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

iv

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

v

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

vi

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.

1

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

identify and eliminate any instances of the live virus that may be

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,

they must be heavily monitored by teams of

veterinarians and maintained under strict sterile protocols. The demand for influenza vaccines

Figure 1: Graphical representation of the

antigen-antibody concept

Figure 2: Structure of an influenza virus particle.

2

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 production-

scale processes because they are more easily scaled.

3

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.

4

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 beta-

propiolactone, which alters the composition of the viruses biological components, and causes the

5

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 pre-

sterilized 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 pre-

sterilized disposable bags as the inoculum container.

Figure 4: GE WAVE Bioreactor

6

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 on-

site 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 pre-

sewage 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.

7

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.

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.

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10 12 14

Dis

trib

ute

d (

mill

ion

s)

Year (2001-2013)

Doses Distributed

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

8

Year Distributed (millions)

Sanofi Market Share (millions)

Hemagglutinin Concentration (g)

2000-01 70.4

2001-02 77.7

2002-03 83.5

2003-04 83.1

2004-05 57

2006-07 81.5

2007-08 102.5

2008-09 112.8 42.3 1903.5

2009-10

2010-11

2011-12

2012-13 134.9

Projected

2013-14 139.4 52.275 2352.375

2014-15 145.2 54.45 2450.25

Actual Data

Projected Values 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.

9

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.

10

Results

Process Flow Diagrams

Figure 5: Overall PFD of process showing phases

11

Figure 6: Seed train phase of the process.

12

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

13

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

14

Figure 9: Ultrafiltration phase, followed by SEC

15

Figure 10: Anion exchange chromatography phase

16

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

17

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 AEC Eq Buffer 299 99.563 0.121

AEC El Buff 613 204.319 0.248

AEC Strip Buffe 306 101.890 0.124

AEC Wash Buffer 300 100.003 0.121

Amm. Sulfate 32 10.562 0.013

TOTAL 1,549 516.337 0.627

Seed Train Material kg/yr kg/batch kg/g MP Media 105 34.861 0.042

Injection Water 476 158.754 0.193

Biomass 0 0.000 0.000

Air 3,168 1,056.072 1.282

TOTAL 3,749 1,249.687 1.517

Production Bioreactor Material kg/yr kg/batch kg/g MP Injection Water 4,625 1,541.792 1.872

Media 78 25.856 0.031

Air 58,229 19,409.791 23.568

TOTAL 62,932 20,977.439 25.472

Clarification and Inactivation Material kg/yr kg/batch kg/g MP H3PO4 (5% w/w) 3,855 1,285.028 1.560

NaOH (0.5 M) 2,661 887.005 1.077

WFI 11,862 3,953.993 4.801

B-Propiolactone 38 12.765 0.015

TOTAL 18,416 6,138.790 7.454

Ultrafiltration and SEC Material kg/yr kg/batch kg/g MP IEX-El-Buff 4,552 1,517.292 1.842

TOTAL 4,552 1,517.292 1.842 Table 3:Bulk Material Analysis by Section

18

Table 4: Stream Analysis

Stream Analysis

Stream Name WCB Vial Thaw S-101 SFR-101M S-102 Source INPUT P-01 INPUT P-02 Destination P-01 P-02 P-02 P-03 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 24.99 25.00 37.00

Pressure (bar) 1.01 1.29 1.01 1.26

Density (g/L) 994.70 994.71 994.70 990.33

Total Enthalpy (kW-h) 0.00 - 0.00 0.00 0.00

Specific Enthalpy (kcal/kg) 0.00 - 0.01 0.00 11.98

Heat Capacity (kcal/kg-°C) 1.00 1.00 1.00 1.00

Component Flowrates (kg/batch) Biomass 0.000 0.000 0.000 0.003

Media 0.001 0.001 0.019 0.006

Water 0.000 0.000 0.000 0.011

TOTAL (kg/batch) 0.001 0.001 0.019 0.020

TOTAL (L/batch) 0.001 0.001 0.019 0.020

Stream Name SFR-102M S-103 SFR-103M S-104 Source INPUT P-03 INPUT P-04 Destination P-03 P-04 P-04 P-05 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 37.00 25.00 37.00

Pressure (bar) 1.01 1.10 1.01 2.95

Density (g/L) 994.70 990.33 994.70 990.33

Total Enthalpy (kW-h) 0.00 0.00 0.00 0.00

Specific Enthalpy (kcal/kg) 0.00 11.98 0.00 11.98



Media 0.060 0.020 0.239 0.078

Water 0.000 0.048 0.000 0.193

TOTAL (kg/batch) 0.060 0.080 0.239 0.318

TOTAL (L/batch) 0.060 0.080 0.240 0.321

Stream Name SFR-104M S-105 Media BBS-101a

Vent 101a

Source INPUT P-05 INPUT P-06 Destination P-05 P-06 P-06 OUTPUT Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 37.00 25.00 37.00

Pressure (bar) 1.01 2.95 1.01 1.01

Density (g/L) 994.70 990.33 994.70 1.67


19




Carb. Dioxide 0.000 0.000 0.000 0.316

Media 0.955 0.310 5.699 0.000

Nitrogen 0.000 0.000 0.000 0.017

Oxygen 0.000 0.000 0.000 0.005

Water 0.000 0.771 0.000 0.000

TOTAL (kg/batch) 0.955 1.273 5.699 0.338

TOTAL (L/batch) 0.960 1.286 5.729 202.082

Stream Name S-106 Media BBS-102a

Vent 102a S-107

Source P-06 INPUT P-07 P-07 Destination P-07 P-07 OUTPUT P-10 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 37.00 25.00 37.00 37.00

Pressure (bar) 1.01 1.01 1.01 1.01

Density (g/L) 990.33 994.70 1.68 990.33





Carb. Dioxide 0.000 0.000 1.864 0.000

Impurities 0.168 0.000 0.000 0.960

Media 1.803 22.926 0.000 4.946

Nitrogen 0.000 0.000 0.085 0.000

Oxygen 0.000 0.000 0.026 0.000

Water 3.925 0.000 0.000 18.762

TOTAL (kg/batch) 6.636 22.926 1.975 27.583

TOTAL (L/batch) 6.700 23.048 1,175.867 27.853

Stream Name S-108 S-109 S-110 S-111 Source INPUT INPUT P-08 P-09 Destination P-08 P-08 P-09 P-10 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.00 25.00 25.00

Pressure (bar) 1.01 1.01 5.75 5.75

Density (g/L) 994.70 994.70 994.70 994.70




Component Flowrates (kg/batch) Injection Water 158.754 0.000 158.754 158.754

Media 0.000 4.963 4.963 4.963

TOTAL (kg/batch) 158.754 4.963 163.717 163.717

TOTAL (L/batch) 159.599 4.989 164.589 164.589

20

Stream Name DBS-101 Air Inlet

DBS-101 Vent S-112 S-201

Source INPUT P-10 P-10 INPUT Destination P-10 OUTPUT P-13 P-11 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 37.00 37.00 25.00

Pressure (bar) 1.01 1.01 1.01 1.01

Density (g/L) 1.18 1.13 990.33 994.70





Carb. Dioxide 0.000 0.693 0.000 0.000

Impurities 0.000 0.000 1.237 0.000

Injection Water 0.000 0.000 158.754 1,541.792

Media 0.000 0.000 2.973 0.000

Nitrogen 810.132 810.324 0.000 0.000

Oxygen 245.940 245.999 0.000 0.000

Water 0.000 0.000 23.618 0.000

TOTAL (kg/batch) 1,056.072 1,057.015 190.607 1,541.792

TOTAL (L/batch) 895,566.922 932,233.615 192.468 1,550.000

Stream Name S-202 S-203 S-205 DBS-201 Air Inlet Source INPUT P-11 P-12 INPUT

Destination P-11 P-12 P-13 P-13 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.00 25.00 25.00

Pressure (bar) 1.01 67.39 67.39 1.01

Density (g/L) 994.70 994.70 994.70 1.18




Component Flowrates (kg/batch) Injection Water 0.000 1,541.792 1,541.792 0.000

Media 25.856 25.856 25.856 0.000

Nitrogen 0.000 0.000 0.000 14,889.598

Oxygen 0.000 0.000 0.000 4,520.193

TOTAL (kg/batch) 25.856 1,567.648 1,567.648 19,409.791

TOTAL (L/batch) 25.994 1,575.994 1,575.994 16,459,833.447

21

Stream Name Vent-5 S-206 S-301 S-302

Source P-13 P-13 P-14 P-15 Destination OUTPUT P-14 P-15 P-16 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 37.00 37.00 37.00 42.13

Pressure (bar) 1.01 1.01 10.51 1.01

Density (g/L) 1.13 990.33 990.33 988.46





Carb. Dioxide 14.520 0.000 0.000 0.000

HAeq 0.000 1.471 1.471 1.419

Impurities 0.000 1.972 1.972 1.903

Injection Water 0.000 1,700.546 1,700.546 1,640.809

Media 0.000 10.447 10.447 10.080

Nitrogen 14,891.250 0.000 0.000 0.000

Oxygen 4,502.315 0.000 0.000 0.000

Water 0.000 36.485 36.485 35.203

TOTAL (kg/batch) 19,408.085 1,762.115 1,762.115 1,689.638

TOTAL (L/batch) 17,117,990.006 1,779.320 1,779.318 1,709.360

Stream Name S-303 S-304 S-305 S-306

Source P-15 P-16 P-16 INPUT Destination OUTPUT P-17 OUTPUT P-17 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 42.13 42.13 42.13 25.00

Pressure (bar) 1.01 1.01 1.01 1.01

Density (g/L) 988.46 988.46 988.46 1,146.00




Component Flowrates (kg/batch) B-Propiolactone 0.000 0.000 0.000 12.765

Biomass 10.970 0.000 0.224 0.000

HAeq 0.052 1.419 0.000 0.000

Impurities 0.069 1.903 0.000 0.000

Injection Water 59.737 1,640.592 0.217 0.000

Media 0.367 10.078 0.001 0.000

Water 1.282 35.199 0.005 0.000

TOTAL (kg/batch) 72.477 1,689.190 0.448 12.765

TOTAL (L/batch) 73.323 1,708.907 0.453 11.138

22


Source P-17 P-18 P-19 P-20 Destination P-18 P-19 P-20 P-21 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 42.09 42.08 42.08 42.08

Pressure (bar) 10.70 24.13 24.13 24.13

Density (g/L) 989.50 989.50 989.50 989.50





HAeq 1.419 1.419 1.419 1.419

Impurities 1.903 1.903 1.903 1.903

Injection Water 1,640.592 1,640.592 1,640.592 1,640.592

Media 10.078 10.078 10.078 10.078

Water 35.199 35.199 35.199 35.199

TOTAL (kg/batch) 1,701.955 1,701.955 1,701.955 1,701.955

TOTAL (L/batch) 1,720.022 1,720.019 1,720.019 1,720.015

Stream Name S-404 S-405 S-406 SEC Elute Buffer Source P-21 P-21 P-23 INPUT

Destination P-22 P-23 P-25 P-24 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 42.48 42.48 42.47 25.00

Pressure (bar) 24.13 24.13 3.44 1.01

Density (g/L) 989.35 989.33 989.34 1,025.59





HAeq 0.007 1.412 1.412 0.000

Impurities 1.828 0.075 0.075 0.000

Injection Water 1,576.277 64.315 64.315 1,424.062

Media 9.683 0.395 0.395 0.000

NaH2PO4 0.000 0.000 0.000 14.869

Sodium Chloride 0.000 0.000 0.000 78.361

Water 33.819 1.380 1.380 0.000

TOTAL (kg/batch) 1,633.878 68.077 68.077 1,517.292

TOTAL (L/batch) 1,651.458 68.811 68.811 1,479.427

23

Stream Name S-407 SEC Waste

Stream S-408 S-409

Source P-24 P-25 P-25 P-26 Destination P-25 OUTPUT P-26 P-31 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.80 25.00 25.00

Pressure (bar) 10.12 3.44 3.44 9.46

Density (g/L) 1,025.59 1,023.92 1,025.18 1,025.18





HAeq 0.000 0.212 1.200 1.200

Impurities 0.000 0.075 0.000 0.000

Injection Water 1,424.062 1,403.596 84.781 84.781

Media 0.000 0.395 0.000 0.000

NaH2PO4 14.869 13.984 0.885 0.885

Sodium Chloride 78.361 73.695 4.665 4.665

Water 0.000 1.380 0.000 0.000

TOTAL (kg/batch) 1,517.292 1,493.837 91.532 91.532

TOTAL (L/batch) 1,479.427 1,458.937 89.284 89.284


Source INPUT INPUT INPUT INPUT Destination P-27 P-28 P-29 P-30 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.00 25.00 25.00

Pressure (bar) 1.01 1.01 1.01 1.01

Density (g/L) 1,003.92 1,053.51 1,048.02 1,012.39





KCl 0.000 0.000 0.000 0.000

KH2PO4 0.000 0.000 0.000 0.000

Na2HPO4 0.110 0.000 0.000 0.110

NaH2PO4 0.000 2.002 0.000 0.000

Sodium Chloride 0.896 10.552 5.777 1.800

TOTAL (kg/batch) 99.563 204.319 101.890 100.003

TOTAL (L/batch) 99.174 193.941 97.221 98.779

24

Stream Name S-505 S-506 S-507 S-508 Source P-27 P-28 P-29 P-30 Destination P-31 P-31 P-31 P-31 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.00 25.00 25.00

Pressure (bar) 9.92 9.92 9.92 9.92

Density (g/L) 999.53 1,025.59 1,022.89 1,003.94





KCl 0.000 0.000 0.000 0.000

KH2PO4 0.000 0.000 0.000 0.000

Na2HPO4 0.110 0.000 0.000 0.110

NaH2PO4 0.000 2.002 0.000 0.000

Sodium Chloride 0.896 10.552 5.777 1.800

TOTAL (kg/batch) 99.563 204.319 101.890 100.003

TOTAL (L/batch) 99.610 199.221 99.610 99.610

Stream Name S-509 AEC Waste S-510 S-511

Source P-31 P-31 INPUT P-32 Destination P-32 OUTPUT P-32 P-33 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.00 25.00 25.00

Pressure (bar) 9.46 9.46 1.01 10.12

Density (g/L) 1,025.29 1,015.26 1,769.00 1,066.95




Component Flowrates (kg/batch) Amm. Sulfate 0.000 0.000 10.562 10.562

HAeq 0.996 0.204 0.000 0.996

Injection Water 95.882 473.426 0.000 95.882

KCl 0.000 0.000 0.000 0.000

KH2PO4 0.000 0.000 0.000 0.000

Na2HPO4 0.000 0.220 0.000 0.000

NaH2PO4 1.001 1.886 0.000 1.001

Sodium Chloride 5.276 18.415 0.000 5.276

TOTAL (kg/batch) 103.156 494.151 10.562 113.718

TOTAL (L/batch) 100.612 486.724 5.971 106.582

25

Stream Name S-601 S-602 UF-601 Filtrate S-603 Source P-33 P-34 P-35 P-35 Destination P-34 P-35 OUTPUT P-36 Stream Properties Activity (U/ml) 0.00 0.00 0.00 0.00

Temperature (°C) 25.00 25.00 31.53 31.53

Pressure (bar) 10.12 2.16 2.16 2.16

Density (g/L) 1,066.95 1,066.95 1,065.25 1,051.60




Component Flowrates (kg/batch) Amm. Sulfate 10.562 10.562 10.129 0.433

HAeq 0.996 0.996 0.004 0.992

Injection Water 95.882 95.882 91.949 3.934

NaH2PO4 1.001 1.001 0.960 0.041

Sodium Chloride 5.276 5.276 5.060 0.216

TOTAL (kg/batch) 113.718 113.718 108.101 5.617

TOTAL (L/batch) 106.582 106.582 101.479 5.341

Stream Name S-604 S-605 To Formulation

Source P-36 P-36 P-37 Destination OUTPUT P-37 OUTPUT Stream Properties Activity (U/ml) 0.00 0.00 0.00 Temperature (°C) 32.28 32.28 32.28 Pressure (bar) 2.16 2.16 2.31 Density (g/L) 1,061.61 1,028.40 1,028.41 Total Enthalpy (kW-h) 0.03 0.01 0.01 Specific Enthalpy (kcal/kg) 6.35 3.43 3.42 Heat Capacity (kcal/kg-°C) 0.87 0.47 0.47 Component Flowrates (kg/batch) Amm. Sulfate 0.351 0.082 0.082 HAeq 0.169 0.824 0.824 Injection Water 3.186 0.747 0.747 NaH2PO4 0.033 0.008 0.008 Sodium Chloride 0.175 0.041 0.041 TOTAL (kg/batch) 3.914 1.702 1.702 TOTAL (L/batch) 3.687 1.655 1.655

26

Equipment List and Specifications

Table 5: Equipment Summary

1. EQUIPMENT SUMMARY (2014 prices)

Name Type Units Standby/ Staggere

d

Size (Capacity)

Material of

Construction Purchase

Cost ($/Unit) DE-101 Dead-End Filter 1 0/0 2.41 m2 SS316 25,000

BBS-101a Rocking Bioreactor Skid

1 0/1 20.00 L CS 176,000

BBS-102a Rocking Bioreactor Skid

1 0/1 100.00 L CS 557,000

DCS-102 Disposable Generic Container Skid

1 0/0 200.00 L CS 1,000

SFR-104 Shake Flask Rack

1 0/1 2.00 L CS 5,000


1 0/1 0.50 L CS 4,000

TTR-101 Test Tube Rack 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

DBS-101 Disposable Bioreactor Skid

1 0/3 700.00 L CS 215,000

DE-201 Dead-End Filter 1 0/3 0.06 m2 SS316 25,000


1 0/3 1,600.00 L CS 1,000

DBS-201 Disposable Bioreactor Skid

1 0/3 3,000.00 L CS 226,000

V-301 Blending Tank 1 0/0 1,977.02 L SS316 215,000

V-303 Blending Tank 1 0/0 1,911.14 L SS316 214,000

DS-301 Disk-Stack Centrifuge

1 0/0 1,587.25 L/h SS316 299,000


UF-401 Ultrafilter 1 0/0 2.50 m2 SS316 29,000

SDLB-401 Skid for Disposable Large Bag

18 0/0 100.00 L SS316 3,000


1 0/0 100.00 L SS316 3,000

C-401 GFL Chromatography Column

1 0/0 688.11 L SS316 629,000

27


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

C-501 PBA Chromatography Column

1 0/0 49.81 L SS316 355,000

V-501 Blending Tank 1 0/0 118.43 L SS316 150,000

UF-601 Ultrafilter 1 0/0 2.50 m2 SS316 29,000


2 0/0 100.00 L SS316 3,000


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


SDLB-402a Skid for Disposable Large Bag

17 0/0 100.00 L SS316 3,000

28

Summary of Capital Requirements and Manufacturing Costs

2. MAJOR EQUIPMENT SPECIFICATIONS AND FOB COST (2014 prices)

Quantity/ Standby/ Staggered

Name Description Unit Cost ($) Cost ($)

1 / 0 / 0 DE-101 Dead-End Filter 25,000 25,000

Filter Area = 2.41 m2 1 / 0 / 1 BBS-101a Rocking Bioreactor Skid 176,000 352,000

Container Volume = 20.00 L 1 / 0 / 1 BBS-102a Rocking Bioreactor Skid 557,000 1,114,000

Container Volume = 100.00 L 1 / 0 / 0 DCS-102 Disposable Generic Container Skid 1,000 1,000

Container Volume = 200.00 L 1 / 0 / 1 SFR-104 Shake Flask Rack 5,000 10,000




Container Volume = 2.00 L 1 / 0 / 3 DBS-101 Disposable Bioreactor Skid 215,000 860,000

Container Volume = 700.00 L 1 / 0 / 3 DE-201 Dead-End Filter 25,000 100,000

Filter Area = 0.06 m2 1 / 0 / 3 DCS-201 Disposable Generic Container Skid 1,000 4,000

Container Volume = 1600.00 L 1 / 0 / 3 DBS-201 Disposable Bioreactor Skid 226,000 904,000

Container Volume = 3000.00 L 1 / 0 / 0 V-301 Blending Tank 215,000 215,000

Vessel Volume = 1977.02 L 1 / 0 / 0 V-303 Blending Tank 214,000 214,000

Vessel Volume = 1911.14 L 1 / 0 / 0 DS-301 Disk-Stack Centrifuge 299,000 299,000

Throughput = 1587.25 L/h 1 / 0 / 0 DE-308 Dead-End Filter 41,000 41,000

Filter Area = 10.00 m2 1 / 0 / 0 UF-401 Ultrafilter 29,000 29,000

Membrane Area = 2.50 m2 18 / 0 / 0 SDLB-401 Skid for Disposable Large Bag 3,000 54,000

Container Volume = 100.00 L 1 / 0 / 0 SDLB-403 Skid for Disposable Large Bag 3,000 3,000

Container Volume = 100.00 L 1 / 0 / 0 C-401 GFL Chromatography Column 629,000 629,000

Column Volume = 688.11 L 1 / 0 / 0 DCS-401 Disposable Generic Container Skid 1,000 1,000

Container Volume = 1644.00 L

29

1 / 0 / 0 DCS-402 Disposable Generic Container Skid 1,000 1,000 Container Volume = 100.00 L 1 / 0 / 0 DCS-501 Disposable Generic Container Skid 1,000 1,000 Container Volume = 111.00 L 1 / 0 / 0 DCS-502 Disposable Generic Container Skid 1,000 1,000 Container Volume = 222.00 L 1 / 0 / 0 DCS-503 Disposable Generic Container Skid 1,000 1,000 Container Volume = 111.00 L 1 / 0 / 0 DCS-504 Disposable Generic Container Skid 1,000 1,000 Container Volume = 111.00 L 1 / 0 / 0 C-501 PBA Chromatography Column 355,000 355,000 Column Volume = 49.81 L 1 / 0 / 0 V-501 Blending Tank 150,000 150,000 Vessel Volume = 118.42 L 1 / 0 / 0 UF-601 Ultrafilter 29,000 29,000 Membrane Area = 2.50 m2 2 / 0 / 0 SDLB-601 Skid for Disposable Large Bag 3,000 6,000 Container Volume = 100.00 L 1 / 0 / 0 DE-601 Dead-End Filter 41,000 41,000 Filter Area = 10.00 m2 1 / 0 / 0 MF-601 Microfilter 26,000 26,000 Membrane Area = 0.05 m2 1 / 0 / 0 DCS-601 Disposable Generic Container Skid 1,000 1,000 Container Volume = 3.00 L 18 / 0 / 0 SDLB-402 Skid for Disposable Large Bag 3,000 54,000 Container Volume = 100.00 L 1 / 0 / 0 DE-102 Dead-End Filter 41,000 41,000 Filter Area = 10.00 m2 17 / 0 / 0 SDLB-402a Skid for Disposable Large Bag 3,000 51,000 Container Volume = 100.00 L Unlisted Equipment 1,408,000 TOTAL 7,042,000

Table 6: Major Equipment Specs and FOB cost

30

3. FIXED CAPITAL ESTIMATE SUMMARY (2014 prices in $)

3A. Total Plant Direct Cost (TPDC) (physical cost) 1. Equipment Purchase Cost 7,042,000

2. Installation 6,167,000

3. Process Piping 2,465,000

4. Instrumentation 2,817,000

5. Insulation 211,000

6. Electrical 704,000

7. Buildings 3,169,000

8. Yard Improvement 1,056,000

9. Auxiliary Facilities 2,817,000

TPDC 26,449,000

3B. Total Plant Indirect Cost (TPIC) 10. Engineering 6,612,000

11. Construction 9,257,000

TPIC 15,869,000

3C. Total Plant Cost (TPC = TPDC+TPIC) TPC 42,318,000

3D. Contractor's Fee & Contingency (CFC) 12. Contractor's Fee 2,116,000

13. Contingency 4,232,000

CFC = 12+13 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 Unit Cost

($/h) Annual Amount

(h) Annual Cost

($) %

Operator 69.00 5,901 407,202 100.00

TOTAL 5,901 407,202 100.00 Table 8: Labor Summary

31

5. MATERIALS COST - PROCESS SUMMARY

Bulk Material Unit Cost ($)

Annual Amount

Annual Cost ($)

%

AEC Eq Buffer 0.000 299 kg 0 0.00 AEC El Buff 0.000 613 kg 0 0.00 AEC Strip Buffe 0.000 306 kg 0 0.00 AEC Wash Buffer 0.000 300 kg 0 0.00 Amm. Sulfate 8.000 32 kg 253 0.04 Media 300.000 182 kg 54,645 8.91 Injection Water 1.000 5,102 kg 5,102 0.83 Biomass 0.000 0 kg 0 0.00 Air 0.000 61,398 kg 0 0.00 HAeq 0.000 0 kg 0 0.00 H3PO4 (5% w/w) 1.535 3,855 kg 5,918 0.97 NaOH (0.5 M) 0.815 2,661 kg 2,170 0.35 WFI 0.300 11,862 kg 3,559 0.58 B-Propiolactone 14.000 38,294 g 536,116 87.46 IEX-El-Buff 1.145 4,552 kg 5,213 0.85 TOTAL 612,975 100.00

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 Units Cost

($) Annual

Amount

Annual Cost ($)

%

20 L Cell Bag 700.000 3 item 2,100 0.91

200 L Bag 300.000 27 item 8,100 3.51

100 L Cell Bag 1,850.000 3 item 5,550 2.40

2000 mL Shake Flask 1.800 1 item 1 0.00

5 mL Test Tube 0.500 3 item 2 0.00

125 mL Shake Flask 1.226 0 item 0 0.00

500 mL Poly Shake Flask 1.160 3 item 3 0.00

Dft Stirred Bioreactor Bag 6,220.000 3 item 18,660 8.08

3000L 8,600.000 3 item 25,800 11.18

Dft DEF Cartridge 1,000.000 9 item 9,000 3.90

Dft Membrane 400.000 0 m2 16 0.01

Dft Large Bag 340.000 168 item 57,120 24.75

Dft Gel Filtration Resin 2,000.000 41 L 82,573 35.77

1 L Plastic Bag 0.200 6,897 item 1,379 0.60

Dft PBA Chrom Resin 1,500.000 4 L 5,603 2.43

MF Membrane (Biotech) 735.835 0 m2 106 0.05

FlexBoy Bag 3.0 L 30.000 3 item 90 0.04

UF 750kDa SE Membrane 981.120 15 m2 14,717 6.38

TOTAL 230,821 100.00 Table 10: Consumables Cost

32

7. WASTE TREATMENT/DISPOSAL COST (2014 prices) - PROCESS SUMMARY

Waste Category Unit Cost

($) Annual

Amount

Annual Cost ($)

%

Solid Waste 0 0.00

Aqueous Liquid 38,714 100.00

S-303 5.000 217 kg 1,087 2.81

S-305 5.000 1 kg 7 0.02

SEC Waste Stream 5.000 4,482 kg 22,408 57.88

AEC Waste 5.000 1,482 kg 7,412 19.15

UF-601 Filtrate 5.000 324 kg 1,622 4.19

S-604 5.000 12 kg 59 0.15

P-17:CIP-1(Pre Rinse) 5.000 1,224 kg 6,120 15.81

Organic Liquid 0 0.00

Emissions 0 0.00

TOTAL 38,714 100.00 Table 11:Waste Treatment/Disposal Costs

8. UTILITIES COST (2014 prices) - PROCESS SUMMARY

Utility Unit Cost

($) Annual

Amount Ref.

Units Annual Cost

($) %

Electricity 0.050 6,200 kW-h 378 53.37

Steam 12.000 1 MT 18 2.52

Cooling Water 0.050 0 MT 0 0.00

Chilled Water 0.400 780 MT 312 44.11

TOTAL 707 100.00 Table 12: Utilities Costs

33

9. ANNUAL OPERATING COST (2014 prices) - PROCESS SUMMARY

Cost Item $ Correlation

Direct Manufacturing Costs Raw Materials 612,975 CRM

Waste Treatment/Disposal 39,000 CWT

Utilities 707 CUT

Operating Labor 407,202 COL

Supervisory and Clerical Labor 73,296

Maintenance and Repairs 2,905,140

Operating Supplies 435,771 .009*FCI

Laboratory/QC/QA 61,080 .15*COL

Patents and Royalties 507,660 .03*COM

TOTAL DMC 5,042,545

Fixed Manufacturing Costs Local Taxes and Insurance 1,557,312 .032*FCI

Plant Overhead Costs 2,040,275 .708*COL+.036*FCI

Depreciation 6,604,671 (DFC-.05DFC)/7

TOTAL FMC 3,597,587

General Manufacturing Costs

Administration Costs 1,158,741 .177*COL

Advertising/Selling 1,838,077 .11*COM

Research and Development 835,490 .05*COM

TOTAL GE 3,832,308

ESTIMATED COSTS 16,701,045 ***

TOTAL COM 12,472,726

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

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

34

Profitability analysis

10. PROFITABILITY ANALYSIS (2014 prices)

A. Direct Fixed Capital 48,666,000 $

B. Working Capital 615,000 $

C. Startup Cost 2,433,000 $

D. Up-Front R&D 835,490 $

E. Up-Front Royalties 507,660 $

F. Total Investment (A+B+C+D+E) 53,058,150 $

G. Investment Charged to This Project 53,058,150 $

H. Revenue/Savings Rates HAeq in 'To Formulation' (Main Revenue) 2,471 g HAeq/yr

I. Revenue/Savings Price HAeq in 'To Formulation' (Main Revenue) 248,889.00 $/g HAeq

J. Revenues/Savings HAeq in 'To Formulation' (Main Revenue) 614,922,153 $/yr

1 Total Revenues 614,922,153 $/yr

2 Total Savings 0 $/yr

K. Annual Operating Cost (AOC) 1 Actual AOC 12,472,726 $/yr

2 Net AOC (K1-J2) 12,472,726 $/yr

L. Unit Production Cost /Revenue Unit Production Cost 5,047.64 $/g MP

Net Unit Production Cost 5,047.64 $/g MP

Unit Production Revenue 248,889.00 $/g MP

M. Gross Profit (J-K) 602,449,427 $/yr

N. Taxes (40%) 240,979,771 $/yr

O. Net Profit (M-N + Depreciation) 368,109,000 $/yr

Gross Margin 97.98 %

Return On Investment 711.81 %

Payback Time 0.14 years

MP = Flow of Component 'HAeq' in Stream 'To Formulation' Table 14: Profitability analysis

35

Feasibility Analysis

CASH FLOW ANALYSIS (thousand $)

Year Capital

Investment Debt

Finance Sales

Revenues Operating

Cost Gross Profit

Loan Payments

Depreciation Taxable Income

Taxes Net Profit Net Cash

Flow

-2 - 14,600 0 0 0 0 0 0 0 0 0 - 14,600

-1 - 19,466 0 0 0 0 0 0 0 0 0 - 19,466

0 - 14,600 0 0 0 0 0 0 0 0 0 - 14,600

1 - 615 0 614,922 12,427 602,449 0 6,605 602,449 240,979 361,469 360,857

2 0 0 614,922 12,427 602,449 0 6,605 602,449 240,979 368,109 368,109

3 0 0 614,922 12,427 602,449 0 6,605 602,449 240,979 368,109 368,109

4 0 0 614,922 12,427 602,449 0 6,605 602,449 240,979 368,109 368,109

5 0 0 614,922 12,427 602,449 0 6,605 602,449 240,979 368,109 368,109

6 0 0 614,922 12,427 602,449 0 6,605 602,449 240,979 368,109 368,109

7 0 0 614,922 12,427 602,449 0 6,605 602,449 240,979 368,109 368,109

8 0 0 614,922 5,822 609,112 0 0 609,112 243,645 365,467 365,467

9 0 0 614,922 5,822 609,112 0 0 609,112 243,645 365,467 365,467

10 3,049 0 614,922 5,822 609,112 0 0 609,112 243,645 365,467 368,516

IRR/NPV SUMMARY

IRR Before Taxes 241.80 % Interest % 7.00 9.00 11.00

IRR After Taxes 189.92 % NPV 2,209,762.00 1,941,165.00 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

36

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.

37

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.

38

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.

39

Figure 15: Proposed floor plan of facility

40

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

41

Appendices

Appendix A: Scheduling Optimization

Figure 16: Single batch Gantt chart

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

times, respectively.

Complete Recipe

P-01 in TTR-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

h56 112 168 224 280 336 392 448 504 560 616 672 728 784 840 896 952 1008 1064 1120 1176 1232 1288h

day3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54day

42

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.

Complete Recipe

Complete Recipe (Batch #2 )








Complete Recipe (Batch #10)






h96 192 288 384 480 576 672 768 864 960 1056 1152 1248 1344 1440 1536 1632 1728 1824 1920 2016 2112 2208 2304 2400 2496 2592h

day4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 108day

43

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

manufacturing the next generation of vaccines: non-egg based platform for influenza vaccine

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