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
cells 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 years 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
D
i
s
t
r
i
b
u
t
e
d

(
m
i
l
l
i
o
n
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
strains
a
:
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 I nformation

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
Heat Capacity (kcal/kg-C) 1.00 1.00 1.00 1.00
Component Flowrates (kg/batch)
Biomass 0.000 0.012 0.000 0.048
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
Total Enthalpy (kW-h) 0.00 0.02 0.00 0.01
19
Specific Enthalpy (kcal/kg) 0.00 11.98 0.00 29.12
Heat Capacity (kcal/kg-C) 1.00 1.00 1.00 0.21
Component Flowrates (kg/batch)
Biomass 0.000 0.193 0.000 0.000
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
Total Enthalpy (kW-h) 0.09 0.00 0.07 0.38
Specific Enthalpy (kcal/kg) 11.98 0.00 29.38 11.98
Heat Capacity (kcal/kg-C) 1.00 1.00 0.21 1.00
Component Flowrates (kg/batch)
Biomass 0.739 0.000 0.000 2.916
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
Total Enthalpy (kW-h) 0.00 0.00 0.00 0.00
Specific Enthalpy (kcal/kg) 0.00 0.00 0.00 0.00
Heat Capacity (kcal/kg-C) 1.00 1.00 1.00 1.00
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
Total Enthalpy (kW-h) 0.00 3.59 2.65 0.00
Specific Enthalpy (kcal/kg) 0.00 2.92 11.98 0.00
Heat Capacity (kcal/kg-C) 0.24 0.24 1.00 1.00
Component Flowrates (kg/batch)
Biomass 0.000 0.000 4.025 0.000
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
Total Enthalpy (kW-h) 0.00 0.00 0.00 0.00
Specific Enthalpy (kcal/kg) 0.00 0.00 0.00 0.00
Heat Capacity (kcal/kg-C) 1.00 1.00 1.00 0.24
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
Total Enthalpy (kW-h) 66.02 24.51 24.50 33.54
Specific Enthalpy (kcal/kg) 2.93 11.97 11.96 17.08
Heat Capacity (kcal/kg-C) 0.24 1.00 1.00 1.00
Component Flowrates (kg/batch)
Biomass 0.000 11.194 11.194 0.224
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
Total Enthalpy (kW-h) 1.44 33.53 0.01 0.00
Specific Enthalpy (kcal/kg) 17.08 17.08 17.09 0.00
Heat Capacity (kcal/kg-C) 1.00 1.00 1.00 0.25
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
Stream Name S-307 S-401 S-402 S-403
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
Total Enthalpy (kW-h) 33.52 33.51 33.51 33.50
Specific Enthalpy (kcal/kg) 16.95 16.94 16.94 16.94
Heat Capacity (kcal/kg-C) 0.99 0.99 0.99 0.99
Component Flowrates (kg/batch)
B-Propiolactone 12.765 12.765 12.765 12.765
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
Total Enthalpy (kW-h) 32.95 1.34 1.34 0.00
Specific Enthalpy (kcal/kg) 17.35 16.99 16.98 0.00
Heat Capacity (kcal/kg-C) 0.99 0.97 0.97 0.97
Component Flowrates (kg/batch)
B-Propiolactone 12.264 0.500 0.500 0.000
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
Total Enthalpy (kW-h) 0.00 1.34 0.00 0.00
Specific Enthalpy (kcal/kg) 0.00 0.77 0.00 0.00
Heat Capacity (kcal/kg-C) 0.97 0.97 0.96 0.96
Component Flowrates (kg/batch)
B-Propiolactone 0.000 0.500 0.000 0.000
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





Stream Name S-501 S-502 S-503 S-504
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
Total Enthalpy (kW-h) 0.00 0.00 0.00 0.00
Specific Enthalpy (kcal/kg) 0.00 0.00 0.00 0.00
Heat Capacity (kcal/kg-C) 0.99 0.97 0.98 0.99
Component Flowrates (kg/batch)
Injection Water 98.557 191.765 96.113 98.092
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
Total Enthalpy (kW-h) 0.00 0.00 0.00 0.00
Specific Enthalpy (kcal/kg) 0.00 0.00 0.00 0.00
Heat Capacity (kcal/kg-C) 0.99 0.97 0.98 0.99
Component Flowrates (kg/batch)
Injection Water 98.557 191.765 96.113 98.092
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
Total Enthalpy (kW-h) 0.00 0.00 0.00 0.00
Specific Enthalpy (kcal/kg) 0.00 0.00 0.00 0.00
Heat Capacity (kcal/kg-C) 0.96 0.98 0.34 0.90
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
Total Enthalpy (kW-h) 0.00 0.00 0.75 0.03
Specific Enthalpy (kcal/kg) 0.00 0.00 5.95 4.90
Heat Capacity (kcal/kg-C) 0.90 0.90 0.91 0.75
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
SFR-103
Shake Flask
Rack
1 0/1 0.50 L CS 4,000
TTR-101 Test Tube Rack 1 0/0 0.01 L CS 0
SFR-101
Shake Flask
Rack
1 0/1 0.13 L CS 4,000
SFR-102
Shake Flask
Rack
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
DCS-201
Disposable
Generic
Container Skid
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
DE-308 Dead-End Filter 1 0/0 10.00 m2 SS316 41,000
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
SDLB-403
Skid for
Disposable Large
Bag
1 0/0 100.00 L SS316 3,000
C-401
GFL
Chromatography
Column
1 0/0 688.11 L SS316 629,000
27
DCS-401
Disposable
Generic
Container Skid
1 0/0 1,644.00 L CS 1,000
DCS-402
Disposable
Generic
Container Skid
1 0/0 100.00 L CS 1,000
DCS-501
Disposable
Generic
Container Skid
1 0/0 111.00 L CS 1,000
DCS-502
Disposable
Generic
Container Skid
1 0/0 222.00 L CS 1,000
DCS-503
Disposable
Generic
Container Skid
1 0/0 111.00 L CS 1,000
DCS-504
Disposable
Generic
Container Skid
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
SDLB-601
Skid for
Disposable Large
Bag
2 0/0 100.00 L SS316 3,000
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
DCS-601
Disposable
Generic
Container Skid
1 0/0 3.00 L CS 1,000
SDLB-402
Skid for
Disposable Large
Bag
18 0/0 100.00 L SS316 3,000
DE-102 Dead-End Filter 1 0/0 10.00 m2 SS316 41,000
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 / 1 SFR-103 Shake Flask Rack 4,000 8,000

Container Volume = 0.50 L

1 / 0 / 1 SFR-101 Shake Flask Rack 4,000 8,000

Container Volume = 0.13 L

1 / 0 / 1 SFR-102 Shake Flask Rack 4,000 8,000

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.

Complet e 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
h 56 112 168 224 280 336 392 448 504 560 616 672 728 784 840 896 952 1008 1064 1120 1176 1232 1288 h
day 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 day
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.
Complet e Recipe
Complet e Recipe (Bat ch #2 )
Complet e Recipe (Bat ch #3 )
Complet e Recipe (Bat ch #4 )
Complet e Recipe (Bat ch #5 )
Complet e Recipe (Bat ch #6 )
Complet e Recipe (Bat ch #7 )
Complet e Recipe (Bat ch #8 )
Complet e Recipe (Bat ch #9 )
Complet e Recipe (Bat ch #10)
Complet e Recipe (Bat ch #11)
Complet e Recipe (Bat ch #12)
Complet e Recipe (Bat ch #13)
Complet e Recipe (Bat ch #14)
Complet e Recipe (Bat ch #15)
h 96 192 288 384 480 576 672 768 864 960 1056 1152 1248 1344 1440 1536 1632 1728 1824 1920 2016 2112 2208 2304 2400 2496 2592 h
day 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 108 day
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