, 51–52 Decline phase, of cell growth, 154–155, 154f Delivery of feed medium, 245–247 Deoxyribonucleic acid. See DNA Derived parameters, 160, 160f Dextran in medium, 116t for microcarriers, 203–204, 205t DH82 cells, 8t DHFR amplification system, 138–141, 140f Differential allosteric regulation, of glucose metabolism, 75–76, 75f Differentiated data, calculated, 169 Diffusion in bioreactors, 212–219, 213f, 214f carrier-mediated, 40–42, 41f channel-mediated, 40–42 facilitated, 40–42 Dihydroxyacetone-phosphate (DHAP), 61 Dilution rate, in perfusion culture, 254, 254f Dimensionless variables, in scale translation, 267–270 Diploid cells, 53–55, 143, 150 Diploid chromosomes, 290 Direct DNA sequencing, 295 Direct measurement, for fedbatch culture, 243 Disc centrifuge, 256, 256f Disposable bag-based centrifuge, 256 Disposable cell culture systems, 199–202 Dissociation, of cells from surface, 53–54, 54f Disulfide-bond formation, in protein therapeutics, 5, 14 DNA as cellular component, 21 location in cell, 28 methylation of, 292–293 mitochondrial, 29–30

INDEX | 313

modifications of, 291–293 packaging into chromatin, 290, 290f synthesis of, 28, 47 DNA–calcium phosphate Co–precipitation, 133–134, 134t DNA microarrays, 294–297, 298t cDNA, 295, 295f, 298t layer-by-layer synthesis in, 295, 295t, 296f oligonucleotide-based, 295–296, 298t for pathway-related data, 173 RNA-seq, 296–297, 298t for two-channel comparison, 296 DNA sequencing, 288–289, 302–305 in cell culture processing, 306–308, 306f direct, 295 evolution of technologies for, 302–303 next generation technologies for, 304–305 Sanger method of, 302–303, 302t, 303f Dog (MDCK) cells, 8t, 9, 19–20, 19t Dominant marker, 136 Doubling time, 154–157, 154t Driving force, for oxygen transfer, 214–216, 220 scaling up and, 277, 279–280 Dry mass, of cells, 148, 148t E E. coli. See Escherichia coli E2F transcription factor, 49 ECM. See Extracellular matrix Eddies, in bioreactors, 274–275, 275f EF-1. See Elongation factor 1 Electron transfer chain, 61, 63–64, 66 Electroporation, 133–134, 134t Electrospray ionization (ESI), 299 Elongation factor 1 (EF-1), 135 Endocytosis, 26–27, 36 Endoplasmic reticulum, 27, 31 expansion of, 33 glycan biosynthesis in, 93 glycosylation in, 90 lipid metabolism in, 83, 88f protein secretion through, 31–35, 35f, 83 rough, 31 smooth, 31 Endosomes, 27 Endothelial cells, volume of, 150t Enzyme(s) in glucose metabolism regulation, 74–76, 75f Michaelis–Menton kinetics of, 41 Epigenetics in cell culture, 291–293 definition of, 291 inhibition of, 293 mechanisms of, 292–293 molecular mechanisms mediating, 291–293 Epigenome, 291–293 Epigenomics, definition of, 291

Epithelial cells movement of, 46–47 use in bioprocessing, 19–20, 19t EPO. See Erythropoietin ER. See Endoplasmic reticulum Erythropoietin (EPO) development of, 5 glycan and, 89 product quality and process robustness, 15 quantity for administration, 11 Escherichia coli genome of, 28, 290 as host system, 12 in Sanger sequencing, 302–303 ESI. See Electrospray ionization Essential amino acids, 81, 108, 108t ESTs. See Expressed sequence tags Ethanol, transport of, 39 Euchromatin, 290 Eukaryotes chromosomes of, 290, 290f gene structure in, 286 genome of, 287–288, 289t European Union, biosimilar approval in, 10, 10t Ex-Cyte, 124 Exocytosis, 39–40 Exons, 286 Exponential phase, of cell growth, 151f, 154–155, 154f Expressed sequence tags (ESTs), 294 Extended fedbatch culture, 235, 235f Extracellular fluid, 100–101, 100t Extracellular ion concentration, 21–22, 21t, 100–101, 100t Extracellular matrix (ECM), 45–46 composition of, 45–46 functions of, 45–46 Extracellular matrix extract, in medium, 123 F Facilitated diffusion, 40–42 F actin, 39 Factor VIII in-process structural alterations to, 16 recombinant, 5 tissue-derived, 5 FADD. See Fas-associated death domain FADH2, from glucose metabolism, 63, 68 FANTOM. See Functional Annotation of the Mammalian genome Fas-associated death domain (FADD), 51–52 Fatty acids in lipid bilayer, 23–24 in medium, 84 metabolism of, 85–86 oxidation of, 36 saturated, 85 transport of, 39, 84, 122 FBS. See Fetal bovine serum

INDEX | 314

Fedbatch culture, 233–247 in bioreactors, 198, 198f, 212 control objective and criteria in, 241–243 control strategies for, 241–243 data processing and plotting of, 160, 160f delivery of feed medium in, 245–247 feeding parameters in, 245 feeding strategy for, 241–245 direct measurement of nutrient consumption, 243 proportional with base addition, 244 proportional with oxygen uptake rate, 245 proportional with turbidity, 244 fortified feed and addition, 235, 235f habitation-conducive components of, 104 for industrial production, 125 intermittent harvest and feed, 198, 198f, 234, 234f lactate and production in, 59 medium for, 233, 237–240 for consumed nutrients, 238–240, 239f, 239t for unconsumed components, 240 with metabolic state manipulation, 236–237, 236f, 236t metabolic stress in, 161, 161t on-line estimation of stoichiometric feeding in, 246–247 productivity and product quality of, 241–243 for stable cell lines, 130 stoichiometric ratio in, 160f, 236–240, 236f, 236t, 239f, 239t, 246–247 types of, 234–237 Fermentation technology, 1 Fermentors, 206–207 large-scale, 264f Ferrous ion, in medium, 111 Fetal bovine serum (FBS), 118 Fetuin, in medium, 123t Fibroblast(s) doubling time of, 154, 154t human vs. chicken embryo, 19 life span in culture, 54 on microcarrier, 203f use in bioprocessing, 19–20, 19t vs. epithelial cells, 20 Fibroblast growth factor, 48 Fibronectin in extracellular matrix, 45 in medium, 123, 123t Filopodia, 38–39, 46 Filtration, 259–260 FL72 cells, 8t Fleming, Sir Alexander, 2 Flippases, 84, 90 Flooding, in bioreactors, 280 Fluid extracellular, 100–101, 100t intracellular, 153–154, 153t Fluidized bed bioreactor, 207 Flux vectors, 179–180

Folding, of proteins, 32–33, 89–91 Follow-up biologics, 3, 9–10 approval in Europe, 10, 10t uncertainty about quality of, 10 Formalin, viral inactivation with, 4, 9 Fortified feed and addition culture, 235, 235f 454 sequencing technology, 304–305 Friction factor, in scale translation, 267 Fructose in glycan biosynthesis, 93–94 in medium, 108 transport of, 41, 71 Fructose 1,6-bisphosphate (F16BP), 75, 75f Fructose 2,6-bisphosphate (F26BP), 77 Fructose-6-phosphate, 67 FS-4 cells, 19t Functional Annotation of the Mammalian genome (FANTOM), 286–287 Fungus, genome of, 288 Fusion proteins, 8 G G1 phase of cell cycle, 47–48, 48f G2 phase of cell cycle, 47–48, 48f G actin, 39 Galactose in glycan biosynthesis, 93–94 in medium, 108 transport of, 71 Gangliosides, in lipid bilayer, 22, 24 GAPDH. See Glyceraldehyde dehydrogenase Gap phase of cell cycle, 47–48, 48f Gas-liquid interface, oxygen transfer through, 212–219, 213f, 214f Gas phase, in bioreactor, 196, 276, 278–279 Gas sparging, 220–229 damage to cells by, 226–228, 226f, 227f, 228f direct, 221 orifice and bubble size in, 222–223 Gelatin, for microcarriers, 203–204, 205t Gene(s), 285–287 abundant, 293, 293t alternative splicing of, 285–286 coding for non-protein RNA, 285–286 coding for proteins, 285–286, 288 coding for RNA, 285–286 definition of, 285 environment and, 291–293 minimum set of, 287 number of, 287, 289t rare, 293–294, 293t structure in eukaryotes, 286 Gene expression, 285 in cell culture processing, 306–308, 306f, 307f epigenetic regulation of, 291–293 proteome profiling of, 299–302, 299f

INDEX | 315

transciptome analysis of, 293–297 Genentech, Vacaville (California) facility, 12 Geneticin, as selection marker, 137t Gene transfer amplification in, 131–132, 138–141, 140f, 141f cell adaptation to, 142, 142f, 143f direction to transcriptionally active region, 141 methods of, 133–134, 134t promoters for, 134–135 selectable marker in, 135–137, 137t for stable cell line, 131–145 for transient expression, 128–129 Genome(s) and complexity of organism, 287 environment and, 291–293 eukaryotic, 28 mammalian, 287–289 mitochondrial, 29–30 mouse, 285–287, 288, 289t, 293 organization of, 287–289, 287f prokaryotic, 28, 287–288 repetitive sequences in, 287–288 species comparison of, 287–288, 289t Genome engineering, 3 Genome scale analysis, 293–297 Genomics, 3, 285–308 cell line, 146, 306–308, 306f, 307f protein (proteome profiling), 299–302 transcriptome, 293–297 Gentamicin, in medium, 117, 117t Geometric-nonsimilar scale translation, 264 Geometric-similar scale translation, 264 Germanium, in medium, 112 Glass, as microcarrier, 203–204, 205t Gluconeogenesis, 67 Glucosaminoglycans, in extracellular matrix, 45–46 Glucose in cellular growth, 149–150 in fedbatch culture, 236–237, 238–240, 239f, 246–247 lactate ratio to, 158–159, 236–237, 236f, 236t in medium, 106–107, 149 metabolism of. See Glucose metabolism specific consumption rate of, 156 transport of, 39–43, 42f, 71–72, 71f, 72t Glucose-6-phosphate, 64, 67 Glucose metabolism, 58–77, 62f, 70f aerobic, 65–66 amino acid metabolism and, 81–82, 82f anaerobic, 58, 65 ATP consumption in, 60–61 ATP production in, 60–64, 66, 70 carbon flow or flux in, 67 carbon production in, 59, 61–67 in culture, 149 in electron transfer chain, 61, 63–64, 66 glutamine and, 80, 80f

insulin in, 120 lactate consumption in, 59, 78–80, 79f lactate conversion in, 58–59, 60, 65–66, 67 lipid metabolism and, 86 metabolic flux analysis of, 186 NADH balance in, 68–69, 69f reaction intermediates in, 67 regulation of, 74–78 differential allosteric, 75–76, 75f growth control and, 77, 77f isozymes in, 74–76, 75f signaling pathways and, 77, 77f transport in, 71–74 Warburg effect in, 65–66 yield of, 60–61, 70 Glucose oxidation, 58–64, 70f Glutamate, metabolism of, 81–82 Glutamine in cellular growth, 150 in medium, 106–108, 149 metabolism of, 58, 59, 67, 81–82 in regulation of glucose metabolism, 80, 80f Glutamine oxidation, 58 Glutamine synthetase (GS) for amplification, 138–141, 141f for glutamine synthesis, 108 Glutathione reduced, in medium, 116 reduction of, 64 GLUT transporter(s), 71, 72t GLUT1 transporter, 40–41, 43, 71, 72t, 107 GLUT2 transporter, 72t GLUT3 transporter, 72t GLUT4 transporter, 43, 71, 72t, 77 GLUT5 transporter, 41, 71, 72t, 107 GLUT6 transporter, 72t GLUT7 transporter, 72t GLUT8 transporter, 72t GLUT9 transporter, 72t GLUT10 transporter, 72t GLUT11 transporter, 72t GLUT12 transporter, 72t Glycan(s) biosynthesis of, 67, 89–96 diversity among species, 95–96 effect/importance of, 89–90 extension in Golgi apparatus, 91–92, 91f, 93–94 and immunogenicity, 95–96 macroheterogeneity of, 89 microheterogeneity of, 89, 92–93, 92f N-linked, 89–92, 90f, 91f, 174, 174f nucleotide sugar precursors of, 93–94, 93f, 94f O-linked, 89 types of, 92–93, 92f visualization of data on, 174, 174f Glyceraldehyde dehydrogenase (GAPDH), 135

INDEX | 316

Glycerol, as backbone of phospholipid, 22, 22f, 24 Glycine as buffer in medium, 115t in medium, 108 Glycoforms, heterogeneity in, 89 Glycolipids, in lipid bilayer, 22, 24 Glycolysis, 58–70 aerobic, 65–66 carbon flow and reaction intermediates in, 67 in culture, 149 lactate consumption in, 78–80 metabolic flux analysis of, 186 pentose phosphate pathway as shunt from, 60, 64–65 regulation of, 74–78 transport and, 71–74 yield of, 60–61, 70 Glycosylation, 5, 12–14, 89–96, 90f diversity among species, 95–96 multiple sites of, 89 N-linked, 89–92, 90f, 91f O-linked, 89, 90 in secretion process, 33 visualization of data on, 174, 174f Glycosyltransferases, 34 Glycylglycine, as buffer in medium, 115t Glyeraldehyde-3-phosphate (G3P), 61 Golgi apparatus, 27, 32–34 classical view of, 32 compartments of, 32 dynamic nature of, 32 glycan extension in, 91–92, 91f, 93–94 glycosylation in, 90 protein secretion through, 32–35, 35f, 83 transport across, 33–34 Green monkey kidney cells, 8t, 9, 19t, 129, 201 Growth autocatalytic, 156 balance equations for, 162–165 cell death consideration in, 157 doubling time of, 154–157, 154t kinetic model of, 162–165 mammalian cell, 154–155, 154f, 154t material balance on, 149–154, 149f, 162–165 medium for. See Medium multiplicative model of, 165 overall synthesis equation for, 149–150 phases of, 151f, 154–155, 154f quantitative description of, 156–161 Growth control apoptosis in, 51–53 cell cycle and, 47–49 contact inhibition in, 46, 48, 53, 54f Hayflick’s phenomenon and, 54 loss, in continuous cell lines, 49 in regulation of glucose metabolism, 77, 77f telomeres and, 54–55, 54f

Growth curve, 154–155, 154f Growth factors in cell cycle, 48, 49 in cell migration, 46–47 in medium, 97–98, 117, 118 Growth hormone. See Human growth hormone Growth rate, 53–55, 53f, 156–157 GS. See Glutamine synthetase GTP-mannose, 67 Guanosine, in medium, 109t H Habitation-conducive components, of medium, 103–104 Hamster cells, 8t, 9, 19t, 20. See also BHK cells; Chinese hamster ovary cells Hayflick’s phenomenon, 54 Heavy metal ions, in medium, 112 HEK 293 cells, 8t, 19t adhesion of, 142 aggregates of, 209, 209f for transient expression, 129 Helicos sequencing technology, 302t Henry’s law, 217–218 Heparin in extracellular matrix, 46 in medium, 123 Hepatocyte(s) cell membrane of, 25, 25t endoplasmic reticulum of, 31 size of, 21, 151 Hepatocyte growth factor, 46–47 HEPES buffer, 115, 115t HepG2 cells, on microcarrier, 204, 204f Heterochromatin, 290 Heterogeneous bioreactor, 196, 196f Hexokinase (HK), in regulation of glucose metabolism, 75 hGH. See Human growth hormone High-mannose glycans, 92, 92f High-molecular-weight supplements, in medium, 117–125 High-performance liquid chromatography (HPLC), for fed-batch culture, 243 High throughput technology for cell line production, 145–146 for DNA microarrays, 296–297 for DNA sequencing, 302–305 for proteome profiling, 299–302, 299f Histone, 28, 290, 290f Histone modification, 292–293 HMG-CoA, 87–88, 88f HMG-CoA reductase (HMGCR), 87–88, 88f HMG-CoA synthase (HMGCS), 87–88, 88f Holding time, in secretion, 32, 34 Hollow fiber bioreactor, 208, 208f, 229 Homeostasis, of cell membranes, 26 Hormone(s) in interstitial fluid, 100

INDEX | 317

in serum, 118 Host systems, 5–6, 12–14, 127–129. See also specific systems Hot spot integration, 141 HPLC, for fedbatch culture, 243 Human growth hormone (hGH) biosimilar, 9–10 quantity for administration, 11 Humira, expiration of patent, 9 Hyaluronic acid, in extracellular matrix, 46 Hybrid glycans, 92 Hybridoma, 5 adhesion of, 142 gene expression in, 306, 306f growth of, 149 stoichiometric ratio and metabolic stress in, 161, 161t therapeutic antibody products from, 7t volume of, 150t Hydrogen in cellular growth, 149–150 transport of, 43–45 Hydrogen peroxide, 116 Hydrolysates, in medium, 124, 240 Hydrostatic pressure, and oxygen transfer, 225 Hydroxylethyl starch (HES), in medium, 116t Hygromycin B, as selection marker, 137t Hyper-producing cell line, 129–133. See also Stable cell lines basic steps for generating, 131–133, 132f characteristics of, 129–131, 130t, 131f gene expression in, 306–308 I IEF. See Isoelectric focusing (IEF) IGF. See Insulin-like growth factor(s) IgG. See Immunoglobuin G Illumina sequencing technology, 302t, 304–305 Immobilization reactors agarose, 209 oxygen transfer in, 229, 229f Immortalization, 54 Immunogenicity, glycans and, 95–96 Immunoglobuin G (IgG) in cellular growth, 151, 151t Fc fragment, in fusion protein, 8 secretion time of, 34f, 34t Impellers, in bioreactors, 206, 265–268, 265t, 266f amount of fluid moved by (pumping), 268–270 constant tip speed, for scale translation, 269–270, 269t velocity of, 268 Inactivated vaccines, 4, 9 Incline settling, 255, 255f India, biosimilars marketed in, 10t Inducible promoters, 134 Industrial cell lines, 8t, 9 Industrial production bioreactors for, 192–193. See also Bioreactors medium for, 125–126

scale translation for, 263–284. See also Scale translation; Scaling up In-process calculations, 169 Insect cell culture, 12–13, 13t Insulin in cell cycle, 48, 49 in glucose metabolism, 120 in glucose transport, 43, 71 in medium, 120–121, 240 mitogenic response to, 120 recombinant, 121 as tissue-derived protein therapeutic, 5 Insulin-like growth factor(s), 48 Insulin-like growth factor-1, in medium, 120–121 Insulin-like growth factor-2, in medium, 120 Integral cell concentration, 159, 159f Integrated data, calculated, 169 Interactive data exploration, 172 Interferon(s), as tissue-derived protein therapeutic, 5 Intermediate(s), of reaction modes, abbreviation of, 189t Intermediate filaments, 37, 38, 38f Intermittent harvest and feed, 198, 198f, 234, 234f Interstitial fluid, 100–101, 100t In-time measurement, for fedbatch culture, 243 Intracellular fluid, 153–154, 153t Introns, 286, 287, 288 Ion(s) bulk, in medium, 111–112, 111t intracellular vs. extracellular concentration of, 21–22, 21t, 100–101, 100t transport of, 43–45 Ion channels, as transport mechanism, 40–42, 41f Iron free vs. bound form of, 45 in intracellular fluid, 153–154 in medium, 111, 112 reactivity of, 45 transport of, 45, 122t. See also Transferrin Iron chelators, 121, 121t Isobaric Tagging for Relative and Absolute Protein Quantitation (iTRAQ), 300–301, 300f, 301f Isoelectric focusing (IEF), 299 Isoleucine, metabolism of, 81–82 Isozymes, in glucose metabolism regulation, 74–76, 75f iTRAQ, 300–301, 300f, 301f J Jenner, Edward, 199 K Karyotype, variable, in cell lines, 143–144 a-Ketoglutarate in amino acid metabolism, 81 in glucose metabolism, 61, 68, 80 in medium, 108 Kinetics

INDEX | 318

in bioreactors, 194–196 of cell cultivation, 147–166 model of growth, 162–165 KL. See Mass transfer coefficient Krebs cycle. See Tricarboxylic acid cycle L Lactate accumulation in culture, 78, 236 in fedbatch culture, 236–237, 246–247 metabolism of, 58–59, 65–66, 78–80 consumption in glucose metabolism, 59, 78–80, 79f correlation with productivity, 58f, 59 production in glucose metabolism, 58–59, 60, 65–66, 67 specific production rate of, 156 transport of, 41–42, 42f, 43, 72–73, 72f Lactate dehydrogenase, 66, 67, 79 Lactate-to-glucose ratio, 158–159, 236–237, 236f, 236t Lag phase, of cell growth, 154, 154f Lamellipodia, 38–39, 46 Laminins in extracellular matrix, 45 in medium, 123, 123t Large scale, translation for. See Scale translation; Scaling up Large-scale fermenter, 264f LDL. See Low-density lipoprotein Lehmann, Jürgen, 210 Length, in scale translation, 264 Leucine, metabolism of, 81–82 Lipid(s) amphipathic, 22–23 catabolism of, 36 in cell membrane, 22–27 as cellular component, 21 functions of, 83 in medium, 84, 125, 240 metabolism of, 83–88 acetyl CoA shuttle in, 86, 86f subcellular localization of, 83 transport of, 84 Lipid bilayer, 22–27, 83, 87 characteristics of, 23 composition of, 22–25 dynamic nature of, 23, 26 function of, 25 permeability of, 23, 39 phase transition of, 23, 23f, 24 transport across, 23, 26–27, 39–47 turnover rate of, 26 Lipofection/lipid-mediated gene transfer, 133–134, 134t Lipoprotein(s) in medium, 105–106 transport of, 84 Liquid chromatography, 2D, 300–302 Liquid chromatography/mass spectroscopy, 300 Liquid phase, in bioreactor, 196, 277–279

Live attenuated vaccines, 4–5 Liver, endoplasmic reticulum of, 31 Localized amplification, in DNA sequencing, 304, 304f Logging data. See also Data processing standardized templates for, 168–169 Low-density lipoprotein (LDL) in medium, 105–106 transport of, 84 Lymphoid cells, use in bioprocessing, 19–20, 19t Lysis, 157 Lysosome, 36 M MA104 cells, 8t Macroheterogeneity, of glcyans, 89 Macromolecules catalytic, in medium, 105–106 transport of, 39–40 Macroporous microcarriers, 203, 204, 204f, 205t, 229 Madin-Darby bovine kidney (MDBK) cells, 8t Madin-Darby canine kidney (MDCK) cells, 8t, 9, 19t Magnesium ions in fedbatch culture, 240 intracellular and extracellular concentrations of, 21, 100, 100t, 153, 153t in medium, 111, 111t Malate-aspartate shuttle, 68–69, 186 MALDI. See Matrix-assisted laser desorption ionization Mammalian cells. See also specific cells bioreactors for, 192–193. See also Bioreactors critical feature of rDNA proteins from, 14 fragility of cells in, 22 genome of, 28 growth of, 154–155, 154f, 154t limitations of, 14 product quality and process robustness in, 14–17 products from, 5–6, 6t transgenic animals in, 12, 14, 14t Mammalian genome, 287–288, 289t Manganese, in medium, 112 Mannose, in glycan biosynthesis, 90–94 Manufacturing plants, 12 Manufacturing processes, 11–12, 12f. See also specific processes and products Mass. See Biomass Mass spectrometry, in proteome profiling, 299–302 Mass transfer coefficient (KL) for carbon dioxide, 282 for oxygen, 216 constant, in scale translation, 269–270, 269t experimental measurement of, 224–225, 224f scaling up and, 277 sparger orifice and bubble size and, 222–223 Material balance on cell growth, 149–154, 149f, 162–165 in fedbatch culture, 236–237, 236f, 236t, 238–240, 239f, 239t,

INDEX | 319

246–247 on oxygen, in bioreactors, 220, 276–280 on perfusion culture, 251–253, 252f, 253f for reaction systems, 175–180. See also Metabolic flux analysis setting up equations, 178–179 systematic way to solve problems, 178 Mathematical model of growth, 162–165 information needed for developing, 163 Monod and Monod derivatives, 163–165, 164f purposes of, 162 Matrigel, in medium, 123 Matrix-assisted laser desorption ionization (MALDI), 299 Matrix operations, 179–180 MDBK cells, 8t MDCK cells, 8t, 9, 19–20, 19t MDR. See Multidrug resistance gene Measurement data, 169 Mechanical/acoustic trapping, 257–258, 258f Mechanical damage protective agents, in medium, 116–117, 116t Medial Golgi, 32, 33, 35f Medium amino acids in, 81, 106–108, 238–240, 246–247 animal component-free, 102–103 antibiotics in, 117, 117t basal, 101, 106–117 buffer systems in, 113–115, 115f, 115t bulk ions in, 111–112, 111t carrier proteins in, 117, 118, 122 cell adhesion molecules in, 122–123, 123t for cell expansion, 97–99, 125 chemically defined, 102, 103 classical, composition of, 58 complex vs. chemically defined, 102 components of basic, 103–117 classes of, 103–106 non-nutritional, 113 stochiometric vs. catalytic macromolecular, 105–106 stochiometric vs. habitation-conducive, 103–104 design for, 97–126 for fedbatch culture, 233, 237–240 for consumed nutrients, 238–240, 239f, 239t for unconsumed components, 240 fundamental influence of, 97 high-molecular-weight and complex supplements in, 117–125 for industrial production, 125–126 insulin and insulin-like growth factors in, 120–121, 240 lipids in, 84, 125, 240 nucleosides in, 109 objectives of, 98 optimal concentration of organic nutrients in, 110, 110f optimization of cell growth environment in, 97–101 osmolarity of, 111–112

for production, 97–99, 125–126 protective agents in, 116–117, 116t protein-free, 103 protein hydrolysates in, 124, 240 serum albumin in, 118, 122 serum-free, 102–103, 108, 109 serum in, 102, 117–119, 240 for stem cells, 97–99, 117 sugars and energy source in, 106–107 tolerance of deviation from optimum, 100–101, 101t trace elements in, 111–112, 112t transferrin in, 105, 116, 118, 121, 240 types of, 101–103 vitamins in, 109, 240 water in, 106 Membrane, cell. See Cell membrane Membrane bioreactor, 208–209 Membrane fusion, 39–40 Membrane potentials, 25–26, 25t, 29–30, 30f, 43–45 Membrane stirred tank, 210, 210f Mercaptoethanol, in medium, 116 Messenger RNA (mRNA), 21, 286 abundant, intermediate, and rare, 293–294, 293t in RNA-seq, 296–297 Metabolic flux analysis (MFA), 173, 173f, 175–187 abbreviation of intermediates in, 189t biomass equations in, 184–185, 185f catabolism reactions for, 188t on cellular system, 181–186 compartmentalization in, 184 example of, 186 general approach to, 182f, 183–185 selecting reactions for, 183, 183f solution and analysis in, 185 utility and limitations of, 181–182 Metabolic state, of culture, 158, 236–237, 236f, 236t Metabolic stress, stoichiometric ratio as indicator of, 161, 161t Metabolism. See also specific types amino acid, 58, 81–82, 81f, 82f central, in cultured cells, 58–59 glucose, 58–70 lactate, 58–59, 65–66, 78–80 lipid, 83–88 transport and transporters in, 71–74 Methane combustion, 176–178 Methionine sulphoximine (MSX), in glutathione synthetase amplification, 138–141 Methotrexate (MTX), in DHFR amplification, 138–141, 140f Methylation DNA, 292–293 histone, 292 Methylcelluloses (MC), in medium, 116t MFA. See Metabolic flux analysis Micelles, 22–23 Michaelis–Menton enzyme kinetics, 41 Microarray analysis. See DNA microarrays

INDEX | 320

Microcarriers, 203–204, 205t characteristics of, 204t macroporous, 203, 204, 204f, 205t, 229 microporous, 203–204, 203f, 205t solid, 203–204, 203f Microencapsulation, 210, 210f Microfiltration, 259–260, 260f Microheterogeneity, of glcyans, 89, 92–93, 92f Microporous microcarriers, 203–204, 203f, 205t Microsphere-induced cell aggregates, 209, 209f Microtubules, 37, 37f, 46 Minerals, in medium, 111–112 Minimum set of genes, 287 Mitochondria, 29–30 acetyl CoA shuttle in, 86 as cell’s power plant, 29 genome of, 29–30 lipid metabolism in, 83, 85, 86, 88f membrane potential of, 29–30, 30f pH of, 29–30 protein content of, 25 size of, 29 transport across, 73–74 Mitochondrial apoptotic pathway, 52–53, 53f Mitochondrial DNA, 29–30 Mitogenic factors, 48, 49 Mitosis, 47–48, 48f Mixing time, in bioreactors, 206, 268–274 distribution of, 273–274, 273f, 274f measurement of, 272, 272f in scale translation, 269–274 vs. starvation time, 272 Molybdenum, in medium, 112 Monkey cells, 4, 8t, 9, 19t, 129, 201 Monocarboxylate transporter (MCT), 42, 43, 72–73, 72f Monod-derivative models, 164–165 Monod models, 164–165, 164f Monolayer, 53 MOPS buffer, 115t Mouse cells, 8t, 9, 19t, 49. See also NSO cells; SP2/0 cells embryonic stem, doubling time of, 154t gene expression in, 306, 306f Mouse genome, 285–287, 288, 289t, 293 Movement, cellular, 46–47 M phase of cell cycle, 47–48, 48f MRC-5 cells, 19t MRCS cells, 8t mRNA. See Messenger RNA MSX, in glutathione synthetase amplification, 138–141 MTX, in DHFR amplification, 138–141, 140f Multidimensional data exploration, 172 Multidrug resistance (MDR) gene, 137 Multiple membrane plate bioreactor, 208–209 Multiple plate system, 199, 201, 201f, 212 Multiplicative model, of growth, 165 Myc, in regulation of glucose metabolism, 77, 77f

Myelin membrane, protein content of, 25 Myeloma cells, 20. See also NSO cells as continuous cell line, 49 for stable expression, 129–130, 130t, 146 stoichiometric ratio and metabolic stress in, 161, 161t N NADH balance of, 68–69, 69f carrier system for, 68–69 in electron transfer chain, 63–64, 66 in glycolysis, 60–64, 66, 68–69, 69f in lactate metabolism, 79–80 in lipid metabolism, 86 in tricarboxylic acid cycle, 61–63, 68–69, 69f NADPH in lipid metabolism, 85 in pentose phosphate pathway, 64–65 Necrosis, 51 Neomycin in gene transfer, 139–140 in medium, 117 Next generation sequencing technologies, 304–305 Nickel, in medium, 112 NimbleGen microarrays, 295–296, 295t Nitrogen in cellular growth, 150 in cultured cell metabolism, 67 metabolic flux analysis of, 182f, 183 N-linked glycosylation, 89–92, 90f, 91f, 174, 174f Non-essential amino acids, 81, 108, 108t Non-nutritional components, of medium, 113 NSO cells, 8t, 19t, 20 as continuous cell line, 49 doubling time of, 154t gene expression in, 307f for stable expression, 130t therapeutic antibody products from, 7t Nuclear envelope, 29 Nuclear membrane, 29 Nuclear pores, 29 Nucleoid, 28 Nucleoside(s), in medium, 109 Nucleosomes, 290 Nucleotides, precursors of glycans, 93–94, 93f, 94f Nucleus, 27–29, 28f Nunc Cell Factories, 201 Nutrient consumption curve, 154–155 Nutrient consumption rate, specific, 156–157 Nutrients, transport of, 43 Nutrient starving time, 271–272, 271t Nystatin, in medium, 117t O OAA. See Oxaloacetate Off-line data, 160, 160f

INDEX | 321

Oligonucleotide-based microarrays (oligoDNA), 295–296, 298t Oligopeptides, transport of, 43 O-linked glycosylation, 89 Omnitrope, 9–10 On-line data, 160, 160f On-line measurement, for fedbatch culture, 243 Organelle(s), 23–24, 27–36, 28f. See also specific organelles Organic nutrients, in medium, optimal concentration of, 110, 110f Osmolarity of interstitial fluid, 100 of medium, 111–112, 125 OTR. See Oxygen transfer rate OUR. See Oxygen uptake rate Oxaloacetate (OAA), in glucose metabolism, 61, 68 Oxidative phosphorylation pathway, 61 Oxygen cellular demand for, 219 concentration in medium, 217–218 consumption of, 219 dissolved concentration of, 214 optimal concentration of, 219 transport of, 39 Oxygen balance, in reactor, 220, 276–280 on gas phase, 276, 278–279 on liquid phase, 277–279 Oxygen starvation time, 271–272, 271t Oxygen supply, for bioreactors, 220–228 by medium recirculation, 221f by silicon tubing/membrane, 220 by sparging, 220–228 damage to cells by, 226–228, 226f, 227f, 228f direct, 221 orifice and bubble size in, 222–223 by surface aeration, 220 Oxygen transfer, in bioreactors, 206–207, 213–231 driving force for, 214–216, 220, 277, 279–280 enhancing or improving, 216, 220, 221 experimental measurement of, 224–225, 224f, 225f hydrostatic pressure and, 225 in immobilization reactor, 229, 229f mass transfer coefficient in, 216 objective of, 219 in plug-flow reactors, 229–230, 230f rate of, 215–216, 220 scaling up and, 275–280, 280f, 280t surface/interfacial area and, 216 through gas-liquid interface, 212–219, 213f, 214f Oxygen transfer rate (OTR), 215–216, 220 balance with oxygen uptake rate, 220, 276–280 scaling up and, 276–280 Oxygen uptake rate (OUR), 160, 160f, 219, 220, 224–225, 225f balance with oxygen transfer rate, 220, 276–280 in fedbatch culture, 245–247 scaling up and, 276–280

P p53 tumor suppressor, in regulation of glucose metabolism, 77 Pancreas, beta cells of, 31 Passage, 53–55 PAT. See Process analytical technology Patents, expiration of, 9–10 Pathway-related data, 173, 173f. See also Metabolic flux analysis PCR. See Polymerase chain reaction PDI. See Protein disulfide isomerase PDQuest, 299 Penicillin(s) declining price of, 2–3 development and production of, 2–4, 2f discovery of, 2 Penicillin G in medium, 117t production outside U.S., 2–3 Pentose phosphate pathway (PPP), 60, 62f, 64–65 carbon flow or flux in, 67 in culture, 149 molecular transformation in, 64–65 oxidative segment of, 64 Peptides, in medium, 108, 123t Peptone, in medium, 124 Perfusion culture, 249–261 analysis of, 251–254 cell culture products from, 249t cell retention in, 251, 254–261 external vs. internal recovery device in, 252 material balance on, 251–253, 252f, 253f recycling factor in, 254, 254f Permeability, of lipid bilayer, 23, 39 Permeases, 39 Peroxisomes, 27, 36 lipid metabolism in, 83, 85, 87, 88f PFK. See Phosphofructokinase PFKFB. See Phosphofructokinase/fructose biphosphate PFR. See Plug-flow reactor pH of bioreactors, scale translation and, 283, 283f of fedbatch culture, 244 of medium, 113–115 of mitochondria, 29–30 Phenotype, epigentic changes in, 291–293 Phosphate in fedbatch culture, 240 intracellular and extracellular concentrations of, 100, 100t, 153, 153t in medium, 104, 111, 111t transport of, 43–45 Phosphatidyl choline, in medium, 125 Phosphatidyl ethanolamine, in medium, 125 Phosphofructokinase (PFK), in regulation of glucose metabolism, 75, 75f Phosphofructokinase/fructose biphosphate (PFKFB), in regulation of glucose metabolism, 75–76, 75f

INDEX | 322

Phospholipids in cell membrane, 22–27 turnover rate of, 26 types of, 22, 22f Phosphorylation of histones, 292–293, 292t in protein therapeutics, 14, 16 Photolithographic synthesis, in microarray analysis, 295, 295t, 296f Physiological state, of culture, 158 Pichia (yeast) cell culture, 12–13, 13t Pinocytosis, 39–40 Piston-flow reactor. See Plug-flow reactor PK. See Pyruvate kinase PK cells, 8t Plants, transgenic, 12 Plasma cells, 20, 33, 33f, 130, 151 Plasma membrane. See Cell membrane Plasmid(s). See also Gene transfer basic elements on, 134–137, 134f free, 135 method of delivery, 133–134, 134t selectable marker for, 135–137, 137t for stable cell line, 131–137 for transient expression, 128–129 Plastic, for microcarriers, 204, 205t Plug-flow reactor (PFR), 193–196, 193f oxygen transfer in, 229–230, 230f reaction and reaction kinetics in, 194–195 tracer concentration response in, 193–195, 194f Pluronic F68, in medium, 116–117, 116t, 123 Pluronic F77, in medium, 117 Pluronic F88, in medium, 116t, 117 PolyA tail, 286 Poly-d-lysine, in medium, 123t Polyethylene glycol (PEG), in medium, 116t Poly-L-lysine, in medium, 123 Polymerase chain reaction (PCR), 304 Polymyxin B, in medium, 117 Polypropylene microcarriers, 205t Polystyrene, for microcarriers, 203–204, 205t Polyvinyl alcohol (PVA), in medium, 116t Polyvinylpyrrolidone (PVP), in medium, 116t Post-process calculations, 169 Post-translational modification analysis of, 299 in endoplasmic reticulum, 31 in protein therapeutics, 5, 12–17 Potassium chloride, in medium, 111–112 Potassium ions intracellular and extracellular concentrations of, 21–22, 100– 101, 100t, 153, 153t in medium, 111–112, 111t transport of, 43–45 PPP. See Pentose phosphate pathway pRB. See Retinoblastoma protein

Process analytical technology (PAT), 167–168 Product accumulation rate, 156, 159 Product concentration profile, 154–155 Product formation. See also specific products in fedbatch culture, 241–243 kinetic model of, 162–165 lactate metabolism and, 58f, 59 quantitative description of, 156–161 specific rate of, 156–157, 159, 165–166 Programmed cell death. See Apoptosis Proline, in medium, 108 Promoter for gene transfer, 134–135 in mammalian genes, 286, 288 for transient expression, 128–129 Pro-oncogenic genes, in regulation of glucose metabolism, 77, 77f Proportional feeding with base addition, 244 with oxygen uptake rate, 245 with turbidity, 244 Protease inhibitors, in serum, 118 Proteases, for dissociation, 53 Proteasome, 36 Protective agents, in medium, 116–117, 116t Protein(s) carrier in medium, 117, 118, 122 transport by, 122, 122t in cell membrane, 25 in cellular growth, 151 in cytoplasm, 27, 27t in cytoskeleton, 37 in extracellular matrix, 45–46 folding of, 32–33, 89–91 gene expression in, 299–302 genes coding for, 285–286, 288 in interstitial fluid, 100 secretion of and cell membrane, 26 in endoplasmic reticulum, 31–35, 35f, 83 in Golgi apparatus, 32–35, 35f, 83 time of, 32, 34, 34f, 34t, 35, 35f Protein C, in-process structural alterations to, 16 Protein disulfide isomerase (PDI), 33 Protein-free medium, 103 Protein hydrolysates, in medium, 124, 240 Protein molecules, as therapeutics, 7–8. See also Protein therapeutics Protein therapeutics, 4–8 alternative technologies for, 12–14 biosimilar or follow-up biologics, 9–10 g-carboxylation in, 12, 14, 16 disulfide-bond formation in, 5, 14 fedbatch culture for, 233, 235f. See also Fedbatch culture from fusion proteins, 8

INDEX | 323

glycosylation in, 5, 12–16, 89–96, 90f growth and advances in, 1 host cells for, 127–129 immunogenicity of, 95–96 industrial cell lines for, 8t, 9 in-process structural alterations to, 15–17, 16t instability in production of, 144 from mammalian cells, 6t manufacturing of, 11–12 from non-mammalian host, 6t phosphorylation in, 14, 16 post-translational modification of, 5, 12–17 product quality and process robustness for, 14–17 quantity for administration, 11 recombinant technology for, 5 stable cell lines for, 127, 129–146 stoichiometric ratio in, 11 tissue-derived, 5 transient expression of, 127–129 Proteoglycans, in extracellular matrix, 45–46 Proteome profiling, 299–302, 299f iTRAQ labeling in, 300–301, 300f, 301f SILAC labeling in, 300–302, 301f 2D liquid chromatography in, 300–302 Proton pumps, 36 Pseudogenes, 286 Pulsatile flow, in microfiltration, 260, 260f Pumping, in bioreactor, 268–270 Purines, in medium, 109, 109t Puromycin, as selection marker, 137t Pyridoxine, in medium, 109 Pyruvate in amino acid metabolism, 81 as controlling node, 67 in glycolysis, 60, 61, 66, 67, 70 lactate conversion to, 79–80 in lipid metabolism, 86 in medium, 107 NADH balance and, 68–69, 69f transport of, 43, 72–73, 72f in tricarboxylic acid cycle, 60, 61 Pyruvate kinase (PK), in regulation of glucose metabolism, 75–76 Q Quality, of cell culture products, 14–17 Quality by Design, 162 Quantitative description, of growth, 156–161 Quasi-steady state, 179 R Radial flow impellers, 206, 265–266, 265t, 266f Rare genes, 293–294, 293t Rate-limiting enzymes, 74 Rate-limiting nutrient, 165 Reaction systems cellular vs. chemical, 175–176

material balance for, 175–180 Reactive oxygen species (ROS) glutathione and, 64, 116 in medium, 116 Reactor state parameters, 160f Read, in DNA sequencing, 297 Recessive marker, 136 Recombinant technology, 5, 192. See also specific applications and products Recovery process, 11–12 Recycling factor, in perfusion culture, 249–261, 254, 254f Remicade, expiration of patent, 9 Repetitive sequences, in genome, 287–288 Reporter gene, 136 Respiratory quotient (RQ), 219, 281 Retention, cell methods of, 254–261 in perfusion culture, 249–261 Retinoblastoma protein (pRB), 48–49 Retrograde transport, 34, 35, 35f Reynolds number, 267–268, 267f RGD peptide, in medium, 123t Riboflavin, in medium, 109 Ribonucleic acid. See RNA Ribose in medium, 107 synthesis of, 67 Ribosomal RNA (rRNA), 21, 27 removal, in microarray analysis, 296–297 Ribosomes, 27, 31 Rice hydrolysate, 124 RNA abundant, intermediate, and rare, 293–294, 293t as cellular component, 21 genes coding for, 285–286 location in cell, 28 non-protein coding, 285–286 synthesis of, 28 RNA-seq, 296–297, 298t Robustness, of cell culture processes, 14–17 Roche sequencer, 302t Roller bottles, 199–201, 200f, 212 ROS. See Reactive oxygen species Rotational cage, 258, 258f Rough endoplasmic reticulum, 31 RQ. See Respiratory quotient rRNA. See Ribosomal RNA Rubidium, in medium, 112 Rushton impellers, 206, 265–266, 265t, 266f S Sacchromyces cerevisiae systems, 12 Saturated fatty acids, 85 Scale translation and carbon dioxide removal, 275, 281–283 and chemical environment, 283, 283f

INDEX | 324

constant parameters in, 269–270, 269t dimensionless variables in, 267–270 and driving force, 279–280 effect of scale on physical behavior, 269–270 geometric nonsimilarity in, 264 geometric similarity in, 264 major effects of scale, 264 and mechanical forces on cells, 274–275, 275f mixing time in, 271–274, 271t nutrient starvation time in, 271–274, 271t objective of, 264 and oxygen transfer, 275–280, 280f, 280t physical and mechanical parameters of, 264 Reynolds number in, 267–268, 267f and superficial gas velocity, 278, 278f Scaling down, 263–264. See also Scale translation Scaling up and carbon dioxide removal, 275, 281–283 and driving force, 279–280 and mechanical forces on cells, 274–275, 275f and oxygen transfer, 275–280, 280f, 280t and superficial gas velocity, 278, 278f Secretion time, 32, 34, 34f, 34t, 35, 35f Sedimentation, 254–255, 254f Selectable marker, in gene transfer, 135–137, 137t Selenate, in medium, 111 Selenium in intracellular fluid, 153 in medium, 112, 116 Senescence, 53–55 Separation methods, 254–261 Sequencing, DNA. See DNA sequencing Serine as backbone of phospholipid, 22, 24 in medium, 108 Serum disadvantages of use, 119 in medium, 102, 117–119, 240 Serum-free medium, 102–103, 108, 109 Settling cyclone, 254–255, 254f SGLT transporters, 71–72 Shotgun liquid chromatography, 300 Sialic acid, in glycan biosynthesis, 93–94 Signaling, cellular, 45–46 Signaling pathways, in regulation of glucose metabolism, 77, 77f Signal recognition particles (SRPs), 32–33, 35, 35f SILAC, 300–302, 301f Silicon tubing/membrane, for oxygen supply, 220 Simple stirred tank bioreactor, 206–207 Single-cell cloning, 131–133 Single molecule detection, in DNA sequencing, 304 Single-use bioreactor, 199 Smooth endoplasmic reticulum, 31 Sodium beta-glycero-phosphate buffer, 115 Sodium bicarbonate buffer, 113–114, 115f, 115t Sodium butyrate, 293

Sodium chloride, in medium, 111–112 Sodium/glucose transporter, 42 Sodium ions, 21–22, 21t intracellular and extracellular concentrations of, 21–22, 21t, 100–101, 101t, 153, 153t in medium, 104, 111–112, 111t transport of, 43–45 Sodium/potassium ATPase transporter, 44–45, 44f Software, for data visualization, 172 Solid microcarriers, 203–204, 203f Solid phase, in bioreactor, 196 SOLiD sequencing technology, 302t Solutes, cellular transport of, 40 Soybean hydrolysate, 124 SP2/0 cells, 19t, 49 for stable expression, 130t therapeutic antibody products from, 7t Sparging, 220–228 damage to cells by, 226–228, 226f, 227f, 228f direct, 221 orifice and bubble size in, 222–223 Specific death rate, 157 Specific growth rate, 156–157 Specific nutrient consumption rate, 156–157 Specific product formation rate, 156–157, 159, 165–166 Specific rate, 156–157, 169 Spectinomycin, in medium, 117t S phase of cell cycle, 47–48, 48f Sphingomyelin, in medium, 125 Spin filter separation, 258, 258f Spin filter stirred tank, 210–211, 211f Spreadsheets, 168, 170–171 SRPs. See Signal recognition particles Stable cell lines, 127, 129–146 adaptation of cells in, 131, 142, 142f, 143f amplification for, 131–132, 138–141, 141f automation and high throughput technology for, 145–146, 145f basic steps for generating, 131–133, 132f gene expression in, 306–308 genomics and, 146 screening for, 131 selection of cells for, 131–132 single-cell cloning for, 131–132 stability of clones selected for, 143–145 stability of product quality in, 144–145 transfection for, 131 Stable Isotope Labeling by Amino Acid in Cell Culture (SILAC), 300–302, 301f Standardized templates, for data logging and processing, 168– 169 Starvation time, in bioreactors, 271–272, 271t State of cultures, 158 Stationary phase, of cell growth, 151f, 154–155, 154f ST cells, 8t Stem cell(s), 4

INDEX | 325

differentiation in culture, 143 medium for, 97–99, 117 mouse embryonic, doubling time of, 154t size of, 21, 151 Stirred tank bioreactor, 193, 193f agitation in, 265–266 mechanism of, 265–266 purpose of, 265 continuous, 193–196, 193f, 212 reaction and reaction kinetics in, 194–196 tracer concentration response in, 193–195, 194f heterogeneous, 196, 196f impellers in, 206, 265–268, 265t, 266f membrane, 210, 210f mixing time in, 206, 268–274 power consumption in, 266–270, 266f simple, 206–207 spin filter, 210–211, 211f velocity of, 268 volumetric flow rate in, 268–270 well-mixed, 193–196 Stock solutions, amino acid, 108 Stoichiometric components, of medium vs. catalytic macromolecular components, 105–106 vs. habitation-conducive components, 103–104 Stoichiometric-limiting nutrient, 165 Stoichiometric matrix, 179–180 Stoichiometric ratio, 11, 156, 158–159 in data processing, 169, 171, 171f in fedbatch culture, 160f, 236–240, 236f, 236t, 239f, 239t, 246–247 as indicator of metabolic stress, 161, 161t of lactate to glucose, 158, 236–237, 236f, 236t of product to substrate, 158 Stoichiometry, of cell cultivation, 147–166 Streptomycin, in medium, 117 Stress, metabolic, stoichiometric ratio as indicator of, 161, 161t Substrate stoichiometric ratio of product to, 158 yield of biomass on, 158 yield of product on, 158 Sugars, in medium, 106–107 Superoxide dismutase, in medium, 116 Superoxide radical, 116 Support systems, cellular, 202–206 Surface aeration, 220 Surface area, and scale translation, 264 Surfactants, in medium, 116–117 Suspension culture, 202, 212 SV40 late promoter, 135 Symporters, 41–42, 42f Syrian hamster cells (BHK), 8t, 9, 19t adhesion of, 142 aggregates of, 209 as continuous cell line, 49 for transient expression, 129

veterinarian vaccines from, 8t T Tangential flow, in microfiltration, 260, 260f Taurine, in medium, 116 TCA. See Tricarboxylic acid cycle Telomerase, 54 Telomeres, 54–55, 54f Temperature, and lipid bilayer, 24 T-flasks, 199 Thiamine pyrophosphate, in medium, 109 3T3 cells, 54 Thymidine, in medium, 109, 109t TIGAR, 77 Tin, in medium, 112 Tissue culture systems, 199–202 Tissue plasminogen activator (tPA) development of, 5 in-process structural alterations to, 16 product quality and process robustness, 15–16 quantity for administration, 11 Titers, increases in, 2–3, 2f TNFa. See Tumor necrosis factor a (TNFa) tPA. See Tissue plasminogen activator Trace elements, in medium, 111–112, 112t Transcription, 28 Transcription factors, 28, 291 Transcriptome analysis, 293–297 in cell culture processing, 306–308, 306f, 307f DNA microarrays for, 295–297, 295t, 298t Transfection. See also Gene transfer for stable cell line, 131–137 Transferrin iron chelators as alternative to, 121, 121t iron transport by, 45, 122t in medium, 105, 116, 118, 121, 240 recombinant, 121 secretion time of, 34, 34t Transgenic animals, 12, 14, 14t Transgenic plants, 12 Trans-Golgi network (TGN), 32, 33, 35, 35f Transient expression, 127–129 host cells for, 129 production life of system, 129 Translation of scale. See Scale translation; Scaling down; Scaling up Transport classes of processes, 40 mechanisms of, 39–47. See also specific mechanisms in metabolism, 71–74 nutrient, 43 Transporters, 40–42. See also specific types Tricarboxylic acid cycle (TCA), 60–64, 62f amino acid metabolism and, 81–82, 82f carbon flow or flux in, 67 in culture, 149

INDEX | 326

glutamine and, 80, 80f lipid metabolism and, 86 NADH balance in, 68–69, 69f regulation of, 74–78 transport and, 71–74 TrichostabinA, 293 TRICINE buffer, 115t Trypsin for dissociation, 53, 212 for proteolysis, 300 Tubular bioreactors, 193–196, 193f oxygen transfer in, 229–230, 230f reaction and reaction kinetics in, 194–196 tracer concentration response in, 193–195, 194f Tumor necrosis factor a (TNFa), in fusion protein, 8 Turbidity, proportional feeding with, 244 2D liquid chromatography, 300–302 U Ubiquitin, 36 Ubiquitination of histones, 292–293, 292t UDP-galactose, 67 UDP-glucose, 67 Unfolded protein response (UPR), 33 Uniporters, 41, 42f, 71 Untranslated regions (UTRs), 286, 288 UPR. See Unfolded protein response Uridine, in medium, 109t Urokinase, as tissue-derived protein therapeutic, 5 UTRs. See Untranslated regions V Vaccine(s) veterinary, 8t, 9 viral. See Viral vaccines Vanadium, in medium, 112 Vector. See Gene transfer Velocity, in bioreactors, 268, 278, 278f Vero cells, 8t, 9, 19t, 201 Vesicle diffusion model, 33 Veterinary vaccines, 8t, 9 Vibromixer, 212, 212f Viral vaccines, 4–5 cell sources for, 20 inactivated, 4, 9 industrial cell lines for, 8t, 9 live attenuated, 4–5 manufacturing of, 11–12 principal, in prevention of human disease, 5t quantity for administration, 11 Viscosity, of medium, 116–117 Vitamin(s), in medium, 109, 240 Vitamin A, in medium, 109 Vitamin B6, in medium, 109 Vitamin B12, in medium, 109 Vitamin C, in medium, 109

Vitamin D, in medium, 109 Vitamin E, in medium, 109, 116 Vitamin K, in medium, 109 Vitronectin, in medium, 123t Volume of cells, variation in, 150–151, 150t, 151f Volumetric flow rate, in bioreactor, 268–270 Volumetric rates, 156–157 W W1-38 cells, 19t Warburg effect, 65–66 Water as cellular component, 21, 148, 153 in medium, 106 Water for injection (WFI), 106 WaveTM, 201 Well-mixed stirred tank reactor, 193–196, 193f Westfalia disc centrifuge, 256 WFI. See Water for injection X XBP-1, 33 Y Yeast (Pichia) cell culture, 12–13, 13t Yield coefficient, 158–159 Z Zeocin, as selection marker, 137t Zinc in intracellular fluid, 153 in medium, 111, 112 Zirconium, in medium, 112 Zwitterionic buffers, 115

INDEX | 327