Biochemical Engineering (CHEE 3024)

Technology Review and Appraisal Report

for
Influenza H5N1 Vaccine Production by using
microcarrier-based MDCK cell culture system

Group 1
Name I.D. Contribution (%)
Angel Mah Xin Yee 015100 30
Brenda Seow Chai Yie 016025 30
Lim Qai Jack 012925 30
Benson Moo 012594 10

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Table of Contents
1. Executive Summary ............................................................................................................... 2
2. Process Background .............................................................................................................. 3
2.1 Brief Introduction ............................................................................................................. 3
2.2 Process Description .......................................................................................................... 3
2.2.1 Pre-treatment ............................................................................................................ 3
2.2.2 Fermentation ............................................................................................................. 3
2.2.3 Separation.................................................................................................................. 4
4. Discussion .............................................................................................................................. 5
4.1 Scaling-up ......................................................................................................................... 5
4.2 Contamination .................................................................................................................. 6
4.2.1 Chemical Contamination (Ryan, 2008) ...................................................................... 6
4.3 Safety ................................................................................................................................ 8
4.3.1 Operating Conditions................................................................................................. 8
4.3.2 Biosafety .................................................................................................................... 8
4.3.3 Chemical Safety ......................................................................................................... 8
4.3.4 Others ........................................................................................................................ 9
4.4 Ethics ................................................................................................................................ 9
4.5 Sustainability .................................................................................................................. 10
4.5.1 Efficiency .................................................................................................................. 10
4.5.2 Energy ...................................................................................................................... 10
4.5.3 Economics ................................................................................................................ 10
4.5.4 Environment ............................................................................................................ 11
4.6 Appraisal ......................................................................................................................... 11
5. Conclusion and Future Study ............................................................................................... 13
6. References ........................................................................................................................... 14

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1. Executive Summary

The conventional egg-based seasonal vaccines production is too slow and inefficient
to meet the global demand during an influenza pandemic. In recent years, the cell-based
technology has been emerging as an alternative approach to replace the egg-based method.
A comprehensive studies on this technology is necessary to understand its mode of operation,
advantages and also the drawbacks.
This report aims to study the cell-based process by producing influenza H5N1 vaccines
using MDCK cells that adhere on microcarriers. The production process mainly involves the
pre-culture of MDCK cells, inoculation of cells into bioreactor, virus infection, purification of
virus particles from the supernatants and finally inactivation of viruses. The final product will
be inactivated whole influenza H5N1 vaccines. A process flow diagram has been drawn to
clearly illustrate the streams and units involved in addition to the operating conditions.
To evaluate the potential of cell-based method in taking over the future vaccine
production, a detailed discussion has been done to identify the critical challenges faced in
scaling-up of process and possible contamination problems. Several measures have been
proposed to encounter the limitations. Safety and ethical issues related to the process are
also described in the text. Besides, the sustainability of the process has been discussed from
different perspectives such as economics, environmental impact, efficiency of the process and
energy demand.
An appraisal is done to make clear comparison between the cell-based and egg-based
approaches. It is found that the cell-based method is more advantageous in large scale
vaccine production due to the capability of scaling-up, monitoring cell growth, and shorter
production time by eliminating the dependency on eggs supply.

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2. Process Background
2.1 Brief Introduction
Influenza H5N1 viruses are killing avian hosts globally and causing zoonotic
transmission to humans, posing the threat of influenza pandemics in humans (World Health
Organization, 2007). An influenza pandemic can occur if the viruses continue to evolve and
develop the ability to induce large scale human-to-human transmission. Therefore,
vaccination is crucial to avoid the occurrence of unforeseen circumstances.
Egg-based method has been dominating the manufacture of inactivated seasonal
influenza vaccines in the past. However, due to the low efficiency of the approach, only a
small fraction of the growing population among the world can be covered by the current
vaccine supply (Tree et al., 2001). In this report, the virus propagation in Madin Darby Canine
Kidney (MDCK) cells will be discussed as an alternative approach which could replace the
conventional egg-based processes.
The technology will aim to produce inactivated whole influenza vaccines due to the
lower regulatory hurdle of inactivate influenza vaccines as compared to that of the live-
attenuated influenza vaccines. Besides, as reported by Nicholson et al. (2001), Treanor et al.
(2001) and Hehme et al. (2004), inactivated whole virus antigens have been more
immunogenic in naive populations than split and subunit vaccine antigens.
2.2 Process Description
2.2.1 Pre-treatment
Cells are pre-cultured in spinner flasks to be inoculated into the lab-scale bioreactors.
Cell culture is carried out in 250 mL spinner flasks containing 100 mL of culture medium at
37⁰C in a 5% CO2 incubator. MDCK cells are grown using Dulbecco's Modified Eagle's Medium
(DMEM) plus 5% fetal bovine serum. The spinner flasks are inoculated with 2 x 105 cells/mL
and the stirring speed is maintained at 45 rpm. (Hu et al., 2011).
2.2.2 Fermentation
2.2.2.1 Growth of cells in Bioreactor
Prior to inoculation of cell cultures, Cytodex 1 microcarriers are hydrated, autoclaved,
and preconditioned according to the manufacturer’s instructions (Pö rtner, 2014). The MDCK
inoculums are then added into the 2L bioreactor with a seeding density of 2 x 10 5 cells/mL.
The agitation speed is maintained at 35–50 rpm. During cell growth, pH, dissolved oxygen and
temperature are maintained at 7, 50% air-saturation, and 37⁰C, respectively. Perfusion rate
is adjusted daily to maintain glucose concentration at around 1 g/L (Hu et al., 2011).
Instead of conventional batch or fed-batch operations, the bioreactor is operated in
perfusion mode, in which the fresh media is constantly fed into the bioreactor while the by-
products that could inhibit cell growth are removed continuously. Toxic metabolites can be
kept in a very low concentration as a result of the continuous exchange of medium. In addition,
only a very small scale of perfusion culture is required as compared with batch culture in order
to obtain the desired amount of product (G., 2002).
2.2.2.2 Virus Infection and Fermentation
When the cells on the microcarriers are fully confluent, the medium is exchanged with
90% of fresh medium. 2 mm/mL of TPCK-trypsin will be added to the medium before viral
replication. The cells will then be infected by H5N1 (NIBRG-14) viruses with a multiplicity of

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infection (MOI) of 0.00001 (Hu et al., 2011). The addition of trypsin is to cleave the precursor
protein of hemagglutinin (HA0) into active hemagglutinin (HA1 and HA2). Only cleaved
hemagglutinin leads to the adsorption of the influenza viruses on cells with subsequent virus
assimilation into the cell, which leads to further replication (Emdmillipore, 2014). Samples
were taken daily to determine cell density and viral assays.
At the end of fermentation process, the animal cells have to be removed from the
microcarriers before the purification stage begins. The microcarriers will be washed with
EDTA-PBS solution and incubated with trypsin-EDTA according to the manufacturer’s
instructions to ease the removal of cells (Pö rtner, 2014).
2.2.3 Separation
2.2.3.1 Virus Purification
The cells and cellular debris are first removed from the supernatants by using 0.65 µm
depth filter. After that, the filtered solution will be concentrated by using a 300K ultrafiltration
membrane to reduce the volume required for downstream equipment (Hu et al., 2011).
In order to digest the DNA, RNA and nucleic acids from the host cells, Benzonase will
be added to the ultrafiltration retentate at a final concentration of 1 Units/ml and the mixture
was incubated 6 hours at 32°C (Kistner et al., 2013). Next, the ultrafiltration retentate is
subjected to a sucrose gradient ultracentrifugation and result in layers of solution. A sucrose
gradient can be prepared by using 0.125 M citrate buffer, pH 7.8 and a 60% sucrose solution
(GE healthcare life science, 2014). The purified whole virions are pooled from the percentage
of sucrose ranging from approximately 28% to 52% (ANDRE and Champluvier, 2011).
After that, the purified whole influenza virus particles will undergo a diafiltration
process to remove sucrose content. The stream will first be concentrated by using a
ultrafiltration membrane and directed to a mixing tank, where the sucrose concentration can
be further reduced by the addition of phosphate buffer. Continuous diafiltration washes out
original low molecular weight species by adding buffer to the sample at substantially the same
rate as filtrate is being generated (Hauschild, Haussmann and Jobst, 2011).
2.2.3.2 Virus Inactivation
For antigen stabilization and inactivation of the purified viruses, formalin (37%
formaldehyde) is added to the diafiltration retentate to a final concentration of 0.025%. The
mixture is then incubated at 32°C for 24 hours. The virus is inactivated by irreversibly cross-
linking primary amine groups in surface proteins with other nearby nitrogen atoms in protein
or DNA through a —CH2— linkage. The formalin will then be filtered out by ultrafiltration
process (Kistner et al., 2013).
The final step of vaccine purification will be sterile filtration. The virus particles are
filtered through a 0.22 μm filter and ready for clinical use, if desired (ANDRE and Champluvier,
2011). The concentration of the inactivated viruses was measured by using plaque assays
method. Plaque assays can be used to quantify infectious virons by measuring the discrete
plaques which indicated the dead zone of cellular in the cell culture.

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4. Discussion
4.1 Scaling-up
Global demand of vaccine is gradually increasing as more viruses are detected. This has
contributed to the growth in cell analysis market significantly. Thus, the technology of
producing vaccine is constantly revised and improved. The crucial aspect in the production of
vaccine is its capability of large scale production to meet the demand. The vaccine production
for the 2016-2017 season is projected to achieve 168 doses of injectable vaccine in the United
States (CDC, 2016).
Scale up can be defined as a procedure for the design and construction of a large-scale
system based on experimental results with small-scale equipment. It is common to experience
some difficulties when scaling up, but it is crucial to maintain same temperature, pH, dissolved
oxygen concentration (DO), medium composition and quality of MDCK culture medium.
Vaccine production with MDCK cells attached to microcarriers is cost-effective for mass
production to meet the growing demand (Barney Z., 2014). They can be easily suspended in
bioreactors and allow better distribution throughout the bioreactor. They have the capability
of culturing large number of adherent cells to seed bioreactors of above 1000L capacity
(Whitford, 2011).
Barney Z. (2014, p. 3) has concluded that stirred, sparged bioreactor is a promising
method to propagate cells on microcarriers in vaccine production. Many process parameters
can be evaluated and manually controlled easily. Furthermore, vaccine production can be
carried out in smaller bioreactor and shorter time due to the adherence on microcarriers. This
makes the handling of bioreactors more convenient and speeds up vaccine production to
allow better response to pandemic threat. However, large scale cell-culture production still
faces challenges in maintaining product quality, besides minimizing contamination issues.
One of the main issues is the quantification of oxygen uptake in large-scale. The
starting point for online monitoring and control makes calculation of oxygen consumption
difficult when considering the dynamics in the gas phase (Zita et al., 2009). This is due to the
mixing effects and tubing delays which tends to filter out the dynamics of oxygen uptake rate.
Zita et al (2009) have managed to come up with models which contain system parameters
that reduces the observation error of oxygen consumption up to 30%. Observer and controller
for the cell culture growth are applicable at both small-scale and large-scale. Stirred speed is
controlled on the basis of measured dissolved oxygen, while oxygen supply is controlled based
on the oxygen concentration between the inlet and outlet (J. E. Baart et al., 2007).
On the other hand, the handling of animal cells in bioreactor can be difficult as the
animal cells have no cell wall as their outer protection layer. The generation of turbulence
due to mechanical agitation in the bioreactor causes the exposure of cells to shear stress
which leads to apoptosis or cell death. In addition, the fluid turbulence will result in the
formation of eddies which directly interact with the adherent animal cells. The yield of cell
will thus become low as the cells will be significantly damaged and results in the formation of
more cell debris (Vaccixcell, 2017). As reported by Croughan, Sayre and Wang (1989), with
the addition of thickening agents such as polymers, the cells can be protected from
turbulences at high agitation due to the increases viscosity.
Moreover, the number of microcarriers used in the bioreactor is also subjected to
constraints. With large amount of microcarriers used, the density of cell culture increases

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which leads to higher degree of collisions either from bead to bead or adherent cells to
mechanical impeller (Vaccixcell, 2017). The collisions again damage the cells, resulting in cell
death and lower cell yield. Hassanzade et al. (2011) have found that the by replacing solid
carriers such as Cytodex 1 by Fibra-cel discs, where the cells are stationary, can lead to higher
virus production. This might be due to the reduction on cell loss caused by bead collisions.
As the manufacture of influenza vaccine requires oxygen, the aeration can be a crucial
factor in industrial scale production. Uneven distribution of oxygen can affect the cell growth
and thus the product yield. This is usually overcome by using a sparger to induce oxygen to
the system uniformly. However, foam formation could be another issue where the
microcarriers accumulate at the liquid-foam interface and thus are lost for the culture. Foam
formation can be prevented by using microspargers which reduce the size of bubbles, or with
the addition of antifoaming agents into the system (Merten, 2014). First option is prefered as
the introduction of chemicals could damage the cells or affect the cells growth.
In short, as the cell-based method is an emerging technology for vaccine production,
there are many limitations to be overcome in large scale production. More studies and
researches have to be conducted to attained higher production yield.

4.2 Contamination
Vaccine has been the cost-effective method to prevent infectious diseases in human for the
past 60 years. Its biological manufacturing process must always be monitored closely to
prevent any undesired variation or contamination in the product. This is crucial in preventing
the recipient to be infected by contaminant. Therefore, the manufacturing process of vaccine
from MDCK cells must be carried out in closed system, in which the isolated production
equipment can ensure no invasion of unwanted outer component. Generally, the cell culture
contaminants can be classified into two categories: chemical contaminants and biological
contaminants.
4.2.1 Chemical Contamination (Ryan, 2008)
Chemical contamination can be defined as the undesirable effects on the culture
system due to the presence of non-living substance. The major source of chemical
contaminants is normally the cell culture media, which contains reagents, water and
additive.
Water is usually used for preparation of culture media and the washing of
equipment. High quality water with the absence of trace metals and dissolved organic
compounds should be used for the processes to minimize the contamination. The
water can be purified by proper treatment such as ultrafiltration and reverse osmosis.
However, due to the aggressive solvent characteristics of water, leaching of metal ions
from vessel wall can occur contaminate the culture medium.
Another potential contaminant is known as endotoxins, which are commonly
found in sera. They are the lipopolysaccharide-containing by-products of gram
negative bacteria. Due to the biologically reactive nature of the molecules, they can
have great influences in vivo on humoral and cellular systems. The level of endotoxins
in the sera can be minimized by preparing and handling the sera in aseptic conditions.
4.2.2 Biological Contamination (Ryan, 2008)
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 For biological contamination, the degree of damage to the system is less if the
contaminants can be detected quickly. The contamination by bacteria, molds and
yeasts is relatively easier to detect and causes less problems. The presence of viruses,
protozoa, insects and mycoplasmas causes more serious problems to the cell culture
as they are hard to be identified.
The biological contamination is normally arises from the use of nonsterile
equipment, media or solutions. Before the process begins, all the equipment must be
sterilized to ensure that there are no living microorganisms that will potentially
contaminate the products. In addition, all the chemicals used in the process such as
formalin and sucrose solution should also be sterilized.
The MDCK cell substrate must have low tumorigenicity, no detectable adventitious agents
and oncogenicity. This can prevent exposure of vaccine recipient to intact cells, cellular
components and residual cellular DNA (VRBPAC, 2008). In order to eliminate the potential of
exposure to adventitious agent from the MDCK cell, the cells should be transferred to serum-
free medium. The use of serum-free medium is vital to prevent adventitious viral
contamination and prion contamination, while removing lot-to-lot variability which is a
common production issue in serum containing medium.
In vitro and in vivo testing can be carried out to detect specific and general
adventitious agent. This issue can be controlled through plasmid rescue and manufacturing
processes (VRBPAC, 2008). Sudden drop of dissolved oxygen level is a good indication of
contamination. Growth rate of contaminating organism can be determined by the rate of
change of dissolved oxygen level and termination of process may be necessary depending on
the severity of contamination.
Multiple filtration steps are usually carried out to ensure the safety of the product.
Downstream purification steps can remove all intact cells, reduces the quantity of host
protein and DNA, followed by sterilisation and inactivation of virus.
Oncogenicity test is a test to evaluate the tendency of cellular component to induce
tumor. This is done on MDCK DNA and cell culture by injecting the vaccine into rodent species
(laboratory mice). Reduction of quantity and size of MDCK DNA and quantity of MDCK
proteins should be carried out based on the standard as stated below:
● More than 90% removal of MDCK cell DNA (or less than 1 ng of MDCK DNA in
one dose)
● More than 90% removal of MDCK protein (or less than 0.5μg per dose)

The risk of one dose of vaccine containing an intact MDCK cell can be reduced to
1.6×10-16 (VRBPAC, 2008). The reduction process is done by introducing benzonase in the
early stage of purification to digest the DNA, RNA and nucleic acid of host cell.
Tumorigenicity test is aim to evaluate whether intact cells can establish a tumor.
Similar test is done on mice as in oncogenicity test to evaluate the tumorigenicity of the
product. Removal of all cells is done through multiple filtration steps, such as depth filter and
ultrafiltration. To ensure the safety of vaccine, the multiple filtration must be able to removing
at least 1021 cells after ultrafiltration membrane of 35 nm.
In MDCK cell culture and virus strain preparation stage, all materials must be

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controlled to minimise the exposure to animal cell components. This can be achieved by
highly characterised MDCK cell banks and vaccine seeds. The cells can be biologically cloned
by limiting dilution. This will develop a uniform population from a genetic parent cell, which
eases the tracking exposure of cells to animal derived components.

4.3 Safety
Safety is one of the essential factors which need to be well considered to reduce the
risk of accidents. Knowledge on plant safety plays an important role in reducing any damages,
accidents or fatalities from happening when working in industry. Hazard analysis should be
conducted to identify the potential risks in the production area and corresponding safety
measures should be taken to minimize the occurrence of accidents. In this section, the main
discussion will be focused on the safety aspects in laboratory, however similar practices can
also be applied in a large scale chemical process plant.
4.3.1 Operating Conditions
The operating temperature and pressure of each units of operation is close to ambient
conditions due to the fact that proteins are heat sensitive. Thus, there is no safety
issue regarding the use of high temperature and pressure in the manufacturing
process.
4.3.2 Biosafety
One of the safety aspects that need to be discussed is the awareness of biological
hazards which refer to organisms or organic matters produced by microorganisms,
allergens, parasites, bacteria and virus that are harmful to human health. In our
biological plant, influenza viruses are involved during the process of producing vaccine.
According to the United States Centers for Disease Control and Prevention (CDC),
influenza viruses that may cause mild disease to human are categorized under
biohazard level 2 (Chosewood and Wilson, 2010). Some of the practices and
procedures are required to be followed before or during working in the industry.
Before entering or exiting the laboratory, workers are advised to wash their
hands to ensure that hands are free from viruses or bacteria. In addition, workers or
visitors are required to wear proper personal protective equipment (PPE) such as
gloves, face mask, laboratory coat, safety boots and goggle. PPE is protective gear that
required keeping the safety of workers while they are performing their jobs. Eating,
drinking and smoking are prohibited in the laboratory due to hygienic and health care
issue.
If the spillage of potentially infectious materials occurs, the work surfaces
should be cleaned immediately with appropriate disinfectant. Besides, the
microbiological waste and the potentially infectious materials have to be
decontaminated or autoclaved prior to the disposal.
4.3.3 Chemical Safety
The lab must be equipped with fume cupboards for working with flammable, volatile,
or toxic chemical solutions such as formalin solution used during the separation
process. All hazardous chemicals must be labelled with standard logos and placed in
proper safety cabinets.

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 Moreover, some actions must be taken immediately when exposure to
chemical solution like formalin. If a personnel has been exposed to formalin solution,
he/she needs to immediately take off the contaminated clothing and wash the skin in
contact with plenty of soap with water or continue rinsing to prevent skin irritation or
eye damage. Victims are also advised to move away from the spot and breathe fresh
air to prevent over inhalation of formalin gas (Fishersci, 2015). The spillage needs to
be cleaned and disposed when hands with gloves and face with mask.
4.3.4 Others
As for safety measure, emergency escape plan is also one of the most important
elements. Emergency escaping plan is an evacuation route for all personnel from a
dangerous situation or accident without any cause of injuries. This should be placed
at the safety notice board and ensure that everyone knows it well. Fire extinguisher,
break glass alarm and first aid box should be provided at reachable corner. Doors with
emergency exit sign are denoted as the closest emergency exit.
Lastly, safety briefing must be conducted in order to improve the knowledge
of workers on how to operate and handle the equipment and procedures of producing
vaccine. The training session should be compulsory for all the workers before they
begin their work.

4.4 Ethics
Ethics are the principle guidelines designed by an organization to its management and
workers to help them lead through their actions with the ethical standards and primary values.
Several aspects of ethical are found through this project such as personal, environmental and
scientific ethics. There are some codes of ethics that will outline the values and mission of the
organization which will be discussed in the content below.
Having an attitude of respectful and cooperative with colleagues or interested parties
is an essential rule when dealing the project of producing vaccines. Colleagues should value
each and other’s ideas and work together. Through this, colleagues will have the same aims
and targets to be achieved and teamwork together to enrich it.
Besides, one of the ethics codes is being honest and dependable. Workers should be
honest and confidence with their works when reporting their results to their superior. Being
a professional worker, they have to be dependable in the workplace which means that no
supervisor is required to supervising them when they are assigned to do any works. Through
the collegiality, self-development and mentorship, they will be capable to contribute to the
development of profession.
In the production of viral vaccines, the honesty is represented by the use of certified
animal cells, growth media and virus strains from reliable sources. As the vaccine is to be
injected to human, any contaminants or impurities present in the final product can may cause
illness or even death.
Moreover, promoting a culture of safety is also an essential rule when working in a
plant to reduce the risk of accidents. For example, providing a safe workplace will indicate the
foundation for the organizational integrity and excellence in strategic and operational
performance (H. Krause, 2006). The workers are required to wear proper personal protective

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equipment (PPE) inside the plant as one of the safety practice.
Furthermore, taking responsibility for workers professional acts is also the one of the
ethics codes which may be relate to environmental aspect. To prevent the potentially
infectious materials and microbiological waste from causing harm to environment, workers
should take up their responsibility to decontaminate and autoclave them before disposal.
Workers are advised to always practice within the specialties authorized on their duty to
achieve the spirit of responsible.
High In order to strive for excellence in a professional practice through this project,
workers are advised to maintaining and improving their skills and knowledge via training
event or continuing education. Finally, workers will always performing at their best with their
passionate to their work to achieve the goals of the company.

4.5 Sustainability
High demand and consumption rates of ecological materials and services to satisfy
societal needs and for the dissipation of emissions are quickly exceeding the capacity that
nature can provide. Thus, sustainability has become an important topic where actions or
modifications are taken to avoid the use up of ecological services. By incorporating a
sustainability approach into the early stage of process design, the demand on goods and
services can be reduced and environmental releases can be prevented or minimized (Ruiz-
Mercado, Smith and Gonzalez, 2012).
According to Gonzalez and Smith (2003), the process sustainability can be evaluated
by using a methodology known as GREENSCOPE, which includes 4 main aspects: Efficiency,
Energy, Economics and Environment.
4.5.1 Efficiency
The efficiency of a process or a unit operation can be reflected in terms of the amount
of material and services that are necessary to generate desired product or complete
a specific process task. High reaction yield and atom economy is desired as there will
be less inerts and wastes presence in the process stream. For the cell-based
production of vaccines, the system is relatively new in which the type of influenza virus
strains will affeact the yield of viral titres (Nair, Lau and Brooks, 2013). However, as
reported by Hu et al. (2008), the growth of NIBRG-14 influenza virus strain MDCK cells
can achieve similar or even higher titers than seasonal influenza viruses in cells or eggs.
4.5.2 Energy
Energy demand of a chemical process plays an important role in the sustainability
performance as it affects the cost of total product, energy goods and services, and also
the emission of heat. During the manufacture of vaccines, the operating conditions
(temperature and pressure) are close to ambient conditions, with the highest
temperature at 37°C and pressure at 2 bar. Thus the heating and cooling utilities
requirement is not high. The main energy consumption will be on the shaft work of
agitator in mixing tanks and also the pumps.
4.5.3 Economics
Economic indicators describing profitability and costs are fundamental to defining a
sustainable process. For vaccine production, about 60 percent of the total production

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 costs are fixed regardless of the production scale. Thus a higher production is more
favorable in terms of the returns. One of the major challenge in vaccine production is
that the manufacturers have to foresee the occurrence of flu season and the type of
virus strain that will be prevalent at almost one year earlier (Baumann, 2009). If the
prediction is inaccurate there will be a huge loses to the company or organization. This
limits the interest of the manufacturers for massive vaccine production and thus there
is lacking of detailed economic analysis of the plant.
4.5.4 Environment
The prevention of negative impacts on environment is always one of the goal in
process design and optimization. By reducing or eliminating the pollutants through
the manufacturing process, the need for expensive further treatment can be reduced
or even removed.
For influenza vaccine production, formalin is used to inactivate the virus
particles which leads to the generation of toxic formaldehyde waste. In the
atmosphere, formaldehyde usually breaks down quickly to create formic acid and
carbon monoxide, which can also be harmful substances. Formaldehyde can cause
harm to the animals by making them sick, affecting their ability to breed, and reducing
their life spans. In addition, formaldehyde is highly toxic to aquatic life.
As the formalin is used in low amount, it is not very harmful and the effect can
be minimized by proper treatment of waste. Meanwhile, the biological wastes such as
cells and cellular debris are not likely to affect the environment if deactivated and
disposed properly.
As a conclusion, the efficiency, energy demand, and environmental performance of vaccine
production poses the potential for sustainable vaccine production. Nevertheless, in order to
evaluate the long term sustainability of this technology and its potential to preserve global
demands, a more comprehensive studies and complete economic analysis for industrial scale
vaccine production are required.

4.6 Appraisal
Vaccine is crucial for the body to build up immunity against virus. Continuous development
and improvement in the production of vaccines has led to cell culture-based vaccine
manufacturing method, which potentially replaces the conventional egg-based approach.
Egg-based vaccine is produced by injecting strains of virus in separate embryonated
chicken eggs. The virus is allowed to spread in the egg for 4 to 6 months. The fluid with fully
grown virus is then removed from the egg and undergo further treatment. The virus will be
purified, inactivated and fragmented. Inactivation is done to prevent the virus from infecting
the vaccine recipient.
Despite the long-standing egg-based manufacturing process, there are significant
drawbacks of the method. The main disadvantage is its long processing time, which could last
up to 10 months (Hennessy, 2010). Virus strains require a few months to be fully grown in the
egg, which contributes to its long lead time. Eggs must be prepared and allowed to grow the
virus months before the actual demand of vaccine, which may differ from year to year. This
indicates that manufacturers will not be able to adjust the production volume according to its

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demand after securing the eggs (Hennessy, 2010). Manufacturers are also unable to produce
the vaccine on short notice, where the demand increases due to the severity of flu (Bsargent,
2011). The production of vaccine by MDCK cells is quicker as it removes the need for
embryonated eggs from biosecure flocks. This method only requires 3 to 6 months as it
eliminates the lead time for the organization of egg supplies. The production of vaccine can
be initiated once the virus strain has been identified. This allows the production of vaccine to
be increased with ease in the event of a pandemic.
One major drawback of the egg-based vaccine is its variable yield. Instability and
variability within each egg may affect the growth of virus and the quality of vaccine (Matthews,
2006). Results of modifications done to the virus to improve its growth rate cannot be seen
until the virus has been removed from the egg. There is a risk of shortage of vaccine as the
slowest growing strain may limit the entire vaccine. MDCK cell-cultured vaccine does not pose
such uncertainty as the cell are pre-cultured in small spinner flask and its growth in the
bioreactor can be monitored by manipulating glucose concentration and maintaining its
temperature, pH and dissolved oxygen.
Risk of contamination is higher in egg-based vaccine as eggs are handled in open
environment. The process is usually carried out in lower biosafety level and the pathogens
cannot be handled effectively. There is risk of contamination of particulates and infection in
the eggs, which may wipe out the entire batch of vaccine. This further delays the availability
of the vaccine. Meanwhile, contamination in MDCK cell-cultured vaccine is reduced by
isolated environment with intensive biosafety (Health Science Authority, 2014). This also
reduces the possibility of virus mutation, which can cause culturing vaccine failure if using
embryonic eggs. However, the quality control becomes more difficult and the cost of
production is higher.
Another downside of egg-based vaccine is its large demand of eggs supply. This is
usually overcome by appointing a dedicated organization for egg supply. However, eggs are
susceptible to avian flu. Avian flu can kill off the egg embryo despite the large number of eggs
available, thus resulting in shortage of vaccine. Birds will suffer severe fatalities and the
number of eggs are further reduced. This does not pose any threat to MDCK cell-cultured
vaccine as it is not affected by avian flu.
The vaccine produced in both methods are similar in terms of safety and effectiveness.
However, the initial purity of vaccine is lower in the egg-based vaccine as it contains egg
protein. This also limits its use on people with egg allergies. Vaccine produced by MDCK cells
are relatively safer in this case as it does not contain egg protein.
The manufacturing cost of MDCK cell-cultured vaccine is higher than egg-based
vaccine due to its expensive biosafe environment to maintain high quality of vaccine (Health
Science Authority, 2014). Volume of bioreactor for MDCK cell-cultured vaccine is also larger,
as its volumetric yield is approximately 4 times lower than egg-based vaccine. This contributes
larger bioreactor and thus higher capital cost in cell-based process. However, MDCK cell-
cultured vaccine is more suitable for large scale production. The sizing of bioreactor and
process parameters can be ramped without incurring too much cost (Rappuoli, 2006). Larger
bioreactor is capable of producing more vaccine, but egg-based vaccine is limited by the
amount of eggs cultured.

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5. Conclusion and Future Study

As a conclusion, the cell-based vaccine production technology poses great potential to replace
the egg-based method. It can be operated in large scale and due to the capability of scaling-
up as compared to egg-based process, ability to monitor cell growth, and shorter production
time by eliminating the use of eggs.
However, there are some limitations that must be overcome before the technology
can be more widely used. The scaling-up of production process encounters challenges such
as turbulence of mixing, shear stress on the cells, eddies, aeration and foaming issues.
Corrective measures should be taken to achieve high yield of products. Meanwhile, aseptic
should be maintained to avoid both chemical and biological contamination. The equipment,
media and chemicals must be sterilized or make sure they are sterile before use.
As for the safety concern, there is no major risk since the operating conditions are
close to ambient conditions. However, extra care should be taken on the handling of live
viruses and toxic chemicals such as formaldehyde. The microbiological wastes and potentially
infectious materials should be decontaminated and autoclaved before disposal to minimize
the adverse effects to environment.
The process is considered sustainable in terms of efficiency, environment, and energy.
Nevertheless, it might not be economically feasible due to the large amount of fixed
production costs. More comprehensive studies are required to determine the economically
feasibility of the process.
For future study, the optimization of process should be attempted to achieve higher
yield and shorter production. Firstly, the performance of cell growth and virus yield in
suspension culture should be studied and compared with the adherent cell culture used in
this study. In addition, more research can be made on the type and composition of culture
media which would enhance the cell growth. Besides, different types of cells and virus strains
can also be used for pilot-scale production to look for better alternatives.

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