Manufacturing the Next Generation of Vaccines: Non-egg Based Platform for Influenza Vaccine

Letter of Transmittal May 2, 2014

Dr. Aydin K. Sunol University of South Florida Department of Chemical and Biomedical Engineering 4202 E. Fowler Ave Tampa, FL 34620

Manufacturing the Next Generation of Vaccines: Non-egg Based Platform for Influenza Vaccine

Dear Dr. Sunol, Enclosed is the report representing our response to theAIChE2014 National Student Design Competition. Our report details the construction of a manufacturing facility for the mass production of trivalent seasonal influenza vaccines that will provide immunization against the 2013-2014 influenza strains announced by the World Health Organization: A/California/7/2009 (H1N1), A/Victoria/361/2011 (H3N2), and B/Massachusetts/2/2012 (B). Designed in accordance with the criteria specified in the NSDC problem statement, the proposed process represents an alternative to the widely employed egg-based vaccine production methods. The currently employed egg-based process has a myriad of associated complications, such as inducing allergic reactions in individuals with egg allergies, and having a production capacity limited to the egg supply, which must come from hens raised under sterile conditions. Additionally, the process requires over six months of preparation time before any vaccine production can begin, with a total of up to nine months before production is finished. This time frame is unacceptable for efficiently combating a highly infectious virus that sees new mutations every year. Our proposed process utilizes cell-culture-derived influenza vaccine (CCIV) production techniques with “live” virus infection of suspension adapted CHO cells. This method provides significant advantages over egg-based methods, including easy scalability, and production times of less than 30 days. These qualities make it an especially attractive candidate for use in response to pandemic situations, where short production times are of the utmost importance. Based on projected demands, our facility would distribute around 54.5 million doses of vaccine, with a net annual profit of $368 million (based on a 2014-2015 sale price of $9.22 per dose, as averaged between government and private sector contracting prices). As outbreaks of the influenza virus represent a serious threat to the overall health of our ever-growing global population, there is a pressing demand for our production methods to constantly improve and adapt. Implementation of the process we describe represents a way to meet that demand more effectively, while simultaneously reducing costs and decreasing production time, making it a desirable alternative for vaccine production.

Sincerely,

Christopher Ludwin Erik Madsen

Title Page

Manufacturing Process for Trivalent Influenza Vaccine Production Using CHO Cells

Christopher Ludwin Erik Madsen

May 2, 2014

TABLE OF CONTENTS

Letter of Transmittal ............................................................................................................ i Title Page ............................................................................................................................ ii TABLE OF CONTENTS ..................................................................................................... i List of Tables ..................................................................................................................... iv List of Figures ..................................................................................................................... v Abstract .............................................................................................................................. vi Introduction ......................................................................................................................... 1 Types of Vaccines and Production Methods .................................................................. 2 Recombinant Vaccine Production................................................................................... 3 Subunit/Split Vaccine Production ................................................................................... 3 Whole Inactivated Virus Vaccines.................................................................................. 4 Disposables ..................................................................................................................... 5 Design Premises and Specifications ................................................................................... 6 Product ............................................................................................................................ 6 Process ............................................................................................................................ 6 Facility ............................................................................................................................ 6 Market Basis ................................................................................................................... 7 Results ............................................................................................................................... 10 Process Flow Diagrams............................................................................................... 10 Material Balances & Stream Analysis Information ................................................ 17 Equipment List and Specifications ............................................................................ 26 Summary of Capital Requirements and Manufacturing Costs .............................. 28 Profitability analysis ..................................................................................................... 34 Feasibility Analysis ..................................................................................................... 35 Safety and Operability Considerations ..................................................................... 36 Conclusions and Recommendations ................................................................................. 40 Appendices ........................................................................................................................ 41 Appendix A: Scheduling Optimization ..................................................................... 41 Appendix B: Assumptions .......................................................................................... 43

List of Tables Table 1: Hemagglutinin content of influenza vaccines....................................................... 7 Table 2: Dosage distribution over time ............................................................................... 8 Table 3:Bulk Material Analysis by Section ...................................................................... 17 Table 4: Stream Analysis .................................................................................................. 18 Table 5: Equipment Summary .......................................................................................... 26 Table 6: Major Equipment Specs and FOB cost ............................................................... 29 Table 7: FCI Summary...................................................................................................... 30 Table 8: Labor Summary .................................................................................................. 30 Table 9: Materials Cost ..................................................................................................... 31 Table 10: Consumables Cost ............................................................................................ 31 Table 11:Waste Treatment/Disposal Costs ....................................................................... 32 Table 12: Utilities Costs.................................................................................................... 32 Table 13: Annual operating costs and correlations used to estimate unknown values (Turton 1998) .................................................................................................................... 33 Table 14: Profitability analysis ......................................................................................... 34 Table 15: Table of Cash Flows ......................................................................................... 35

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List of Figures Figure 1: Graphical representation of the antigen-antibody concept .................................. 1 Figure 2: Structure of an influenza virus particle. .............................................................. 1 Figure 3: Experimental cell growth model. Note the fastest growth occurs during .5E6 and 2.6E6 cells/mL ............................................................................................................. 4 Figure 4: Graph of doses distributed ................................................................................... 7 Figure 5: Overall PFD of process showing phases ........................................................... 10 Figure 6: Seed train phase of the process.......................................................................... 11 Figure 7: Production bioreactor phase of the process, S-112 continues from seed train .. 12 Figure 8: Clarification stage, followed by inactivation. B-propiolactone is introduced through S-306.................................................................................................................... 13 Figure 9: Ultrafiltration phase, followed by SEC ............................................................. 14 Figure 10: Anion exchange chromatography phase.......................................................... 15 Figure 11: Secondary UF phase and further concentration of product solution ............... 16 Figure 12: Discounted cumulative cash flow.................................................................... 35 Figure 13: Cash flow diagram........................................................................................... 35 Figure 14: Comparison of different vaccines and their respective BSLs ......................... 37 Figure 15: Proposed floor plan of facility ......................................................................... 39 Figure 16: Single batch Gantt chart .................................................................................. 41 Figure 17: Gantt chart for running multiple theoretical batches ....................................... 42

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Abstract The influenza virus represents a constant threat to the health and wellbeing of general society. Most influenza vaccines are currently produced through a method that involves live virus cultivation in the embryonic cells of millions of fertilized chicken eggs. This method is costly, limited by the supply of prepared eggs, and is burdened by lengthy processing times, with preparation and production taking as long as nine months. With the global population constantly growing and influenza strains mutating every year, there is a pressing need for the development of alternative production methods that are faster and more cost efficient, and thus better prepared to deal with the threat of a pandemic influenza outbreak. Herein we propose a non-egg based manufacturing facility for mass production of a trivalent inactivated influenza vaccine. The facility will produce a vaccine providing immunization against the three strains recommended by the World Health Organization for 2013-2014 trivalent vaccine production: A/California/7/2009 (H1N1), A/Victoria/361/2011 (H3N2), and B/Massachusetts/2/2012 (B). The facility avoids lengthy preparation times by utilizing cultured CHO host cells to cultivate the virus. These cells can be thawed from vials in a working cell bank and cultured to production level volumes within 11 days. Possibility of contamination is reduced through incorporation of pre-sterilized disposable technology throughout the process, reducing downtime and lowering the financial costs associated with using equipment that needs sterilization and validation between cycles. The projected annual demand is 145.2 million doses with our company controlling a market share of %37.5 for a total of 54.5 million doses at a sale price of $9.22 per dose as determined by averaging 2014-2015 CDC pricing data for private sector and government contracts. Economic analysis performed using a 10 year plant life with 7 year depreciation (straight line) indicate a NPV on the order of $2.2 billion dollars using a discounting factor of 7%. Our conclusions suggest that this facility represents an efficient and cost-effective alternative capable of replacing or supplementing current influenza vaccine production methods.

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Introduction Influenza is a well-known viral disease that can be found all over the globe. The highly infectious nature of influenza can make it a serious threat when an outbreak occurs. In the US alone, there are over 30,000 deaths every year as a result of health complications associated with contraction of the influenza virus. Particularly, younger children and the elderly are at increased risk. One of the many challenges in treating and preventing influenza outbreaks lies in the mutable tendency of the virus. The biological structure of the virus is constantly changing and producing different strains almost every year, sometimes species-specific influenza strains will mutate such that they develop the ability to infect humans, giving rise to strains like “swine flu” or “avian flu”. The high propensity for variation in the viral strains that can circulate the population add a great degree of difficulty to the preparation and development of vaccines, especially when a new strain of the virus emerges. The influenza vaccine works by introducing inactivated portions of the influenza strains into the human body. The immune response of the body then starts developing complementary antibodies to the inactivated virus strains, which enable them to Figure 1: Graphical representation of the identify and eliminate any instances of the live virus that may be antigen-antibody concept encountered later. These inactivated portions of the virus act as antigens and promote the production of the desired antibodies. The typical seasonal influenza vaccine is what is known as a trivalent vaccine. This means that it contains antigens for three main influenza strains. These vaccines are prepared every year based on recommendations by the Center for Disease Control (CDC) as to what particular strains are projected to be most common throughout the population in a that particular year. Two of the most common influenza surface antigens used for vaccine production are hemagglutinin (HA) and neuraminidase (NA) shown in Figure 2. Hemagglutinin is a glycoprotein that binds the virus to the cell. Neuraminidase is an enzyme that releases the replicated viruses from the infected cell’s surface. The current production process is over seventy years old, and consists of growing the live virus in large quantities of fertilized chicken eggs, followed by inactivation and processing of the virus. One of the major complications with this method is that the eggs must be prepared months in advance. In addition to the lengthy preparation time, the capacity to produce vaccines is limited to the supply of available eggs, thus creating a need for over-production of eggs in order to be prepared for an influenza pandemic. An additional complication lies in the fact that, since the chickens that produce the eggs are susceptible to avian flu themselves, Figure 2: Structure of an influenza virus particle. they must be heavily monitored by teams of veterinarians and maintained under strict sterile protocols. The demand for influenza vaccines 1

can vary greatly from year to year, and can be difficult to predict. In order to meet the projected demands of the population and be prepared for any unforeseen surges in demand that may occur, such as in the event of a pandemic, vaccines must be produced in excessive quantities otherwise a shortage will occur, which has happened on many occasions in the past. Since the finished vaccines cannot be stored for long periods of time, the unused vaccines must be thrown away at the end of the season. In the event that the vaccination rate for the season is actually much lower than predicted, large amounts of vaccines can end up being produced only to be discarded. The aforementioned complications compound upon each other and ultimately result in a process that by current industry standards is archaic, costly, inefficient, and bloated in its resource consumption and waste production. Types of Vaccines and Production Methods There are several routes available to produce a vaccine that will initiate an immune response and development of desired antibodies in the patient. The goal of any vaccine is to produce the most effective immune response when administered to the patient, while at the same time minimizing possible negative side effects. Like most viruses, influenza is covered in surface glycoproteins which are used for communication between cell surfaces. The most abundant of these are the two proteins Hemagglutinin (HA) and Neuraminidase (NA). Although both play a part for in vivo development of an immune response to a particular viral strain, Hemagglutinin has been found to produce a much more active immune response and initiate a greater production of antibodies within exposed patients, and thus is usually the most desired protein in influenza vaccine development and in standardization of vaccine compositions. As previously mentioned, there are several available methods used to produce and formulate a serum that will deliver an appropriate dosage of these proteins. Currently, industry is shifting towards a cell-culture-derived influenza vaccine (CCIV) production techniques. This method provides significant advantages over egg-based methods, including easy scalability, and production times of less than 30 days. These qualities make it an especially attractive candidate for use in response to pandemic situations, where short production times are of the utmost importance. The two main approaches to CCIV are recombinant antigen production and “live” virus infection. Both techniques yield antigenic components used for vaccine formulation. Recombinant antigen production produces pre-specified antigens (typically HA), whereas “live” virus infection produces the entire virus. Each has distinct advantages and disadvantages. Common cell lines used in research such as SF-9 insect cells and Chinese Hamster Ovary (CHO) cells are ideally suited for both methods. Use of well-known cell lines aide production-scale process development, because known media-based cell growth kinetics offer scalable results from optimized bench- top research. Currently, there remains uncertainty in the media conditions for optimal cell kinetics and product yield. The development of chemically-defined media is key to process optimization. Therefore, a process that incorporates chemically defined media is important for long-term maximization of facility potential. Additionally, cell types such as CHO cells may be anchorage dependent. Anchorage-dependent cells require micro-carries: microscopic beads to which cells can anchor themselves. Developments in CHO cell research have led to cell lines that are not anchorage-dependent and capable of growing suspended in media. Suspension adapted CHO cells are ideal for productionscale processes because they are more easily scaled.

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Recombinant Vaccine Production Recombinant protein production involves changing the genetics of a specific cell line so that they essentially become programmed to use their biological production “machinery” to produce the desired protein. This is also what a virus essentially does in order to replicate itself, viruses are incapable of self-replication, thus they “hijack” the cells they infect by injecting genetic material into the cell, which the cell assembles into more and more viruses until it bursts and releases the viruses into solution, where they go on to infect other cells. In recombinant production the desired protein is produced either as an extracellular product, where it is excreted from the cells into the growth medium, or as an intracellular product, where it remains within the cell and ultimately must be harvested by lysis of the cell. These conditions are determined by the cell line and the type of protein that will be produced. When manufacturing influenza vaccines recombinant antigen production of HA requires development of a cell line that has been transfected by a vector containing an HA coding sequence along with a promoter sequence that allows for control over HA production. The cell line is then adapted to the production media. During the production-scale process the cell line is grown until it has reached production size at which point the promoter is introduced to initiate the production of HA. The culture solution is harvested through mechanically or chemically breaking apart the cells so that the product can be recovered. Downstream purification is then accomplished through a various techniques centered around separating out the Hemagglutinin from the protein slurry. Production of a known antigenic component produces the safest vaccine. The downside of this method is the requirement of the antigenic coding sequence and the time it takes to develop an adaptable cell line, which is not ideally suited to respond to a pandemic from a highly mutable virus like influenza.

Subunit/Split Vaccine Production The subunit or ‘split’ vaccine production method is a commonly employed method and the downstream purification aspect is very similar to the one by the egg-based production methods today. A small vial of a predetermined cell line is thawed out from a working cell bank, and then “passaged” into a larger production level volume. The passaging phase is based around the growth kinetics of the cells, which have a lag phase, logarithmic growth phase, and death phase. The idea of the passaging process is to keep the cells at a concentration such that they are always in the logarithmic growth phase. Figure 3 below illustrates an example of a cell growth curve for Chinese Hamster Ovary cell, modeled using a Gaussian distribution to include the cell death phase.

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Figure 3: Experimental cell growth model. Note the fastest growth occurs during .5E6 and 2.6E6 cells/mL

During passaging, the cell culture is transferred to a new, larger vessel, and diluted with new media back to the beginning concentration, where it is again allowed to culture until it reaches the peak concentration of the log growth phase and is again diluted. After this, it is transferred to a production level bioreactor, where the live virus infects the cells and begins to replicate until most of the cells are destroyed by the virus. At this point in time, the virus is harvested, and inactivated. The inactivation technique and further processing are essentially what separate this method from the whole virus vaccine production one. First the solution is treated with a buffer. This is then followed by the addition of a detergent, which cleaves the desired surface glycoproteins off of the virus, such as the Hemagglutinin and Neuriminidase. Further downstream processing and purification is employed to then separate these proteins out of the rest of the protein slurry. This method is effective, but results in a difficult purification strategy that is often costly, because many of the proteins that are in solution then have a similar composition, molecular size, and chemical behavior. This is countered by implication of more thorough separating techniques such as multiple ion exchange chromatography columns, but again the costs associated with this, as well as the product recovery, are not optimal. Additionally, the chemicals and detergents used will sometimes destroy or attenuate the desired products, resulting in a decreased immune response in those who are administered the vaccine. Whole Inactivated Virus Vaccines The initial phases of the whole inactivated virus production process are extremely similar to that of the subunit vaccine. The selected cell line is cultured and passaged to a suitable volume corresponding to a desired production capacity. The specific strain of influenza that the vaccine is to provide immunity to is then introduced to the solution at an optimal multiplicity of infection (MOI) which the ratio of virus particles to the ratio of cells. Common MOIs are around .1 to .001. Another factor at this phase is the TOI or time of infection. This is the optimal time in the cell growth cycle to introduce the virus, and is usually selected to be somewhere towards the end of the logarithmic growth phase. After the virus is infected and allowed to replicate as with the subunit method, the production processes then begin to diverge. With the whole virus method, the slurry is first clarified to remove larger solid particulates, usually through unit operations such a disk stack centrifugation (a large, continuously operating centrifuge with many rotating conical plates) and depth filtration (filtration step consisting of a series of high surface area filters that are often composed of a porous, fibrous material that allows high liquid flowrates while simultaneously retaining any larger particles). The key step in the process occurs next, with inactivation. Inactivation of the virus involves addition of a chemical such as betapropiolactone, which alters the composition of the viruses biological components, and causes the

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virus to lose its ability to replicate itself, and thus shuts down the infectivity of the viral particles. The full mechanism of beta-propiolactone is not known, but is thought to involve a combination of membrane fusion disruption of the virus and viral genetic alteration. The viral particles are now completely inactivated, but still retain most of their structure and surface proteins. Separating them from the solution is quite simple, as they have a much larger size than most of the other components of the protein slurry, and can be extracted out through utilization of methods such as size exclusion chromatography. Size exclusion chromatography (SEC) is also known as gel filtration, and is a commonly employed technique in laboratories. It uses a porous gel as a stationary medium, through which the solution is passed, and the larger viral particles diffuse through much slower than the rest of the components of the solution, thus eluting all of the waste products first, and allowing subsequent collection of the desired fractions. SEC is often coupled with an ion exchange chromatography step to achieve a great degree of purification in relatively few steps. Ion exchange chromatography involves the use of a column packed with a resin that contains a specific ion that interacts with the desired protein in a buffer solution dependent on the protein being separated out. It works by binding to the desired product, thus retaining it within the column, and eluting out the unwanted waste portions of the solution. After the waste is eluted, a different buffer solution, often containing a chemical such as imidazole, is passed through the column, where the imidazole out-competes the desired compound for the ion sites within the resin and essentially switching places with the product, thus eluting a buffer solution containing the product at high purity levels. Finally the solution is the concentrated down and passed to the formulation stage of the process, where quality control takes place, and the concentration is standardized and prepared for packaging and distribution. Recent studies indicate that the finalized whole virus vaccines have the greatest immunogenic efficiency and most consistent performance as compared to recombinant and subunit vaccines. This is most likely the results of the integrity of the virus being preserved, and thus a more “full spectrum” immune response is achieved that more closely corresponds to what happens during exposure to the live virus. Disposables Another industry movement has been towards disposable process equipment. Disposables replace costly and time consuming clean in place/ steam in place protocols by offering presterilized process equipment for each batch process. Research shows that the increased operational costs from the disposable components is offset by the reduction in initial investment costs and the reduction in process time over the life of the facility. Examples of disposable process equipment ranges from seed train bioreactors to ion exchange chromatography columns. Shown below in Figure 4 is an example of the WAVE bioreactor developed by GE, which uses presterilized disposable bags as the inoculum container.

Figure 4: GE WAVE Bioreactor

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Design Premises and Specifications The overall objective of the following design is intended to create a viable non-egg based influenza vaccine production process and facility. The following are product, process, and facility design considerations/ specifications as dictated by the AIChE Contest Problem. Product The influenza vaccine is the final product of the proposed process. A single vaccine dose is designed to be trivalent, composed of three moieties each representing the equivalent of 15 micrograms of HA antigen. Furthermore, the strain-derived HA antigens is in accordance with the seasonally reported WHO recommendations every year before production takes place. Process The proposed process is non-egg based, specifically using CHO cells. The scope of the process design begins with vial thaw and ends with product purification. Product formulation is not considered in the design. The process follows Good Manufacturing Practice (GMP), including proper sterilization techniques by SIP/CIP protocol and the integration of pre-sterilized single use disposables. Additionally, the media is chemically defined, produced from granulated powder and chosen to support the specified cell-line. The cell line is banked as a 1 ml vial containing 1E6 viable cells/ml. The seed train is a batch process along with the production bioreactor. However, the production bioreactor is capable of operating as a fed-batch reactor. The seed train and production bioreactor are scaled based on typical cell growth curve. Finally, in the downstream processing the CHO cell culture broth has an assumed density of 1.06 g/ml, and the broth is centrifuged/filtered to remove biomass Facility The facility is designed to produce a single product according to a seasonal timeline. The capacity is assumed to be set to the North American market share of Sanofi-Pastuer based on historical trends. Additionally, the facility is designed to scale-up production in case of a pandemic. The facility is considered animal free with chemically defined media assembled onsite from powder contents. Ultimately, the facility should be equipped to freeze dry the product and prepare it for shipping. In terms of safety and environmental impact, waste is treated in presewage kill tanks. All costing data is defined as follows: Cost Data: Electricity: $0.05/kWhr Sewer: $5.00/thousand gallons Water: $0.543 per 1000 liters Water for Injection: $1000 per 1000 liters All prices are delivered to your site and are in current year’s dollars.

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Market Basis The initial considerations in the design concern facility throughput. The basis of these calculations relies on the market data for GSK which holds nearly a 35% market share. Historical trends show an approximately linear increase in influenza vaccine production/distribution over the past ten to fifteen years as shown in Figure 4.

Doses Distributed 160

Distributed (millions)

140 120 100 80 60 40 20 0 0

2

4

6

8

10

12

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Year (2001-2013) Figure 4: Graph of doses distributed

Using the HA antigen quantities per dose (45 mcg) from Table 1, we arrive at the projected total quantity of HA (~2.5 kg) required from the process to respond to a pandemic as shown in Table 2. INGREDIENT

QUANTITY (PER DOSE) FLUZONE 0.25 ML DOSE

FLUZONE 0.5 ML DOSE

Active Substance: Influenza virus, inactivated strainsa:

22.5 mcg HA total

45 mcg HA total

A (H1N1)

7.5 mcg HA

15 mcg HA

A (H3N2)

7.5 mcg HA

15 mcg HA

B

7.5 mcg HA

15 mcg HA

Table 1: Hemagglutinin content of influenza vaccines

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Year 200001 200102 200203 200304 200405 200607 200708 200809 200910 201011 201112 201213 Projected 201314 201415

Distributed Sanofi Market (millions) Share (millions)

Hemagglutinin Concentration (g)

70.4 77.7 83.5 83.1 57 81.5 102.5 112.8

42.3

1903.5

139.4

52.275

2352.375

145.2 Actual Data Projected Values

54.45

2450.25

134.9

Table 2: Dosage distribution over time

Although the above calculations are based only off of HA concentration, the vaccine will consist of the entire inactivated virus. The quantity of which will be measured through assay to arrive at a specified titer. This is significant in terms of downstream processing as it calls for the isolation of the whole virus from the process fluid. In other words, Influenza virus is approximately 250 kDa, so size exclusion chromatography and filters were designed accordingly.

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Whole-virus vaccine allows for fast response to pandemic outbreak of mutant strains. Optimal growth for cell culturing occurs in the log phase of growth as shown by the upward slope in Graph 2. To keep growth within this range, the concentration must be maintained in the bounds of the log phase of the curve. Table 3 uses the bounds form the growth curve (5E4 cells/ml – 1.8E6 cells/ml) to scale the seed train to achieve the necessary cell quantity for the production bioreactor. The downstream processing consists of the inactivation and isolation of the influenza virus from the effluent reactor stream. The general separation process was outlined using the following heuristics. Heuristics: 1. Remove the most plentiful impurities first. 2. Remove the easiest-to-remove impurities first. 3. Make the most difficult and expensive separations last. 4. Select processes that make use of the greatest differences in the properties of the product and its impurities. 5. Select and sequence processes that exploit different separation driving forces. Equipment was then selected that achieved the goals of these separations. For example, the first step in the downstream processing is clarification, which makes use of a centrifuge to remove large debris like cell fragments.

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Results Process Flow Diagrams

Figure 5: Overall PFD of process showing phases

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Figure 6: Seed train phase of the process.

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Figure 7: Production bioreactor phase of the process, S-112 continues from seed train

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Figure 8: Clarification stage, followed by inactivation. B-propiolactone is introduced through S-306

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Figure 9: Ultrafiltration phase, followed by SEC

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Figure 10: Anion exchange chromatography phase

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Figure 11: Secondary UF phase and further concentration of product solution

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Material Balances & Stream Analysis Information

Bulk Material Analysis by Section SECTIONS IN: Main Branch Anion Exchange Chromatography Material

kg/yr

kg/batch

kg/g MP

299 613 306 300 32 1,549

99.563 204.319 101.890 100.003 10.562 516.337

0.121 0.248 0.124 0.121 0.013 0.627

Seed Train Material

kg/yr

kg/batch

kg/g MP

Media Injection Water Biomass Air TOTAL

105 476 0 3,168 3,749

34.861 158.754 0.000 1,056.072 1,249.687

0.042 0.193 0.000 1.282 1.517

Production Bioreactor Material

kg/yr

kg/batch

kg/g MP

Injection Water Media Air TOTAL

4,625 78 58,229 62,932

1,541.792 25.856 19,409.791 20,977.439

1.872 0.031 23.568 25.472

Clarification and Inactivation Material

kg/yr

kg/batch

kg/g MP

H3PO4 (5% w/w) NaOH (0.5 M) WFI B-Propiolactone TOTAL

3,855 2,661 11,862 38 18,416

1,285.028 887.005 3,953.993 12.765 6,138.790

1.560 1.077 4.801 0.015 7.454

Ultrafiltration and SEC Material

kg/yr

kg/batch

kg/g MP

4,552 4,552

1,517.292 1,517.292

1.842 1.842

AEC Eq Buffer AEC El Buff AEC Strip Buffe AEC Wash Buffer Amm. Sulfate TOTAL

IEX-El-Buff TOTAL Table 3:Bulk Material Analysis by Section

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Table 4: Stream Analysis

Stream Analysis Stream Name Source Destination Stream Properties

WCB Vial Thaw INPUT P-01

S-101 P-01 P-02

SFR-101M INPUT P-02

S-102 P-02 P-03

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 24.99 1.29 994.71 - 0.00 - 0.01 1.00

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 37.00 1.26 990.33 0.00 11.98 1.00

0.000 0.001 0.000 0.001 0.001

0.000 0.001 0.000 0.001 0.001

0.000 0.019 0.000 0.019 0.019

0.003 0.006 0.011 0.020 0.020

SFR-102M INPUT P-03

S-103 P-03 P-04

SFR-103M INPUT P-04

S-104 P-04 P-05

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 37.00 1.10 990.33 0.00 11.98 1.00

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 37.00 2.95 990.33 0.00 11.98 1.00

0.000 0.060 0.000 0.060 0.060

0.012 0.020 0.048 0.080 0.080

0.000 0.239 0.000 0.239 0.240

0.048 0.078 0.193 0.318 0.321

Stream Name Source Destination Stream Properties

SFR-104M INPUT P-05

S-105 P-05 P-06

Media BBS101a INPUT P-06

Vent 101a P-06 OUTPUT

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h)

0.00 25.00 1.01 994.70 0.00

0.00 37.00 2.95 990.33 0.02

0.00 25.00 1.01 994.70 0.00

0.00 37.00 1.01 1.67 0.01

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Component Flowrates (kg/batch) Biomass Media Water TOTAL (kg/batch) TOTAL (L/batch)

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Component Flowrates (kg/batch) Biomass Media Water TOTAL (kg/batch) TOTAL (L/batch)

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Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

0.00 1.00

11.98 1.00

0.00 1.00

29.12 0.21

0.000 0.000 0.955 0.000 0.000 0.000 0.955 0.960

0.193 0.000 0.310 0.000 0.000 0.771 1.273 1.286

0.000 0.000 5.699 0.000 0.000 0.000 5.699 5.729

0.000 0.316 0.000 0.017 0.005 0.000 0.338 202.082

Stream Name Source Destination Stream Properties

S-106 P-06 P-07

Media BBS102a INPUT P-07

Vent 102a P-07 OUTPUT

S-107 P-07 P-10

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

0.00 37.00 1.01 990.33 0.09 11.98 1.00

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 37.00 1.01 1.68 0.07 29.38 0.21

0.00 37.00 1.01 990.33 0.38 11.98 1.00

0.739 0.000 0.168 1.803 0.000 0.000 3.925 6.636 6.700

0.000 0.000 0.000 22.926 0.000 0.000 0.000 22.926 23.048

0.000 1.864 0.000 0.000 0.085 0.026 0.000 1.975 1,175.867

2.916 0.000 0.960 4.946 0.000 0.000 18.762 27.583 27.853

S-108 INPUT P-08

S-109 INPUT P-08

S-110 P-08 P-09

S-111 P-09 P-10

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 25.00 5.75 994.70 0.00 0.00 1.00

0.00 25.00 5.75 994.70 0.00 0.00 1.00

158.754 0.000 158.754 159.599

0.000 4.963 4.963 4.989

158.754 4.963 163.717 164.589

158.754 4.963 163.717 164.589

Component Flowrates (kg/batch) Biomass Carb. Dioxide Media Nitrogen Oxygen Water TOTAL (kg/batch) TOTAL (L/batch)

Component Flowrates (kg/batch) Biomass Carb. Dioxide Impurities Media Nitrogen Oxygen Water TOTAL (kg/batch) TOTAL (L/batch)

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Component Flowrates (kg/batch) Injection Water Media TOTAL (kg/batch) TOTAL (L/batch)

19

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

DBS-101 Air DBS-101 Vent Inlet INPUT P-10 P-10 OUTPUT

S-112 P-10 P-13

S-201 INPUT P-11

0.00 25.00 1.01 1.18 0.00 0.00 0.24

0.00 37.00 1.01 1.13 3.59 2.92 0.24

0.00 37.00 1.01 990.33 2.65 11.98 1.00

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.000 0.000 0.000 0.000 0.000 810.132 245.940 0.000 1,056.072 895,566.922

0.000 0.693 0.000 0.000 0.000 810.324 245.999 0.000 1,057.015 932,233.615

4.025 0.000 1.237 158.754 2.973 0.000 0.000 23.618 190.607 192.468

0.000 0.000 0.000 1,541.792 0.000 0.000 0.000 0.000 1,541.792 1,550.000

S-202 INPUT P-11

S-203 P-11 P-12

S-205 P-12 P-13

DBS-201 Air Inlet INPUT P-13

0.00 25.00 1.01 994.70 0.00 0.00 1.00

0.00 25.00 67.39 994.70 0.00 0.00 1.00

0.00 25.00 67.39 994.70 0.00 0.00 1.00

0.00 25.00 1.01 1.18 0.00 0.00 0.24

0.000 25.856 0.000 0.000 25.856 25.994

1,541.792 25.856 0.000 0.000 1,567.648 1,575.994

1,541.792 25.856 0.000 0.000 1,567.648 1,575.994

0.000 0.000 14,889.598 4,520.193 19,409.791 16,459,833.447

Component Flowrates (kg/batch) Biomass Carb. Dioxide Impurities Injection Water Media Nitrogen Oxygen Water TOTAL (kg/batch) TOTAL (L/batch)

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Component Flowrates (kg/batch) Injection Water Media Nitrogen Oxygen TOTAL (kg/batch) TOTAL (L/batch)

20

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Vent-5 P-13 OUTPUT

S-206 P-13 P-14

S-301 P-14 P-15

S-302 P-15 P-16

0.00 37.00 1.01 1.13 66.02 2.93 0.24

0.00 37.00 1.01 990.33 24.51 11.97 1.00

0.00 37.00 10.51 990.33 24.50 11.96 1.00

0.00 42.13 1.01 988.46 33.54 17.08 1.00

0.000 14.520 0.000 0.000 0.000 0.000 14,891.250 4,502.315 0.000 19,408.085 17,117,990.006

11.194 0.000 1.471 1.972 1,700.546 10.447 0.000 0.000 36.485 1,762.115 1,779.320

11.194 0.000 1.471 1.972 1,700.546 10.447 0.000 0.000 36.485 1,762.115 1,779.318

0.224 0.000 1.419 1.903 1,640.809 10.080 0.000 0.000 35.203 1,689.638 1,709.360

S-303 P-15 OUTPUT

S-304 P-16 P-17

S-305 P-16 OUTPUT

S-306 INPUT P-17

0.00 42.13 1.01 988.46 1.44 17.08 1.00

0.00 42.13 1.01 988.46 33.53 17.08 1.00

0.00 42.13 1.01 988.46 0.01 17.09 1.00

0.00 25.00 1.01 1,146.00 0.00 0.00 0.25

0.000 10.970 0.052 0.069 59.737 0.367 1.282 72.477 73.323

0.000 0.000 1.419 1.903 1,640.592 10.078 35.199 1,689.190 1,708.907

0.000 0.224 0.000 0.000 0.217 0.001 0.005 0.448 0.453

12.765 0.000 0.000 0.000 0.000 0.000 0.000 12.765 11.138

Component Flowrates (kg/batch) Biomass Carb. Dioxide HAeq Impurities Injection Water Media Nitrogen Oxygen Water TOTAL (kg/batch) TOTAL (L/batch)

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Component Flowrates (kg/batch) B-Propiolactone Biomass HAeq Impurities Injection Water Media Water TOTAL (kg/batch) TOTAL (L/batch)

21

Stream Name Source Destination Stream Properties

S-307 P-17 P-18

S-401 P-18 P-19

S-402 P-19 P-20

S-403 P-20 P-21

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

0.00 42.09 10.70 989.50 33.52 16.95 0.99

0.00 42.08 24.13 989.50 33.51 16.94 0.99

0.00 42.08 24.13 989.50 33.51 16.94 0.99

0.00 42.08 24.13 989.50 33.50 16.94 0.99

12.765 1.419 1.903 1,640.592 10.078 35.199 1,701.955 1,720.022

12.765 1.419 1.903 1,640.592 10.078 35.199 1,701.955 1,720.019

12.765 1.419 1.903 1,640.592 10.078 35.199 1,701.955 1,720.019

12.765 1.419 1.903 1,640.592 10.078 35.199 1,701.955 1,720.015

Stream Name Source Destination Stream Properties

S-404 P-21 P-22

S-405 P-21 P-23

S-406 P-23 P-25

SEC Elute Buffer INPUT P-24

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

0.00 42.48 24.13 989.35 32.95 17.35 0.99

0.00 42.48 24.13 989.33 1.34 16.99 0.97

0.00 42.47 3.44 989.34 1.34 16.98 0.97

0.00 25.00 1.01 1,025.59 0.00 0.00 0.97

12.264 0.007 1.828 1,576.277 9.683 0.000 0.000 33.819 1,633.878 1,651.458

0.500 1.412 0.075 64.315 0.395 0.000 0.000 1.380 68.077 68.811

0.500 1.412 0.075 64.315 0.395 0.000 0.000 1.380 68.077 68.811

0.000 0.000 0.000 1,424.062 0.000 14.869 78.361 0.000 1,517.292 1,479.427

Component Flowrates (kg/batch) B-Propiolactone HAeq Impurities Injection Water Media Water TOTAL (kg/batch) TOTAL (L/batch)

Component Flowrates (kg/batch) B-Propiolactone HAeq Impurities Injection Water Media NaH2PO4 Sodium Chloride Water TOTAL (kg/batch) TOTAL (L/batch)

22

P-24 P-25

SEC Waste Stream P-25 OUTPUT

0.00 25.00 10.12 1,025.59 0.00 0.00 0.97

0.00 25.80 3.44 1,023.92 1.34 0.77 0.97

0.00 25.00 3.44 1,025.18 0.00 0.00 0.96

0.00 25.00 9.46 1,025.18 0.00 0.00 0.96

0.000 0.000 0.000 1,424.062 0.000 14.869 78.361 0.000 1,517.292 1,479.427

0.500 0.212 0.075 1,403.596 0.395 13.984 73.695 1.380 1,493.837 1,458.937

0.000 1.200 0.000 84.781 0.000 0.885 4.665 0.000 91.532 89.284

0.000 1.200 0.000 84.781 0.000 0.885 4.665 0.000 91.532 89.284

Stream Name Source Destination Stream Properties

S-501 INPUT P-27

S-502 INPUT P-28

S-503 INPUT P-29

S-504 INPUT P-30

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

0.00 25.00 1.01 1,003.92 0.00 0.00 0.99

0.00 25.00 1.01 1,053.51 0.00 0.00 0.97

0.00 25.00 1.01 1,048.02 0.00 0.00 0.98

0.00 25.00 1.01 1,012.39 0.00 0.00 0.99

98.557 0.000 0.000 0.110 0.000 0.896 99.563 99.174

191.765 0.000 0.000 0.000 2.002 10.552 204.319 193.941

96.113 0.000 0.000 0.000 0.000 5.777 101.890 97.221

98.092 0.000 0.000 0.110 0.000 1.800 100.003 98.779

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

S-407

S-408

S-409

P-25 P-26

P-26 P-31

Component Flowrates (kg/batch) B-Propiolactone HAeq Impurities Injection Water Media NaH2PO4 Sodium Chloride Water TOTAL (kg/batch) TOTAL (L/batch)

Component Flowrates (kg/batch) Injection Water KCl KH2PO4 Na2HPO4 NaH2PO4 Sodium Chloride TOTAL (kg/batch) TOTAL (L/batch)

23

Stream Name Source Destination Stream Properties

S-505 P-27 P-31

S-506 P-28 P-31

S-507 P-29 P-31

S-508 P-30 P-31

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

0.00 25.00 9.92 999.53 0.00 0.00 0.99

0.00 25.00 9.92 1,025.59 0.00 0.00 0.97

0.00 25.00 9.92 1,022.89 0.00 0.00 0.98

0.00 25.00 9.92 1,003.94 0.00 0.00 0.99

Injection Water KCl KH2PO4 Na2HPO4 NaH2PO4 Sodium Chloride TOTAL (kg/batch) TOTAL (L/batch)

98.557 0.000 0.000 0.110 0.000 0.896 99.563 99.610

191.765 0.000 0.000 0.000 2.002 10.552 204.319 199.221

96.113 0.000 0.000 0.000 0.000 5.777 101.890 99.610

98.092 0.000 0.000 0.110 0.000 1.800 100.003 99.610

Stream Name Source Destination Stream Properties

S-509 P-31 P-32

AEC Waste P-31 OUTPUT

S-510 INPUT P-32

S-511 P-32 P-33

0.00 25.00 9.46 1,025.29 0.00 0.00 0.96

0.00 25.00 9.46 1,015.26 0.00 0.00 0.98

0.00 25.00 1.01 1,769.00 0.00 0.00 0.34

0.00 25.00 10.12 1,066.95 0.00 0.00 0.90

0.000 0.996 95.882 0.000 0.000 0.000 1.001 5.276 103.156 100.612

0.000 0.204 473.426 0.000 0.000 0.220 1.886 18.415 494.151 486.724

10.562 0.000 0.000 0.000 0.000 0.000 0.000 0.000 10.562 5.971

10.562 0.996 95.882 0.000 0.000 0.000 1.001 5.276 113.718 106.582

Component Flowrates (kg/batch)

Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

Component Flowrates (kg/batch) Amm. Sulfate HAeq Injection Water KCl KH2PO4 Na2HPO4 NaH2PO4 Sodium Chloride TOTAL (kg/batch) TOTAL (L/batch)

24

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

S-601 P-33 P-34

S-602 UF-601 Filtrate P-34 P-35 P-35 OUTPUT

S-603 P-35 P-36

0.00 25.00 10.12 1,066.95 0.00 0.00 0.90

0.00 25.00 2.16 1,066.95 0.00 0.00 0.90

0.00 31.53 2.16 1,065.25 0.75 5.95 0.91

0.00 31.53 2.16 1,051.60 0.03 4.90 0.75

10.562 0.996 95.882 1.001 5.276 113.718 106.582

10.562 0.996 95.882 1.001 5.276 113.718 106.582

10.129 0.004 91.949 0.960 5.060 108.101 101.479

0.433 0.992 3.934 0.041 0.216 5.617 5.341

Component Flowrates (kg/batch) Amm. Sulfate HAeq Injection Water NaH2PO4 Sodium Chloride TOTAL (kg/batch) TOTAL (L/batch)

Stream Name Source Destination Stream Properties Activity (U/ml) Temperature (°C) Pressure (bar) Density (g/L) Total Enthalpy (kW-h) Specific Enthalpy (kcal/kg) Heat Capacity (kcal/kg-°C)

S-604 P-36 OUTPUT

S-605 To Formulation P-36 P-37 P-37 OUTPUT

0.00 32.28 2.16 1,061.61 0.03 6.35 0.87

0.00 32.28 2.16 1,028.40 0.01 3.43 0.47

0.00 32.28 2.31 1,028.41 0.01 3.42 0.47

0.351 0.169 3.186 0.033 0.175 3.914 3.687

0.082 0.824 0.747 0.008 0.041 1.702 1.655

0.082 0.824 0.747 0.008 0.041 1.702 1.655

Component Flowrates (kg/batch) Amm. Sulfate HAeq Injection Water NaH2PO4 Sodium Chloride TOTAL (kg/batch) TOTAL (L/batch)

25

Equipment List and Specifications Table 5: Equipment Summary

1. EQUIPMENT SUMMARY (2014 prices) Name

Type

DE-101

Dead-End Filter Rocking Bioreactor Skid Rocking Bioreactor Skid Disposable Generic Container Skid Shake Flask Rack Shake Flask Rack Test Tube Rack Shake Flask Rack Shake Flask Rack Disposable Bioreactor Skid Dead-End Filter Disposable Generic Container Skid Disposable Bioreactor Skid Blending Tank Blending Tank Disk-Stack Centrifuge Dead-End Filter Ultrafilter Skid for Disposable Large Bag Skid for Disposable Large Bag GFL Chromatography Column

BBS-101a BBS-102a DCS-102 SFR-104 SFR-103 TTR-101 SFR-101 SFR-102 DBS-101 DE-201 DCS-201 DBS-201 V-301 V-303 DS-301 DE-308 UF-401 SDLB-401

SDLB-403

C-401

1

Standby/ Staggere 0/0 d

1

0/1

1

Units

Size (Capacity) 2.41 m2

Material of Purchase Construction Cost ($/Unit) SS316

25,000

20.00 L

CS

176,000

0/1

100.00 L

CS

557,000

1

0/0

200.00 L

CS

1,000

1

0/1

2.00 L

CS

5,000

1

0/1

0.50 L

CS

4,000

1

0/0

0.01 L

CS

0

1

0/1

0.13 L

CS

4,000

1

0/1

2.00 L

CS

4,000

1

0/3

700.00 L

CS

215,000

1

0/3

SS316

25,000

1

0/3

1,600.00 L

CS

1,000

1

0/3

3,000.00 L

CS

226,000

1 1

0/0 0/0

1,977.02 L 1,911.14 L

SS316 SS316

215,000 214,000

1

0/0

1,587.25 L/h

SS316

299,000

1 1

0/0 0/0

10.00 m2 2.50 m2

SS316 SS316

41,000 29,000

18

0/0

100.00 L

SS316

3,000

1

0/0

100.00 L

SS316

3,000

1

0/0

688.11 L

SS316

629,000

0.06 m2

26

DCS-401

DCS-402

DCS-501

DCS-502

DCS-503

DCS-504

C-501

Disposable Generic Container Skid Disposable Generic Container Skid Disposable Generic Container Skid Disposable Generic Container Skid Disposable Generic Container Skid Disposable Generic Container Skid PBA Chromatography Column

1

0/0

1,644.00 L

CS

1,000

1

0/0

100.00 L

CS

1,000

1

0/0

111.00 L

CS

1,000

1

0/0

222.00 L

CS

1,000

1

0/0

111.00 L

CS

1,000

1

0/0

111.00 L

CS

1,000

1

0/0

49.81 L

SS316

355,000

118.43 L

SS316

150,000

SS316

29,000

SS316

3,000

V-501

Blending Tank

1

0/0

UF-601

Ultrafilter

1

0/0

SDLB-601

Skid for Disposable Large Bag

2

0/0

DE-601

Dead-End Filter

1

0/0

10.00 m2

SS316

41,000

MF-601

Microfilter

1

0/0

0.05 m2

SS316

26,000

1

0/0

3.00 L

CS

1,000

18

0/0

100.00 L

SS316

3,000

SS316

41,000

SS316

3,000

DCS-601

SDLB-402

Disposable Generic Container Skid Skid for Disposable Large Bag

DE-102

Dead-End Filter

1

0/0

SDLB-402a

Skid for Disposable Large Bag

17

0/0

2.50 m2

100.00 L

10.00 m2

100.00 L

27

Summary of Capital Requirements and Manufacturing Costs

2. MAJOR EQUIPMENT SPECIFICATIONS AND FOB COST (2014 prices) Quantity/ Standby/ Staggered

Name

Description

1/0/0

DE-101

1/0/1

BBS-101a

1/0/1

BBS-102a

1/0/0

DCS-102

1/0/1

SFR-104

1/0/1

SFR-103

1/0/1

SFR-101

1/0/1

SFR-102

1/0/3

DBS-101

1/0/3

DE-201

1/0/3

DCS-201

1/0/3

DBS-201

1/0/0

V-301

1/0/0

V-303

1/0/0

DS-301

1/0/0

DE-308

1/0/0

UF-401

18 / 0 / 0

SDLB-401

1/0/0

SDLB-403

1/0/0

C-401

1/0/0

DCS-401

Dead-End Filter Filter Area = 2.41 m2 Rocking Bioreactor Skid Container Volume = 20.00 L Rocking Bioreactor Skid Container Volume = 100.00 L Disposable Generic Container Skid Container Volume = 200.00 L Shake Flask Rack Container Volume = 2.00 L Shake Flask Rack Container Volume = 0.50 L Shake Flask Rack Container Volume = 0.13 L Shake Flask Rack Container Volume = 2.00 L Disposable Bioreactor Skid Container Volume = 700.00 L Dead-End Filter Filter Area = 0.06 m2 Disposable Generic Container Skid Container Volume = 1600.00 L Disposable Bioreactor Skid Container Volume = 3000.00 L Blending Tank Vessel Volume = 1977.02 L Blending Tank Vessel Volume = 1911.14 L Disk-Stack Centrifuge Throughput = 1587.25 L/h Dead-End Filter Filter Area = 10.00 m2 Ultrafilter Membrane Area = 2.50 m2 Skid for Disposable Large Bag Container Volume = 100.00 L Skid for Disposable Large Bag Container Volume = 100.00 L GFL Chromatography Column Column Volume = 688.11 L Disposable Generic Container Skid Container Volume = 1644.00 L

Unit Cost ($)

Cost ($)

25,000

25,000

176,000

352,000

557,000

1,114,000

1,000

1,000

5,000

10,000

4,000

8,000

4,000

8,000

4,000

8,000

215,000

860,000

25,000

100,000

1,000

4,000

226,000

904,000

215,000

215,000

214,000

214,000

299,000

299,000

41,000

41,000

29,000

29,000

3,000

54,000

3,000

3,000

629,000

629,000

1,000

1,000

28

1/0/0

DCS-402

1/0/0

DCS-501

1/0/0

DCS-502

1/0/0

DCS-503

1/0/0

DCS-504

1/0/0

C-501

1/0/0

V-501

1/0/0

UF-601

2/0/0

SDLB-601

1/0/0

DE-601

1/0/0

MF-601

1/0/0

DCS-601

18 / 0 / 0

SDLB-402

1/0/0

DE-102

17 / 0 / 0

SDLB-402a

Disposable Generic Container Skid Container Volume = 100.00 L Disposable Generic Container Skid Container Volume = 111.00 L Disposable Generic Container Skid Container Volume = 222.00 L Disposable Generic Container Skid Container Volume = 111.00 L Disposable Generic Container Skid Container Volume = 111.00 L PBA Chromatography Column Column Volume = 49.81 L Blending Tank Vessel Volume = 118.42 L Ultrafilter Membrane Area = 2.50 m2 Skid for Disposable Large Bag Container Volume = 100.00 L Dead-End Filter Filter Area = 10.00 m2 Microfilter Membrane Area = 0.05 m2 Disposable Generic Container Skid Container Volume = 3.00 L Skid for Disposable Large Bag Container Volume = 100.00 L Dead-End Filter Filter Area = 10.00 m2 Skid for Disposable Large Bag Container Volume = 100.00 L Unlisted Equipment

1,000

1,000

1,000

1,000

1,000

1,000

1,000

1,000

1,000

1,000

355,000

355,000

150,000

150,000

29,000

29,000

3,000

6,000

41,000

41,000

26,000

26,000

1,000

1,000

3,000

54,000

41,000

41,000

3,000

51,000

TOTAL

1,408,000 7,042,000

Table 6: Major Equipment Specs and FOB cost

29

3. FIXED CAPITAL ESTIMATE SUMMARY (2014 prices in $) 3A. Total Plant Direct Cost (TPDC) (physical cost) 1. Equipment Purchase Cost 2. Installation 3. Process Piping 4. Instrumentation 5. Insulation 6. Electrical 7. Buildings 8. Yard Improvement 9. Auxiliary Facilities TPDC

7,042,000 6,167,000 2,465,000 2,817,000 211,000 704,000 3,169,000 1,056,000 2,817,000 26,449,000

3B. Total Plant Indirect Cost (TPIC) 10. Engineering 11. Construction TPIC

6,612,000 9,257,000 15,869,000

3C. Total Plant Cost (TPC = TPDC+TPIC) TPC

42,318,000

3D. Contractor's Fee & Contingency (CFC) 12. Contractor's Fee 13. Contingency CFC = 12+13

2,116,000 4,232,000 6,348,000

3E. Direct Fixed Capital Cost (DFC = TPC+CFC) DFC

48,666,000 Table 7: FCI Summary

4. LABOR COST - PROCESS SUMMARY Labor Type Operator TOTAL

Unit Cost Annual Amount ($/h) (h) 69.00

5,901 5,901

Annual Cost ($)

%

407,202 407,202

100.00 100.00

Table 8: Labor Summary

30

5. MATERIALS COST - PROCESS SUMMARY Bulk Material AEC Eq Buffer AEC El Buff AEC Strip Buffe AEC Wash Buffer Amm. Sulfate Media Injection Water Biomass Air HAeq H3PO4 (5% w/w) NaOH (0.5 M) WFI B-Propiolactone IEX-El-Buff TOTAL

Unit Cost ($) 0.000 0.000 0.000 0.000 8.000 300.000 1.000 0.000 0.000 0.000 1.535 0.815 0.300 14.000 1.145

Annual Amount

299 613 306 300 32 182 5,102 0 61,398 0 3,855 2,661 11,862 38,294 4,552

kg kg kg kg kg kg kg kg kg kg kg kg kg g kg

Annual Cost ($)

%

0 0 0 0 253 54,645 5,102 0 0 0 5,918 2,170 3,559 536,116 5,213 612,975

0.00 0.00 0.00 0.00 0.04 8.91 0.83 0.00 0.00 0.00 0.97 0.35 0.58 87.46 0.85 100.00

NOTE: AEC Buffers mixtures are composed of listed components and accounted for via their individual costs here Table 9: Materials Cost

6. VARIOUS CONSUMABLES COST (2014 prices) - PROCESS SUMMARY Consumable 20 L Cell Bag 200 L Bag 100 L Cell Bag 2000 mL Shake Flask 5 mL Test Tube 125 mL Shake Flask 500 mL Poly Shake Flask Dft Stirred Bioreactor Bag 3000L Dft DEF Cartridge Dft Membrane Dft Large Bag Dft Gel Filtration Resin 1 L Plastic Bag Dft PBA Chrom Resin MF Membrane (Biotech) FlexBoy Bag 3.0 L UF 750kDa SE Membrane TOTAL

Units Cost ($)

Annual Amount

700.000 300.000 1,850.000 1.800 0.500 1.226 1.160 6,220.000 8,600.000 1,000.000 400.000 340.000 2,000.000 0.200 1,500.000 735.835 30.000 981.120

3 27 3 1 3 0 3 3 3 9 0 168 41 6,897 4 0 3 15

item item item item item item item item item item m2 item L item L m2 item m2

Annual Cost ($)

%

2,100 8,100 5,550 1 2 0 3 18,660 25,800 9,000 16 57,120 82,573 1,379 5,603 106 90 14,717 230,821

0.91 3.51 2.40 0.00 0.00 0.00 0.00 8.08 11.18 3.90 0.01 24.75 35.77 0.60 2.43 0.05 0.04 6.38 100.00

Table 10: Consumables Cost

31

7. WASTE TREATMENT/DISPOSAL COST (2014 prices) - PROCESS SUMMARY Waste Category Solid Waste Aqueous Liquid S-303 S-305 SEC Waste Stream AEC Waste UF-601 Filtrate S-604 P-17:CIP-1(Pre Rinse) Organic Liquid Emissions TOTAL

Unit Cost ($) 5.000 5.000 5.000 5.000 5.000 5.000 5.000

Annual Amount 217 1 4,482 1,482 324 12 1,224

kg kg kg kg kg kg kg

Annual Cost ($)

%

0 38,714 1,087 7 22,408 7,412 1,622 59 6,120 0 0 38,714

0.00 100.00 2.81 0.02 57.88 19.15 4.19 0.15 15.81 0.00 0.00 100.00

Table 11:Waste Treatment/Disposal Costs

8. UTILITIES COST (2014 prices) - PROCESS SUMMARY Utility Electricity Steam Cooling Water Chilled Water TOTAL

Unit Cost ($)

Annual Amount

Ref. Units

Annual Cost ($)

%

0.050 12.000 0.050 0.400

6,200 1 0 780

kW-h MT MT MT

378 18 0 312 707

53.37 2.52 0.00 44.11 100.00

Table 12: Utilities Costs

32

9. ANNUAL OPERATING COST (2014 prices) - PROCESS SUMMARY Cost Item Direct Manufacturing Costs Raw Materials Waste Treatment/Disposal Utilities Operating Labor Supervisory and Clerical Labor Maintenance and Repairs Operating Supplies Laboratory/QC/QA Patents and Royalties TOTAL DMC

$

Correlation

612,975 39,000 707 407,202 73,296 2,905,140 435,771 61,080 507,660 5,042,545

CRM CWT CUT COL

.009*FCI .15*COL .03*COM

Fixed Manufacturing Costs Local Taxes and Insurance Plant Overhead Costs Depreciation TOTAL FMC

1,557,312 2,040,275 6,604,671 3,597,587

.032*FCI .708*COL+.036*FCI (DFC-.05DFC)/7

1,158,741 1,838,077 835,490 3,832,308 16,701,045 12,472,726

.177*COL .11*COM .05*COM

General Manufacturing Costs Administration Costs Advertising/Selling Research and Development TOTAL GE ESTIMATED COSTS TOTAL COM

***

Table 13: Annual operating costs and correlations used to estimate unknown values (Turton 1998)

***CRM+CWT+CUT+2.215COL+.19COM+.246FCI = COM

33

Profitability analysis

10. PROFITABILITY ANALYSIS (2014 prices) A. B. C. D. E. F. G.

Direct Fixed Capital Working Capital Startup Cost Up-Front R&D Up-Front Royalties Total Investment (A+B+C+D+E) Investment Charged to This Project

H.

Revenue/Savings Rates HAeq in 'To Formulation' (Main Revenue)

I.

2,471 g HAeq/yr

Revenue/Savings Price HAeq in 'To Formulation' (Main Revenue)

J.

Revenues/Savings

1 2

HAeq in 'To Formulation' (Main Revenue) Total Revenues Total Savings

K.

Annual Operating Cost (AOC)

1 2

Actual AOC Net AOC (K1-J2)

L.

Unit Production Cost /Revenue

248,889.00 $/g HAeq

614,922,153 $/yr 614,922,153 $/yr 0 $/yr

12,472,726 $/yr 12,472,726 $/yr

Unit Production Cost Net Unit Production Cost Unit Production Revenue M. N. O.

48,666,000 $ 615,000 $ 2,433,000 $ 835,490 $ 507,660 $ 53,058,150 $ 53,058,150 $

5,047.64 $/g MP 5,047.64 $/g MP 248,889.00 $/g MP

Gross Profit (J-K) Taxes (40%) Net Profit (M-N + Depreciation)

602,449,427 $/yr 240,979,771 $/yr 368,109,000 $/yr

Gross Margin Return On Investment Payback Time MP = Flow of Component 'HAeq' in Stream 'To Formulation'

97.98 % 711.81 % 0.14 years

Table 14: Profitability analysis

34

Feasibility Analysis CASH FLOW ANALYSIS (thousand $)

Year -2 -1 0 1 2 3 4 5 6 7 8 9 10

Capital Investment

Debt Sales Operating Finance Revenues Cost

- 14,600 - 19,466 - 14,600 - 615 0 0 0 0 0 0 0 0 3,049

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 614,922 614,922 614,922 614,922 614,922 614,922 614,922 614,922 614,922 614,922

0 0 0 12,427 12,427 12,427 12,427 12,427 12,427 12,427 5,822 5,822 5,822

Gross Loan Depreciation Profit Payments 0 0 0 602,449 602,449 602,449 602,449 602,449 602,449 602,449 609,112 609,112 609,112

0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 6,605 6,605 6,605 6,605 6,605 6,605 6,605 0 0 0

Taxable Income

Taxes

Net Profit

Net Cash Flow

0 0 0 602,449 602,449 602,449 602,449 602,449 602,449 602,449 609,112 609,112 609,112

0 0 0 240,979 240,979 240,979 240,979 240,979 240,979 240,979 243,645 243,645 243,645

0 0 0 361,469 368,109 368,109 368,109 368,109 368,109 368,109 365,467 365,467 365,467

- 14,600 - 19,466 - 14,600 360,857 368,109 368,109 368,109 368,109 368,109 368,109 365,467 365,467 368,516

IRR/NPV SUMMARY IRR Before Taxes IRR After Taxes

241.80 % 189.92 %

Interest % NPV

7.00 2,209,762.00

9.00 1,941,165.00

11.00 1,713,411.00

Depreciation Method: Straight-Line DFC Salvage Fraction: 0.050

Table 15: Table of Cash Flows

Figure 13: Cash flow diagram

Figure 12: Discounted cumulative cash flow

35

Safety and Operability Considerations

Safety, Health, and Environmental Considerations

Influenza vaccine manufacture safety and health considerations are defined by the FDA. The WHO discusses a two-step approach for production-scale vaccine development. The first step requires identification of hazards. The second step outlines risk management techniques. Hazard Identification Hazard identification is dependent on the vaccine strain and production method. There are several considerations for an inactivated CCIV method detailed as follows. The use of ‘wild’ strain types of viruses for pandemic vaccine production has the possibility of presenting a high level biosafety risks. The level of risk is dependent on the virus strain. The high volumes/titers in the production-scale process further increases the risks. In CCIV production hazards, such as potential spills and contaminated waste disposal, are present during viral input and product removal from the production bioreactor. On a lesser note, but nonetheless import, viral mutations during passaging may also pose a risk. Risk Assessment Potential to harm personnel: Personnel should be limited in exposure to high titer process materials. Any individuals performing labor on the process should be vaccinated against seasonal influenza strains and any strains they may be exposed to. Antiviral treatment is available in case of infection by strains of focus. Environmental protection: Several species of animals are susceptible endemic infection by Influenza A such as farm animals and shorebirds. Sporadic infection is prevalent in a variety of other animals. Of all the animals, pigs are the most susceptible. Because of their receptor content they may be infected by virtually any strain. These concerns are particularly relevant to facility location and personnel contact. Facility construction is ideally limited to areas isolated from endemic species. Personnel are instructed to avoid endemic species for a minimum of 14 days following exposure in the workplace. The disposal of high titer waste will defer to local safety regulations regarding the disposal of waste designated as infectious. Ideally, decontamination should occur on site. Facility Requirements The biosafety level (BSL) required by the facility is dependent on the virus strain used. Table 1 lists the BSL requirements for specified virus strains.

36

Figure 14: Comparison of different vaccines and their respective BSLs (cite req:http://www.who.int/biologicals/publications/trs/areas/vaccines/influenza/Annex%205%20human%20pandemic%20influenza.pdf)

The following list designates BSL-3 measures required for facilities and personnel involved with influenza vaccine manufacture as dictated by the WHO. BSL-3 Facility:  

Biosafety cabinets employing negative relative pressure should be employed when possible; HEPA filtration of air should be employed prior to exhaust ventilation out of the facility or into public areas.

37



Incorporation of positively pressure work environments with negative pressure sink areas built into the ventilation system.

Decontamination should be performed in accordance with the following criteria:  

Any waste generated from BSL-2+ areas (such as those working with pandemic influenza); All manufacturing and quality control areas should be decontaminated at the end of an annual production cycle via cleaning and verification of effective decontamination of areas.

Personnel:  

  

Personal protective equipment (PPE) should consist of laboratory clothing covering all of the skin (Tyvek overalls, for example) and should be worn in the BSL-2+ areas working with production of pandemic influenza vaccine. Should the tasks being performed not be containable by primary containment protocol, respiratory protective equipment, like N95, FFP3 or similar respiration devices should be worn. Any minimum specifications for the filtration capabilities of such equipment should be observed, and all masks should be correctly sized for the user. All workers must sign a written document expressing their understanding that they must not contact any farm animals or birds for at least 14 days after their last time at the facility. All personnel should receive vaccination against seasonal influenza strains using inactivated virus. The workers must have antiviral treatment available if it is needed.

Quality Control of Decontamination: 



Cleaning and decontamination methods need to be validated periodically as part of a master validation plan to demonstrate that the protocols, reagents and equipment used are effective in the inactivation of pandemic influenza virus on facility and equipment surfaces, garments of personnel and waste materials, and within cell growth and storage containers. Once decontamination procedures for influenza virus have been fully described and validated, there is no need to repeat them for each new strain. Validation studies using influenza viruses may be supplemented by studies with biological (for example bacterial) markers selected to be more difficult to inactivate than influenza. Methods employed in cleaning and contamination should be validated as per a predetermined plan in order to demonstrate the procedures and equipment used are sufficient to inactivate the pandemic influenza virus that may be present on surfaces and equipment in the facility. Studies on validation for influenza strains may also be supplemented with data from bacteria that may be more resilient than the strains in question.

A proposed floor plan to facilitate BSL-3 protocols is shown in Figure 15.

38

Figure 15: Proposed floor plan of facility

39

Conclusions and Recommendations

Pressure on large biopharmaceutical companies involved in the manufacture of influenza vaccine is growing. Traditional egg-based technologies are not able to efficiently keep up with an expanding population and the increased response requirements that a pandemic of such a magnitude would elicit. Using CCIV technology is a feasible option that has already emerged on the market place. CHO cells elicit favorable kinetics and scalability incorporable into an animal free facility. Antigenicity is actually improved from traditional egg-based techniques through ‘live’ virus infection with beta-propiolactone inactivation at a comparable yield and presents a technically and economically viable alternative. Economically, the plant is highly profitable boasting a NPV of $2.2 billion over a 10 year plant life for a 7% discounting factor. A relatively low capital investment of $53 million is achieved through the incorporation of single-use disposable equipment. Recommendations • Gather experimental data in order to: - Effectively assess and optimize separations - Accurately quantify viral protein production - Further characterize protein slurry composition • Possible investigation into related techniques: - viral “splitting” - recombinant production profitability • More extensive experimental optimization of cell line and media for this specific process

40

Appendices Appendix A: Scheduling Optimization

day

3

h

56

6 112

9 168

224

12 280

15 336

18 392

21 448

504

24 560

27 616

30 672

728

33 784

36 840

39 896

42 952

1008

45 1064

48 1120

51 1176

1232

54

day

1288

h

Complete Recipe P-01 in T T R-101 P-02 in SFR-101 P-03 in SFR-102 P-04 in SFR-103 P-05 in SFR-104 P-06 in BBS-101a P-07 in BBS-102a P-08 in DCS-102 P-10 in DBS-101 P-09 in DE-101 P-11 in DCS-201 P-13 in DBS-201 P-12 in DE-201 P-14 in V-301 P-15 in DS-301 P-17 in V-303 P-16 in DE-308 P-18 in SDLB-401 P-19 in DE-102 P-20 in SDLB-402 P-21 in UF-401 P-22 in SDLB-402a P-23 in SDLB-403 P-25 in C-401 P-24 in DCS-401 P-26 in DCS-402 P-31 in C-501 P-28 in DCS-502 P-32 in V-501 P-30 in DCS-504 P-29 in DCS-503 P-27 in DCS-501 P-33 in DE-601 P-34 in SDLB-601 P-35 in UF-601 P-36 in MF-601 P-37 in DCS-601

Figure 16: Single batch Gantt chart

Bottlenecking factor: seed and production bioreactors, with 290 hr and 296.45 hr operating times, respectively.

41

day

4

8

12

16

20

24

28

32

36

40

h

96

192

288

384

480

576

672

768

864

960

44

48

52

56

60

64

68

72

76

80

84

88

92

96

100

104

108

1056 1152 1248 1344 1440 1536 1632 1728 1824 1920 2016 2112 2208 2304 2400 2496 2592

day h

Complete Recipe Complete Recipe (Batch #2 ) Complete Recipe (Batch #3 ) Complete Recipe (Batch #4 ) Complete Recipe (Batch #5 ) Complete Recipe (Batch #6 ) Complete Recipe (Batch #7 ) Complete Recipe (Batch #8 ) Complete Recipe (Batch #9 ) Complete Recipe (Batch #10) Complete Recipe (Batch #11) Complete Recipe (Batch #12) Complete Recipe (Batch #13) Complete Recipe (Batch #14) Complete Recipe (Batch #15)

Figure 17: Gantt chart for running multiple theoretical batches

After debottlenecking, subsequent batches can be carried out within about 74 hours after the start of the previous batch through utilization of parallel sets of equipment in the seed train phase.

42

Appendix B: Assumptions     

Titer yields for recombinant and whole virus are approximately equal Experimental growth kinetics data are directly scalable to production-scale process Suspension adapted CHO cells are commercially available No viral mutation in seed train and production bioreactor Experimental data for the separating units is scalable

43