BIOCHEMICAL ENGINEERING (EP 432)

ASSIGNMENT TITLE: Production of Influenza vaccine

NAME STUDENT ID

Ainggararuban Ganeshan 1001642979

Somigha Parthipan 1001541417

Leena Jaiyashre Parana Chandran 1001644779

DATE OF SUBMISSION: 24th July 2020

LECTURER: Dr. Kiew Peck Loo

SCHOOL OF ENGINEERING
FACULTY OF ENGINEERING, TECHNOLOGY & BUILT
ENVIRONMENT

2020

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TABLE OF CONTENT

No. Title Page

1.0 JUSTIFICATIONS FOR THE PRODUCTION OF INFLUENZA 3
VACCINE
2.0 INTRODUCTION TO INFLUENZA VACCINE PRODUCTION 4
3.0 BIOREACTOR FOR INFLUENZA VACCINE PRODUCTION 8
3.1 Design Aspect of Bioreactor 8
3.2 Operation, Monitoring and Control of Bioreactor 11
3.3 Operating Mode of Bioreactor 13
3.4 Type of Bioreactor 15
4.0 STERILIZATION METHOD 16
5.0 RECOVERY AND PURIFICATION OF PRODUCT 17
5.1 Recovery of Product 17
5.2 Purification of Product 18
6.0 REFERENCE 19

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1.0 JUSTIFICATIONS FOR THE PRODUCTION OF INFLUENZA VACCINE

Influenza is a disorder that can eventually lead to hospitalization and sometimes even death.
Every flu season is different, and influenza illness can affect people differently, but every year
millions of people get flu, hundreds of thousands are hospitalized, and thousands to thousands of
people die each year from flu-related causes (Centres for Disease Control and Prevention, 2020).
Influenza is a respiratory infection which can cause severe complications, especially in young
children, older adults and people with certain medical conditions (Mayo Clinic staff, 2019). A
sudden onset of fever, cough (usually dry), headache, muscle and joint pain, severe malaise
(feeling unwell), sore throat, and a runny nose characterize seasonal influenza. The cough may be
severe and may last 2 weeks or more. Most people recover within one week without requiring
medical attention from fever and other symptoms but influenza can cause severe illness or death
especially in people at high risk which includes but not limited to young children, elderly people
and those with impaired immunity such as those with acquired immune deficiency syndrome
(AIDS).

There are 4 types of seasonal influenza viruses, type A, type B, type C and type D (World
Health Organization, 2018). Influenza A and B viruses are circulating and trigger seasonal disease
outbreak and this research focuses mainly on producing a vaccine targeting those two types. The
descriptions of the types of viruses are as follows:

a) Influenza A viruses are further classified into subtypes according to the combinations of
the hemagglutinin (HA) and the neuraminidase (NA), the proteins on the surface of the
virus. Currently circulating in humans are subtype A(H1N1) and A(H3N2) influenza
viruses. The A(H1N1) is also written as A(H1N1)pdm09 as it caused the pandemic in 2009
and subsequently replaced the seasonal influenza A(H1N1) virus which had circulated
prior to 2009. Only influenza type A viruses are known to have caused pandemics.

b) Influenza B viruses are not classified into subtypes, but can be broken down into lineages.
Currently circulating influenza type B viruses belong to either B/Yamagata or B/Victoria
lineage.

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 c) Influenza C virus is detected less frequently and usually causes mild infections, thus does
not present public health importance.

d) Influenza D viruses primarily affect cattle and are not known to infect or cause illness in
people.

2.0 INTRODUCTION TO INFLUENZA VACCINE PRODUCTION

Seasonal flu is caused by the Influenza group of viruses and are continuously mutating
especially Influenza A and B. This makes the production of suitable vaccines to be imperative and
to be done in a short amount of time to be able to be delivered, before the virus mutates again. The
process of vaccine development is described as follows (“WHO | Pandemic influenza vaccine
manufacturing process and timeline,” 2015):

a) Identification of a new virus.

Laboratories around the world routinely collect samples of circulating influenza viruses
and submit these to WHO Collaborating Centres for Reference and Research on Influenza
for analysis.
b) Preparation of the vaccine strain (called vaccine virus).
The virus is combined with a normal laboratory virus strain to make the vaccine virus less
harmful and better able to grow in hen 's eggs (the manufacturing process most
manufacturers use), and the two are allowed to grow together. After a while, a hybrid is
created that contains the laboratory strain's internal components, and the pandemic strain
outer components.
c) Verification of the vaccine strain.
After its preparation, the hybrid virus needs to be tested to make sure that it truly produces
the outer proteins of the pandemic strain.
d) Preparation of reagents to test the vaccine.
WHO Collaborating Centres produce standardized reagents that are given to all vaccine
manufacturers to enable them to measure how much virus they are producing, and to ensure
they are all packaging the correct dose of vaccine

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 There are various methods employed in the biochemical industry for the production of
Influenza vaccine, with the four major methods being egg based, cell based, recombinant protein
and plant based as shown in Figure 2.1.

FIGURE 2.1 Four major methods for Influenza vaccine production.

[Retrieved from (Connelly, 2015)]

The most common method is the egg based production which has remained relatively the
same since its inception in the 1970s and dominants the global vaccine market at 88% (Pandey et

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al., 2019). The advantages and disadvantages of each production method is shown in Table 2.1.
Vaccine strains that match circulating influenza viruses for the upcoming flu season are selected
by the World Health Organization (WHO) Global Influenza Surveillance and Response System
(GISRS). High yielding vaccine strains for egg- or cell-based production are generated by either
classic or reverse genetic reassortment. These adapted viruses go into mass production, either in
embryonated chicken eggs or MDCK cells with a production timeline of approximately six to
eight months. In recombinant HA (rHA) vaccines, the HA sequence is cloned into baculovirus and
expressed by insect cells, significantly shortening production time. The timeline of production of
various methods are shown in Figure 2.2.

TABLE 2.1 Advantages and disadvantages of various methods of production of

Influenza vaccine.

Method of Advantages Disadvantages

Production

Egg based General, can be used for various Longer period – 6 months
types of influenza virus.
Avian flu may affect production of
Well established method. eggs, which reduces the amount the
vaccine which can be produced.
Cheaper to produce.

Cell based Faster start-up of production. Longer period – 6 months

Ease of scale-up due to abundance

availability of mammalian cells.

Large amounts may be frozen and

kept in advance.

Recombinant Shorter period – 2 months. Less established, bigger room for error
protein in production.
No need to work with live virus,
reduces the risk involved.

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 Plant based Shorter period – 1 month. Less established, bigger room for error
in production.
Requires lower dosage and faster
production.

FIGURE 2.2 Timeline of current influenza vaccine production methods.

[Retrieved from (Chen et al., 2020)]

From the various methods of production, the cell-based approach was chosen to product
the Influenza vaccine to meet demand. Many organizations have made progress to move away
from the monopoly of egg based production as it has disadvantages such as allergies and the
instability of eggs when avian flu hits, and this effort has been gaining momentum from
governments globally (Chen et al., 2020). Cell based has a faster start-up of production, has a ease
of scale-up due to abundance availability of mammalian cells and large amounts may be frozen
and kept in advance.

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 Firstly, the target Influenza virus is mixed with a laboratory strain to get a hybrid which
contains the genetic material required for vaccine production, which has a lowered potency while
reducing the risk of contamination and dangers. This vaccine strain of the virus is then left to infect
a batch of mammalian cells which increases the amount of it. Next, the virus is harvested and
tested for required antigens using reagents specified by WHO to ensure the uniform efficiency of
it throughout the world. Once the potency is confirmed with the reagent, mass production of the
vaccine is done using a bioreactor followed by a purification process. The pure vaccine is then
tested for sterility, immunogenicity and safety before entering storage. The main advantage of cell-
based production is the ability to mass produce and freeze to keep it for a long period, enabling to
be transported to various parts of the world.

3.0 BIOREACTOR FOR INFLUENZA VACCINE PRODUCTION

3.1 Design Aspects of Bioreactor

The bioreactor is the key component of any biochemical process where enzymes, microbial,
mammalian, or plant cell systems are used to generate a wide variety of useful biological product
s. A bioreactor has the purpose of preparing a stable environment to meet the needs of the
biological reaction system so that a high yield of the bioprocess is achieved. To structure a proper
bioreactor for a specific bioprocess, intensive researches on the biological systems, for example,
cell development and metobolism, hereditary manipulation, and protein or other product
articulation are expected to comprehend the cells' necessity on their physical and chemical
environment. A variety of bioreactor types and configurations have thus been exploited and
developed along with the advances in the understanding of biological systems. Two particular
assortments of knowledge, to be specific, molecular biology and process engineering, are included
and the bioreactor is the core of the bioprocess, an efficient science-based approach to studying
bioreactors is needed.

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FIGURE 3.1 Process and scope of bioreactor engineering. [Retrieved from (Si-
Jing Wang,2007)]

As shown in Figure 3.1, the bioreactor really is the core of various biological processes. To
consider a bioreactor system, the last goal of this biological process must be distinguished, which
is frequently dictated by the market interest for a specific product or useful biotransformation
process. In light of the quick advances in recombinant DNA innovation and genome sequencing,
a similar product or biological process might be accomplished by various biological systems:
microorganisms, plant cells, animal cells, or enzymes. Their genetics articulations, metabolic
control, and bioreaction pathways all should be comprehended (Si-Jing Wang et al., 2007).

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 Bioreactors give numerous advantages in vaccine producing contrasted with static culture or
roller bottle technology. While numerous antibodies are as yet made using these more older
technologies, a few studies have indicated that manufacturing can be significantly improved by
utilizing bioreactors. For one, bioreactors give a bigger vessel to manufacture and thus can deal
with a larger batches one after another (at times 2,000 liters per batch). This huge scope production
diminishes costs by bringing down the total number of batches necessary to meet demand.
Specifically as the flu antibody is moved from egg-based production to cell culture-based
production, the requirement for huge scope virus manufacturing will be progressively essential
(Brandy Sargent, 2012).

Single - use bioreactors

Single use systems offer another alluring alternative in the area of bioreactors with
comparative benefits and some extra advantages that are especially appropriate to virus production.
They offer comparative development and efficiency to stainless steel bioreactors but are more
flexible. Single use systems have a quicker turnaround time between batches in light of the fact
that there is significantly less cleaning and validation necessary than with stainless steel tanks.
Cleaning and validation can be exorbitant and time consuming in the manufacturing process and
can delay the following manufacturing run.

Closed-system bioreactors

In any biological systems, there is consistently a danger of contamination, which can adversely
influence the quality, and all the more critically, safety of the products. Numerous bioreactors now
offer closed-system operation rather than regular open-system bioreactors, to guarantee the
sterility of their substance. Oxygen and nutrients can be acquainted with culture while waste, for
example, cell extracts can be expelled from culture through filtered pipes. Contrasted with open-
systemcounterparts, closed system bioreactors decrease the risks of fluid splashing and inadvertent
transfer of contaminants from the outside environment into bioreactors and better protecr the
operators. They likewise decline machine down time, leading to cost saving.

Automated Sensing

Another significant part of using bioreactors for biomanufacturing is sensing. To accomplish

high yield, host mammalian cells must be beneficial to help virus replication. Cells, being

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biological agents, are exceptionally sensitive to their environment. Having great chemical sensors
estimating temperature, oxygen, pH, nutrients, for example, glucose and amino acids, and cell
waste is therefore significant. Sensors are commonly more dependable in multi-use bioreactors
than single-use bioreactors. This is on the grounds that costly and progressively precise pH
electrodes are commonly not fused into disposable single-use bioreactor bags. To limit per-use
cost while considering the bag flattening assembly and delivery process, single-use bioreactors are
regularly utilizing non-invasive strategies like electromagnetic waves sensing with radio-
recurrence recognizable tags, and optic fibres installed into patches to distinguish for chemical
changes in their cellular contents (Andy Tay, 2020).

3.2 Operation, Monitoring and Control of Bioreactor

Monitoring and control of a bioreactor is a fundamental part of a process. In a mechanical

scale processes, monitoring a process could set aside a fortune as undesired reaction can be kept
away from or reduced. Strong demand for different applications invigorated progress in bioreactor
structure configuration to design explicit purposes, for example, solid-state fermentation
bioreactors utilized in the anaerobic membrane bioreactors for wastewater treatment, classic tank
bioreactors utilized in the fermentation business, and the as of the recently developed inexpensive
single-use bioreactors for the small scale production of high-esteem biological pharmaceuticals. It
is necessary to control the bioreactor's operating parameters in order to favor the desired functions
of the living cells or enzymes. Dissolved oxygen concentration, pH, temperature, mixing, and
supplementation of nutrients all need to be controlled and optimized (Si-Jing Wang et al., 2007).

The media design and improvement can be founded on an essential information on

stoichiometry and experimental data, including monitoring the composition changes of the media,
intermediates, productss, and nutrients. Stoichiometric calculations give quantitative connections
between yields of biomass and product synthesis, maintenance necessity and energy production.
Complementing stoichiometric information for the design of a bioreactor, a kinetic study will
uncover the biological response rates, including cell development, substrate consumption, product
synthesis and side-effect arrangement rates. Numerous enzymatic responses are involved, and
inhibitions caused by products, results, or even substrate at high concentrations are frequently
observed.
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 The chosen bioreactor ought to be additionally described and the operational mode ought to
be optimized. The bioreactor's qualities and operational mode likewise extraordinarily influence
the biological performances. A productive bioreactor system depends enormously on its control
and support systems. Regardless of how significant the bioreactor system is, it must be intently
and proficiently coordinated into the entire production system. In this way, different procedure
necessities and requirements should also be considered.

To increase virus production yields through process optimization, three key factors need to be
considered:

1) Cell concentration and metabolic/physiological status of the cells at time of infection.As a

general principle, cell focus characterizes final virus titers. Be that as it may, it is fundamental to
perform infections on healthy cells, with no confinement of key nutrients, not inhibited by gathered
by products, for example, lactate and ammonia, and in a suitable growth status

2) Ratio of irresistible particles to viable cells, to be specific variety of disease at the hour of
contamination. Since infection transport to the target cell in the way of life medium is administered
by diffusion, an ideal measure of virus particles per cell ought to be vaccinated to check
degradation/inactivation of irresistible virions before they arrive at their host cell. Likewise, for
most infections, an excessively high number of infection particles per cell can advance replication
of defective interfering particles (DIPs), which diminishes greatest virus yields. This is of specific
significance in consistent developments utilizing falls of stirred tank bioreactors (STR), where
decreases in virus titers for long development times have been seen as a result of DIP
accumulation.

3) Residence time (RT) of virus particles within the bioreactor and time point of harvest. The
RT can be defined as the time that a cell or a virus particle remains inside the bioreactor and is
characteristic for the cultivation mode. In closed systems operated in batch cultivation mode, the
RT is identical for all particles and equivalent to the harvest time. For viral vaccines, where potency
depends totally or partially on infectivity (e.g., live attenuated vaccines, viral vectors), a short RT
is beneficial. When batch knowledge is transferred to continuous systems, the picture is more
complex as not all particles spend the same time inside the continuously operated bioreactor. In

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continuous bioreactors, an important concept is the RT distribution, which is essentially a
statistical approach to describe the probability of particles to leave the bioreactor. Nevertheless, a
good approximation is given by the average RT that, in continuous STRs, equals the inverse of the
dilution rate (RT = 1/D) (Felipe Tapia et al., 2016).

3.3 Possible Type of Bioreactors

Bioreactors provide many benefits in vaccine manufacturing compared to static culture.

Bioreactor provides a larger vessel for manufacturing and in turn can handle much larger batches
at one time. Bioreactors also offer the advantage of optimal culture conditions. Cells lines used for
virus production are notoriously difficult to grow and bioreactors offer more options for optimizing
growing conditions to maintain cell health and increase productivity. There are two possible
bioreactors that were discussed which are stirred-tank bioreactor and hollow fibre bioreactor.
After lengthy discussions among the committee, we have decided to go with the hollow fibre
bioreactor (HFB).

Hollow fibre bioreactors, modelled after the mammalian circulatory system, provide the
most in-vivo way in which to expand cells in any laboratory (Fibre Cell System, 2018). Since the
cells are attached to a porous support (the hollow fibre) rather than a non-porous plastic dish,
nutrients are delivered from the bottom layer of cells on upwards. Splitting of the cells is not
required and cultures can be maintained for many months of continuous production. When the
secreted protein is retained in the extra-capillary space it will accumulate to a concentration of up
to 100 times higher than with conventional flask or roller bottle culture.

Cells are normally seeded into the EC bioreactor space via a port at the top. The fresh
medium is pumped continuously through the fibre lumen. When the cell population increases, this
medium feed rate is gradually increased to meet nutritional needs and to prevent metabolic waste
from building up (Hirschel et al., 2011). Depending on the average pore size of the semi-permeable
hollow fibre membrane, greater molecular weighted components such as secreted protein (> 10
KD) or viruses are prevented from crossing into IC space. For the duration of the crop, therefore,
concentrated product is usually harvested at a steady rate from EC space.

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 Hollow fibres can be constructed from cellulosic, polysulfone, polypropylene, or
polyethylene materials, thus allowing a choice depending on the characteristics needed for optimal
protein or virus production. The vast majority of HFB utilize cellulosic fibres for uniform cell
expansion and production of mammalian cell-secreted products.

FIGURE 3.3.1 Basic hollow fiber bioreactor design and the HF Primer™ small-scale
bioreactor [Retrieved from (Mark Hirschel, 2011)]

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 FIGURE 3.3.2 Working principle of a hollow fibre bioreactor.

[Retrieved from (Fibre Cell System, 2018)]

3.4 Bioreactor Operating Mode

3.4.1 Perfusion mode

Perfusion operates in a similar concept to continuous, but differs slightly in which the
flowrate of inlet is variable and changes depending on demand. It is a process that continuously
replenishes nutrients for the cell culture while removing metabolic wastes at the same time, with
fresh media is provided to the cells at the same rate as the spent media is removed (Shevitz &
Bonham-Carter, 2011). Due to complex or faulty equipment and scale-up problems associated with
cell preservation, perfusion has been performed in cell culture since the 1980's but with minimal
acceptance. Separate advances in cell line engineering, media composition and bioreactor
architecture have resulted in several increases in batch and fed batch titers, thereby reducing the
need for perfusion technology.

Recently, however, the need for alternative manufacturing strategies that can boost
efficiency and productivity while reducing costs has led to renewed interest in perfusion
technology. These benefits include higher yield, increased speed, cost savings, efficient facility
utilization, better scalability and improved product quality and stability. Perfusion bioreactors
culture cells over much longer periods, even months, by continuously feeding the cells with fresh
media and removing spent media while keeping cells in culture. In perfusion there are different
ways to keep the cells in culture while removing spent media (Sargent, 2016). One way is to keep
the cells in the bioreactor by using capillary fibres or membranes, which the cells bind to which is
applied in the case of HFB.

Benefits of perfusion culture include:

a) Stability: Perfusion culture has a proven track record of success in manufacturing unstable
proteins including coagulation factors and enzymes and vaccines.
b) Product Quality: A key advantage here is that you can maintain high cell density and
optimal steady state conditions, thereby lowering levels of impurities and enabling high
and consistent product quality. In addition product quality is improved because of

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 decreased product hold time and there is less intermediate testing needed. Ideally perfusion
systems are run as closed systems, which minimizes bioburden risk
c) Flexibility: Perfusion bioreactors are smaller in size and are typically compatible with
disposable products. This enables rapid capacity increase or decrease with little impact to
overall facility footprint. This size advantage is important because it means that facilities
don’t need a significant increase in space to increase production.
d) Less manual operations and a high level of automation: Automation reduces the amount
of manual operations, thus freeing operator time for other tasks and reducing the risk of
operator error.

4.0 STERILIZATION METHOD

Sterilization refers to any process that destroys, kills or deactivates all life forms (including
micro-organisms such as fungi , bacteria , viruses, spores, unicellular eukaryotic species such as
plasmodium, etc.) and other biological agents such as prions found in a specific surface, substance
or fluid, such as food or biological media. As such, there are various methods employed in the
sterilization process of influenza vaccines to obtain a pure and desired concoction that delivers the
impact with no major side effects. These methods include bombardment of UV rays, application
of antiseptics and heating (Taylor, 2009).

The method chosen after due diligent discussion and consideration by both engineers and
the whole consultation team, we have decided to go with the ultraviolet (UV) inactivation method.
Many studies require the whole virus to be handled in a biosafety level 2 setting. A potential
solution for managing this problem is pathogen inactivation without affecting its antigenicity
(Mathew et al., 2018). The inactivation was complete when a UV dose of 0.09 J/cm2 for 3 × 30 s
was used and no change in antigenicity and integrity was observed. UV irradiation has been known
as an effective method to inactivate viruses for a long time and it preserves the integrity of the
immunological epitopes. The absorption of UV causes photochemical damage in viruses as UV
induces adjacent pyrimidine. In conclusion, UV inactivation with the reported dose could be an
effective and simple inactivation method to inactivate the Influenza virus to a safe level. It is very
convenient and takes less than 2 min to inactivate the virus completely.ne nucleotide dimerization.
This can create mutations, finally inhibiting virus replication.

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5.0 RECOVERY AND PURIFICATION OF PRODUCT

5.1 Recovery of Product

The downstream processing (DSP) is becoming an important factor in the race for higher
overall productivity and decreased cost of goods in the production of influenza vaccine . The
general aim of downstream processing (DSP) is the recovery and purification of biological
products from process- and product-related impurities. Process-related impurities might originate
from cell culture reagents and additives, from the purification process, or from the cell substrate.
Examples for virus particle-related impurities include free envelope proteins, virus aggregates or
empty capsids, and virus particles that contain nucleic acid sequences other than the intended
genome. Naturally, the requirements on product purity and product safety depend on the
particular application. Vaccines and viral vectors need to meet the stringent guidelines of
regulatory authorities.

The cell disruption was carried out by mechanical lysis using a high pressure mechanical
homogenizer. Before that, the cells were washed once to eliminate traces of culture media and
reduce the viscosity of the cell suspension. This was to prevent the formation of cell
conglomerates that could clog the homogenizer’s valve. High pressure homogenizer is used for
this cell disruption process due to greater yield of viable product.

For influenza virus vaccine purification, the unit operation used for clarification is the
depth filtration using depth filters. Depth filters is used to separate cell debris and other solids in
extracellular mammalian culture. Depth filters used in bioprocessing are typically composed of a
fibrous bed of cellulose or polypropylene fibers along with a filter aid (e.g., diatomaceous earth)
and a binder that is used to create flat sheets of filter medium. The filter aids provide a high
surface area to the filter.

Purification of virus particles based on these unique characteristics and removal of

contaminants according to the regulatory guidelines can only be achieved by a combination of
different unit operations. In this study, there are few unit operations used for DSP of viral
vaccines.

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5.2 Purification of product

The unit operation chosen for purification of influenza vaccine is the Monolithic Convective
Interaction Media Chromatography (CIM). CIM is a special type of chromathographic column for
the purification of influenza vaccine production. They have a single block of a homogeneous
stationary phase with many interconnected channels (Podgornik et al. 2014) and is inserted into
the chromatographic housing. They are characterized by highly inter-connected network of
channels of sponge. The binding sites are situated at inside the channels where there is no dead
end pores and no diffusion limitations. The performance of the CIM is same at lower and higher
flow rates. The channels of the CIM is large around (1-2µm) which is optimal for molecules like
virus, virus like particles and DNA to flow through the channels and bind to the binding sites of
the chromatograph.

Due to the various advantages of CIM, it is considered to be the best unit operation for
purification of influenza vaccine. In CIM, the separation of virus dominantly happens by the
convective flow through channels having a diameter of more than 1,000nm. This allows high flow
velocity and therefore high throughput purifications. Monolithic columns have high porosity due
to their large interconnected channels. Optimization of pore size and pore distribution can be done
over a wide range by changing the porogen composition, porogen to monomer ratio and
polymerization temperature (Strancar et al. 2002). Owing to their high bed porosity, the pressure
drop in monoliths is lower. Monolithic columns have a low absolute surface area, but a high
adsorption area due to their porous structure (Jangbauer et al. 2008) allowing a high dynamic
binding capacity (DBC). Another main advantage of monoliths is their flow-independent DBC
allowing straight-forward scalability. The yield of purification recovery is 90% in CIM. Other than
that, Due to the high flow velocity and low pressure drop, the overall process time using monoliths
is low. Monoliths can be customized to the needs of the user, since there are various stationary
phase chemistries, active binding sites and shapes and sizes (disks, tubes) available. Although the
shape of the monolith per se does not present a significant advantage, a minor advantage to be
noted is that monolithic disks do not have a specific flow direction, enabling back-flush to
encounter clogging problems. Monolithic supports are easy to scale up without the need for column
modifications . Monolithic disks can be stacked up to four disks in the same housing, increasing
efficiency and capacity without compromising resolution.

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