VACCINE

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Faculty of Pharmacy
QUALITY CONTROL ASSINGMENT
VACCINE PRODUCTION

Submitted by: Faizah Khalid

Roll no.: L1F17PHMD0174
Section: C
Submitted to: Sir Hassan
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INTRODUCTION:
 World Health Organization (WHO) defines a vaccine as follows:

“A vaccine is a biological preparation that improves immunity to a particular disease. A

vaccine typically contains an agent that resembles a disease-causing microorganism and is
often made from weakened or killed forms of the microbe, its toxins, or one of its surface
proteins. The agent stimulates the body's immune system to recognize the agent as foreign,
destroy it, and "remember" it so that the immune system can more easily recognize and
destroy any of these microorganisms that it later encounters.”

 Vaccines are proteins, polysaccharides, or pathogenic nucleic acids that are used to
stimulate immunity, to eliminate or weaken pathogens or
pathogenic products.
 In other words, vaccines are biological preparations that
help to acquire immunity against diseases. Vaccines are
usually a virus or bacterium-based preparations that fight
against diseases, caused by viruses or bacteria.

MECHANISM OF VACCINES:
 The function of vaccines is built on immunity. Immunity
identifies germs as foreign invaders and reacts by producing proteins called antibodies.
Antibodies destroy the germs and memorize the invaders. They remain in the
bloodstream and can detect if the same germs enter the body again to infect it. Since
antibodies have already once destroyed them earlier, they can annihilate them before
infecting the body
 Antibody is a protein that immune system has created in the presence of specific antigen.
Antibodies help to counter the effects of antigens, by attaching to them. Antigens are
substances that stimulate the immune system to create antibodies.
 Immunization means making the body resistance against germs (germ = pathogenic
microorganism). This happens with the help of antigens that stimulates the antibody
production and cell-mediated immunity. When the immunity has been acquired, the
body is resistant against specific antigens.

Schematic representation of the mechanisms of vaccination and immune defense against viruses.
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 Active and passive immunity are part of ADAPTIVE IMMUNE SYSTEM.

ACTIVE IMMUN ITY:

 If the immunity is based on body's own cell activity (antibodies are made in the
body), it is called active immunity. Active immunity can be achieved by natural
infection or artificially with vaccination. Active immunizations, in other words,
vaccinations biggest advantage is that once acquired protection can last years.
 Immunity can be humoral or cell-mediated and these are often working together.
Humoral immunity is mediated by B-lymphocytes, while cell-mediated immunity
by T-lymphocytes
 B-lymphocytes can divide to memory cells and cells that form antibodies. T-
lymphocytes can recognize and destroy cells infected by a virus and help B-
lymphocytes to create specific antibodies. Both lymphocytes can detect antigens.
 PASSIVE IMMUNITY:
 Resistance is acquired with passive immunization, if antibodies are created
somewhere else. Passive immunity happens naturally through maternal
antibodies or through artificially made monoclonal antibodies. Protection trough
passive immunization usually lasts only few months. Sometimes adjuvants are
needed to help enhance the immunity.

ACTIVE IMMUNIY PASSIVE IMMUNITY

It is developed due to contact with pathogen or antigen
It has no side effects
It is slow but long-lasting
It takes time to develop a response
Eg. Vaccination for polio etc
It is developed when readymade antibodies are injected into
the body
It may cause a reaction
It is fast but last for only few days
It is used when the immune response has to be faster
Eg. Administration of tetanus antitoxins etc
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HISTORY OF VACCINES:

 The use of vaccination to immunize people against infectious diseases has influenced the
world for the past 200 years. It is one of the most remarkable achievements in the history
of medicine.
 Early form of vaccination was developed in 1100's China. This method is called
variolation, and it arrived at Europe around 1700's.
 Most vaccines invented before 20th century, were often created by accident. Scientists
tested different techniques and substances very freely because there were not much
restrictions or regulations concerning it.

TYPES OF VACCINES:
There are four categories of vaccines in clinical trials: 

1. Whole virus
2. Protein subunit
3. Viral vector 
4. Nucleic acid (RNA and DNA)
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i. WHOLE PATHOGEN VACCINES

 Whole virus vaccines use a weakened (attenuated) or deactivated form of the pathogen
that causes a disease to trigger protective immunity to it. 
 There are two types of whole virus vaccines:
i. LIVE ATTENUATED VACCINES
 Live-attenuated vaccines resemble the natural infection, but in the weakened form. It is
made of attenuated form of the germ that causes a disease, but retains the ability to infect
and replicate in the vaccinated host.
 The weakened form is achieved by
passaging the pathogen into tissue cultures
repeatedly. The weakest variants of the
pathogen are then selected from each
repetition.
 Live-attenuated vaccine can give a lifetime
protection against germs that are
responsible for the disease, only from one
or two doses to the body.
 These vaccines can be used against for
example measles, smallpox and yellow fever, which are caused by a virus.
 Tuberculosis vaccine is a bacterium based live-attenuated vaccine. It needs to be kept
in cool, so delivery and storing might be an issue.
 Also, people with weakened immune systems might not be able to receive this type of
vaccine because it contains a small amount of the attenuated live virus
 Some viruses cannot be attenuated enough to be given to humans without a risk of getting
the disease, for example HIV virus (Human Immunodeficiency Virus).

 ADVANTAGES AND DISADVANTAGES:

 Strong immune response
 Immune response involves B cells and T
cells
 Relatively simple to manufacture
 Unsuitable for people with compromised
immune systems
 May trigger disease in very rare cases
 Relatively temperature sensitive, so
careful storage necessary
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ii. INACTIVATED VACCINES

 Inactivated vaccines contain whole bacteria or viruses which have been killed or have
been altered, so that they cannot replicate.
 Because inactivated vaccines do not contain any live bacteria or viruses, they cannot
cause the diseases against which they protect, even in people with severely weakened
immune systems.
 However, inactivated vaccines do not always create such a strong or long-lasting
immune response as live attenuated vaccines. 
 In the inactivated vaccines the pathogen is inactivated by chemical or physical means or
combination of both. Unfortunately, inactivation process could destroy the antigen
conformation required to elicit protective immunity.
 It needs to be taken periodically over time to get permanent immunity.
 This type of vaccine can protect against for example hepatitis A and rabies, which are
caused by a virus. Typhoid vaccine is a bacterium based inactivated vaccine
 ADVANTAGES AND DISADVANTAGES:
 Well-established technology
 Suitable for people with compromised immune systems
 No live components, so no risk of the vaccine triggering disease
 Relatively simple to manufacture
 Relatively stable
 Booster shots may be required
MANUFACTURING OF WHOLE PATHOGEN VACCINES:

 Different viruses require slightly different production processes, meaning separate

equipment and facilities are needed for each one.
 For instance:
 The influenza virus is grown inside fertilized chicken eggs - which must
themselves be sourced from specialized sterile laying facilities.
 Polio virus is grown in dishes of cells, which require different handling, while
bacteria-based vaccines are grown in vast bioreactors.
 Growing live pathogens also means stringent precautions must be taken to avoid the virus
escaping and making vaccine plant workers sick.
 Once large amounts of virus or bacteria have been grown, they must then be isolated,
purified and attenuated or inactivated, depending on the vaccine.  Each of these steps
requires specific equipment, reagents, and stringent procedures to avoid, and check for,
contamination, which can further increase costs.
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ii. SUBUNIT VACCINES

 Subunit vaccine is a type of vaccine that uses only the protein part of the germ and does
not contain its genetic material.
 To prepare subunit vaccines, one must have identified the key antigenic target to include
in the vaccine.
 Immunogens of these vaccines are composed of bacterial proteins, polysaccharides or
conjugates of both.
 This type of vaccine gives a strong immune response because it targets the key parts of
the germ.
 There are similarities with inactivated vaccines. Subunit vaccine can also be given to
infants and people with weakened immunity system. It needs to be taken several times
over period to keep the protection against diseases. Adjuvants are often added to subunit
vaccines. As a result, common local reactions (such as a sore arm) may be more
noticeable and frequent with these types of vaccines
 There are several types: protein subunit vaccines contain specific isolated proteins from
viral or bacterial pathogens; polysaccharide vaccines contain chains of sugar molecules
(polysaccharides) found in the cell walls of some bacteria; conjugate subunit vaccines
bind a polysaccharide chain to a carrier protein to try and boost the immune response.
ONLY PROTEIN SUBUNIT VACCINES ARE BEING DEVELOPED AGAINST
THE VIRUS THAT CAUSES COVID-19.
 This type of vaccine is used to protect against hepatitis B and pneumococcal disease
 ADVANTAGES AND DISADVANTAGES OF SUBUNIT VACCINES:
 Well-established technology
 Suitable for people with compromised immune systems
 No live components, so no risk of the vaccine triggering disease
 Relatively stable
 Relatively complex to manufacture
 Adjuvants and booster shots may be required
 Determining the best antigen combination takes time

 There are 5 types:

i. RECOMBINANT PROTEIN VACCINES
 Protein subunit vaccines contain specific isolated proteins from viral or bacterial
pathogens
 Recombinant vaccines are made using bacterial or yeast cells to manufacture the
vaccine. A small piece of DNA is taken from the virus or bacterium against which we
want to protect and inserted into the manufacturing cells.
 For example, to make the hepatitis B vaccine, part of the DNA from the hepatitis B virus
is inserted into the DNA of yeast cells. These yeast cells are then able to produce one of
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the surface proteins from the hepatitis B virus, and this is purified and used as the active
ingredient in the vaccine. 

ii. TOXOID VACCINES

 Some bacteria release toxins (poisonous proteins) when they attack the body, and it is the
toxins rather than the bacteria itself that we want to be protected against.
 The immune system recognizes these toxins in the same way that it recognizes other
antigens on the surface of the bacteria, and is able to mount an immune response to
them. Some vaccines are made with inactivated versions of these toxins. They are called
‘toxoids’ because they look like toxins but are not poisonous. They trigger a strong
immune response.  
 Toxoid is a chemically inactivated toxin.
 A toxoid vaccine does not give a lifetime protection, thus booster shots might be needed.
 This type of vaccine protects against diphtheria and tetanus. These diseases are caused
by a bacterium.

iii. CONJUGATE VACCINES

 Conjugate subunit vaccines bind a polysaccharide chain to a carrier protein to try and
boost the immune response.
 ‘Conjugate’ means ‘connected’ or ‘joined’. With some bacteria, to get protection from a
vaccine you need to train the immune system to respond to polysaccharides (complex
sugars on the surface of bacteria) rather than proteins.  But in the early days of
polysaccharide vaccines it was found that they did not work well in babies and young
children.
 Researchers discovered that they worked much better if the polysaccharide was attached
(conjugated) to something else that creates a strong immune response. In most conjugate
vaccines, the polysaccharide is attached to diphtheria or tetanus toxoid protein (see
‘Toxoid vaccines’ above). The immune system recognizes these proteins very easily and
this helps to generate a stronger immune response to the polysaccharide. 
 On product information sheets the diphtheria toxoid is often called ‘CRM197 carrier
protein’, because it is almost the same as diphtheria toxoid but not quite.  
iv. VIRUS LIKE PARTICLES

 Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-
infectious because they contain no viral genetic material. They can be naturally occurring
or synthesized through the individual expression of viral structural proteins, which can
then self-assemble into the virus-like structure.

 In some cases, the antigens in a VLP vaccine are the viral structural proteins themselves.
Alternatively, the VLPs can be manufactured to present antigens from another pathogen
on the surface or even multiple pathogens at once.
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 As each VLP has multiple copies of an antigen on its surface it is more effective at
stimulating an immune response that a single copy. In some cases, the structural proteins
of the VLP can act as adjuvants, helping to strengthen the immune response to the
primary target antigen. 

 A handful of VLP-based vaccines are currently used worldwide are Hepatitis B vaccine
and HPV vaccine

v. OMV VACCINES

 Outer Membrane Vesicles (OMVs) are naturally produced by bacteria and are essentially
a bleb of the bacterial outer cell membrane.

 This contains many of the antigens found on the cell membrane but is a non-infectious
particle.

 In the lab these OMVs can be harvested from bacteria to use as vaccines. The OMVs can
also be modified so that toxic antigens are removed and antigens suitable for stimulating
an immune response can be kept. OMVs also naturally act as adjuvants. This is a newer
vaccine technology so there are few licensed examples is MenB vaccine (meningococcal
B vaccine)

MANUFACTURING OF SUBUNIT PROTEINS:

 All subunit vaccines are made using living organisms, such as bacteria and yeast, which
require substrates on which to grow them, and strict hygiene to avoid contamination with
other organisms. This makes them more expensive to produce than chemically-
synthesized vaccines, such as RNA vaccines.
 The precise manufacturing method depends on the type of subunit vaccine being
produced:
 Protein subunit vaccines, such as the recombinant hepatitis B vaccine, are made
by inserting the genetic code for the antigen into yeast cells, which are relatively
easy to grow and capable of synthesizing large amounts of protein. The yeast is
grown in large fermentation tanks, and then split open, allowing the antigen to be
harvested. This purified protein is then added to other vaccine components, such
as preservatives to keep it stable, and adjuvants to boost the immune response –
in this case alum.
 For Polysaccharide, the polysaccharide is produced by growing bacteria in
industrial bioreactors, before splitting them open and harvesting the
polysaccharide from their cell walls.
 In the case of Conjugate Vaccines, the protein that the polysaccharide is
attached to must also be prepared by growing a different type of bacteria in
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separate bioreactors. Once its proteins are harvested, they are chemically attached
to the polysaccharide, and then the remaining vaccine components added.

iii. NUCLEIC ACID VACCINES

 Nucleic acid vaccines use genetic material from a disease-causing virus or bacterium (a
pathogen) to stimulate an immune response against it.

 Depending on the vaccine, the genetic material could be DNA or RNA; in both cases it
provides the instructions for making a specific protein from the pathogen, which the
immune system will recognize as foreign (an antigen). Once inserted into host cells, this
genetic material is read by the cell’s own protein-making machinery and used to
manufacture antigens, which then trigger an immune response.

 This is a relatively new technology, so although DNA and RNA vaccines are being
developed against various diseases, including HIV, Zika virus and COVID-19

 ADVANTAGES AND DISADVANTAGES:

 Immune response involves B cells and T cells

 No live components, so no risk of the vaccine triggering disease

 Relatively easy to manufacture

 Some RNA vaccines require ultra-cold storage

 Booster shots may be required

i. RNA VACCINES
 RNA vaccines use mRNA (messenger RNA) inside a lipid (fat) membrane. This fatty
cover both protects the mRNA when it first enters the body, and also helps it to get
inside cells by fusing with the cell membrane.
 Once the mRNA is inside the cell, machinery inside the cell translates it into the
antigen protein. This mRNA typically lasts a few days, but in that time sufficient
antigen is made to stimulate an immune response. It is then naturally broken down and
removed by the body. RNA vaccines are not capable of combining with the human
genetic code (DNA).
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 The Pfizer BioNTech and the Moderna COVID-19 vaccines are both RNA

vaccines. 
ii. DNA VACCINES
 DNA is more stable than mRNA so doesn’t require the same initial protection. DNA
vaccines are typically administered along with a technique called electroporation.
 This uses low level electronic waves to allow the bodies’ cells to take up the DNA
vaccine. DNA must be translated to mRNA within the cell nucleus before it can
subsequently be translated to protein antigens which stimulate an immune response.
 There are currently no licensed DNA vaccines, but there are many in development.
 Several DNA vaccines are licensed for animal use, including a horse vaccine against
West Nile virus.

MANUFACTURING OF NUCLEIC ACID VACCINES:

 Once a pathogen’s genome has been sequenced, it is relatively quick and easy to design a
vaccine against any of its proteins.

 For instance, Moderna’s RNA vaccine against COVID-19 entered clinical trials within

two months of the SARS-CoV-2 genome being sequenced. This speed could be
particularly important in the face of new emerging epidemic, pandemic pathogens or
pathogens which are rapidly mutating.

 Both DNA and RNA vaccines are relatively easy to produce, but the manufacturing
process differs slightly between them. Once DNA encoding the antigen has been
chemically synthesized, it is inserted into a bacterial plasmid with the help of specific
enzymes - a relatively straightforward procedure.

 Multiple copies of the plasmid are then produced within giant vats of rapidly dividing
bacteria, before being isolated and purified. RNA vaccines are easier to synthesize
because this can be done chemically, from a template in the lab, without the need for any
bacteria or cells.
 In both cases, vaccines for different antigens could be manufactured within the same
facilities, further reducing costs. This is not possible for most conventional vaccines.

iv. VIRAL VECTORED VACCINES

 Viral vector-based vaccines differ from most conventional vaccines in that they don’t
actually contain antigens, but rather use the body’s own cells to produce them.
 They do this by using a modified virus (the vector) to deliver genetic code for antigen, in
the case of COVID-19 spike proteins found on the surface of the virus, into human cells.
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 By infecting cells and instructing them to make large amounts of antigen, which then
trigger an immune response, the vaccine mimics what happens during natural infection
with certain pathogens - especially viruses.
 This has the advantage of triggering a strong cellular immune response by T cells as well
the production of antibodies by B cells.
 An example of a viral vector vaccine is the rVSV-ZEBOV vaccine against Ebola.
 ADVANTAGES AND DISADVANTAGES:
 Well-established technology
 Strong immune response 
 Immune response involves B cells and T cells
 Previous exposure to the vector could reduce effectiveness
 Relatively complex to manufacture

i. REPLICATING 
 Replicating viral vectors retain the ability to make new viral particles alongside
delivering the vaccine antigen when used as a vaccine delivery platform. It produces a
stronger immune response. A single vaccine may be enough to give protection. 
 Replicating viral vectors are typically selected so that the viruses themselves are
harmless, or are attenuated, so whilst they are infecting the host, they cannot cause
disease. Despite this, as there is still viral replication going on there is an increased
chance of mild adverse events (reactions) with these vaccines.
 A vaccine to prevent Ebola called Ervebo (rVSV-ZEBOV) uses a recombinant vesicular
stomatitis virus.

ii. NON-REPLICATING
 Non-replicating viral vectors do not retain the ability to make new viral particles during
the process of delivering the vaccine antigen to the cell. This is because key viral genes
that enable the virus to replicate have been removed in the lab.
 This has the advantage that the vaccine cannot cause disease and adverse events
associated with viral vector replication are reduced. However, vaccine antigen can only
be produced as long as the initial vaccine remains in infected cells (a few days). This
means the immune response is generally weaker than with replicating viral vectors and
booster doses are likely to be required.
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 The Oxford-AstraZeneca COVID-19 vaccine which was approved for emergency use

by the MHRA in December 2020 also uses a non-replicating viral vector called
ChAdOx1. 

MANUFACTURING OF VIRAL VECTOR BASED VACCINES:

 A major bottleneck for viral vector vaccine production is scalability. Traditionally, viral
vectors are grown in cells that are attached to a substrate, rather than in free-floating cells
- but this is difficult to do on a large scale.
 Suspension cell lines are now being developed, which would enable viral vectors to be
grown in large bioreactors.
 Assembling the vector vaccine is also a complex process, involving multiple steps and
components, each of which increases the risk of contamination.
 Extensive testing is therefore required after every step, increasing costs.

INGREDIENTS OF VACCINES:
Each vaccine component serves a specific
purpose, and each ingredient is tested in the
manufacturing process. All ingredients are tested
for safety.

ANTIGEN
 All vaccines contain an active component
(the antigen) which generates an immune
response, or the blueprint for making the
active component. The antigen may be a
small part of the disease-causing
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organism, like a protein or sugar, or it may be the whole organism in a weakened or

inactive form.

PRESERVATIVES
 Preservatives prevent the vaccine from becoming contaminated once the vial has been
opened, if it will be used for vaccinating more than one person. Some vaccines don’t
have preservatives because they are stored in one-dose vials and are discarded after the
single dose is administered. The most commonly used preservative is 2-phenoxyethanol.
It has been used for many years in a number of vaccines, is used in a range of baby care
products and is safe for use in vaccines, as it has little toxicity in humans.

STABILIZERS
 Stabilizers prevent chemical
reactions from occurring within
the vaccine and keep the vaccine
components from sticking to the
vaccine vial. 
 Stabilizers can be sugars (lactose,
sucrose), amino acids (glycine),
gelatin, and proteins (recombinant
human albumin, derived from
yeast).

SURFACTANTS

 Surfactants keep all the ingredients in the vaccine blended together. They prevent settling
and clumping of elements that are in the liquid form of the vaccine. They are also often
used in foods like ice cream.

RESIDUALS

 Residuals are tiny amounts of various substances used during manufacturing or

production of vaccines that are not active ingredients in the completed vaccine.

 Substances will vary depending on the manufacturing process used and may include egg
proteins, yeast or antibiotics.

DILUENT
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 A diluent is a liquid used to dilute a vaccine to the correct concentration immediately

prior to use. The most commonly used diluent is sterile water.

ADJUVANT
 Some vaccines also contain adjuvants. An adjuvant improves the immune response to the
vaccine, sometimes by keeping the vaccine at the injection site for a little longer or by
stimulating local immune cells.
 The adjuvant may be a tiny amount of aluminium salts (like aluminium phosphate,
aluminium hydroxide or potassium aluminium sulphate). Aluminium has been shown not
to cause any long-term health problems, and humans ingest aluminium regularly through
eating and drinking.

VACCINE DEVELOPMENT:
 Research to discover new vaccine antigens and novel approaches to immunization
usually takes several years, and costs tens of millions of dollars. Once a discovery is
made, several developments must be undertaken to reach the licensing stage. Those
developments include:
 PROCESS DEVELOPMENT to produce an economically viable vaccine,
consistently, in a manner that satisfies regulators. It is divided into bulk
manufacturing and product finishing.
o Bulk manufacturing involves the culture of live organisms, followed by
separation and purification of the desired antigen.
o Finishing involves the formulation with either adjuvant and / or stabilizer
and the filling of vials or syringes
 CLINICAL DEVELOPMENT to demonstrate the safety and measure the
protective effect of the vaccine in humans. It involves the iterative process of
testing a vaccine candidate in a progressively larger number of human subjects.
 ASSAY DEVELOPMENT to develop the appropriate tests to ascertain the
purity, potency and stability of the vaccine under development. It is required
because the vaccine candidate will be novel and will, therefore, require specific
tests to identify it and characterize the product to the satisfaction of the regulators.
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Different types of development necessary to reach the vaccine licensing stage

CLINICAL DEVELOPMENT
 If the vaccine triggers an immune response, it is then tested in human clinical trials in
three phases:
1. PHASE I
 Early safety and immunogenicity trials that involve ≤100 subjects and can be completed
in under one year
2. PHASE II
 Safety, dose ranging, and immunogenicity trials that involve several hundred subjects
and that take 1–3 years to complete
3. PHASE III
 Large-scale safety and efficacy trials involving thousands of subjects and requiring 3–5
years to complete.
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The four phases of clinical development of vaccines

  During phase two and phase three trials, the volunteers and the scientists
conducting the study are shielded from knowing which volunteers had received the
vaccine being tested or the comparator product. This is called “blinding” and is
necessary to assure that neither the volunteers nor the scientists are influenced in their
assessment of safety or effectiveness by knowing who got which product.

4. PHASE IV
 Further monitoring takes place in an ongoing way after the vaccine is introduced. There
are systems to monitor the safety and effectiveness of all vaccines. This enables scientists
to keep track of vaccine impact and safety even as they are used in a large number of
people, over a long time frame. These data are used to adjust the policies for vaccine use
to optimize their impact, and they also allow the vaccine to be safely tracked throughout
its use. 
 Once a vaccine is in use, it must be continuously monitored to make sure it
continues to be safe.

VACCINE MANUFACTURING
 The manufacture of vaccines is achieved from the propagation of living organisms. Some
of these may be dangerous human pathogens. Therefore, the manufacture of vaccines is
conducted in a highly regulated and controlled environment. GMP requirements vary
between countries, but the fundamentals are common:
 Ensure that products are safe for use in humans
 Ensure that the identity, strength, quality and purity of products consistently meet
regulatory specifications.
 In general, the production of vaccines entails four basic steps:

i. PROPAGATION

entails the multiplication (or amplification) of the living organism used in the vaccine
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ii. ISOLATION entails the separation of the living organism from the cells or growth
media used in the propagation step
iii. PURIFICATION removes all materials that may be adhering to the isolated organisms,
or selectively separates the portion of the living organism to be used in the vaccine
iv. FORMULATION involves the mixing of the purified product in solutions to obtain a
desired concentration. It may also include the addition of preservatives to some vaccines,
to ensure the sterility of the product over a longer period of time, or to prevent cross-
contamination during dose extraction from vials.
 At the end of the manufacturing process, vaccines are typically filled in vials or syringes
and packaged for shipping to healthcare providers

STEPS IN VACCINE PRODUCTION:

A.
SELECTING THE STRAINS FOR VACCINE PRODUCTION
i. THE SEED (STRAIN):
 Manufacturing begins with small amounts of a specific virus (seed). Viruses or Bacteria
used in manufacture shall be derived from a Seed Lot System.
 The virus must be free of impurities, including other similar viruses and even variations
of the same type of virus.
 The seed must be kept under "ideal" conditions, usually frozen, that prevent the virus
from becoming either stronger or weaker than desired.
 Stored in small glass or plastic containers.
ii. SELECTING THE SEED (STRAIN)
 The choice of the seed depends on a number of factors including the efficacy of the
resulting vaccine, and its secondary effects. If possible, the bacterial strain or cell line
should be obtained from a recognized culture collection with an established and
documented provenance.
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 Alternatively, if the chosen vaccine strain is an “in house” clinical isolate, it will be
necessary to compile a complete history of the strain, including details of its isolation,
identification, and maintenance for product registration.
B. GROWING THE MICRORGANISMS:
 The initial production involves generation of the antigen from the microbe.
 For this the virus or microbe is grown either on primary cells such as chicken eggs (e.g.
in influenza) or on cell lines or cultured human cells (e.g. Hepatitis A).
 Bacteria against which the vaccines are developed may be grown in bioreactors (e.g.
Haemophilus influenzae type b).
 The antigen may also be a toxin or toxoid from the organism (e.g. Diphtheria or
tetanus) or it may be part of the microorganism as well.
 Proteins or parts from the organism can be generated in yeast, bacteria, or cell cultures.
 Bacteria or viruses may be weakened using chemicals or heat to make the vaccine (e.g.
polio vaccine).
C. PURIFICATION AND ISOLATION OF THE ANTIGENS:
 After the antigen is generated, it is isolated from the cells used to generate it.
 For weakened or attenuated viruses no further purification may be required. Recombinant
proteins need many operations involving ultra-filtration and column chromatography
for purification before they are ready for administration.

D. INACTIVATION OF ORGANISMS
 Viruses can be lipid-coated (enveloped) or non-enveloped.
 Virus inactivation involves dismantling a virus’s ability to infect cells without actually
eliminating the virus.
 Virus inactivation works by one of the following two mechanisms:
 By attacking the viral envelope or capsid and destroying its ability to infect or
interact with cells.
 By disrupting the viral DNA or RNA and preventing replication.

There are following techniques for antigen inactivation:

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Solvent/detergent (S/D) inactivation (Effective with lipid-coated viruses)

Pasteurization (Effective for both non-lipid and lipid coated viruses)
Acidic pH inactivation(Low pH Treatment) ( Most effective with lipid-coated
viruses)
Ultraviolet (UV) inactivation

E. QUALITY CONTROL:

 INCREASE IN VIRULENCE TESTS - With live vaccines, there is concern that the
organism might be shed from the host and transmitted to contact animals, causing disease if it
retains residual virulence or reverts to virulence. All live vaccines should be tested for virulence
by means of passage studies.

ASSESSING RISK TO THE ENVIRONMENT - The ability of each live vaccine to shed,
to spread to contact target and non-target animals, and to persist in the environment must be
evaluated to provide information for assessing the risk of the vaccine to the environment, taking
into account human health.

INTERFERENCE TESTS - For products with two or more antigenic components, tests must
confirm that there is no interference between individual components, that is, one component
causing a decrease in the protective immunological response to another component.

CONSISTENCY OF PRODUCTION - Prior to marketing approval of any new product,

each establishment should produce in its facilities three consecutive production batches/serials of
completed product to evaluate the consistency of production.

STABILITY TESTS - Stability studies (based on an acceptable potency test) are required to
establish the validity of the expiry date that appears on the product package.
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F. LOT RELEASE:

BATCH/SERIAL RELEASE FOR DISTRIBUTION:

 Prior to release, the manufacturer must test each batch/serial for purity, safety, and
potency.
1. BATCH/SERIAL PURITY TEST:
 Purity is determined by testing for a variety of contaminants.
 Tests to detect contaminants are performed on: master seeds, primary cells, MCSs
(Master cell stock), ingredients of animal origin if not subjected to sterilization (e.g. fetal
bovine serum, bovine albumin, or trypsin), and each batch of final product prior to
release.
2. BATCH/SERIAL SAFETY TEST:
 Batches are considered satisfactory if local and systemic reactions to vaccination with the
batch to be released are in line with those described in the registration dossier and
product literature.
3. BATCH/SERIAL POTENCY TEST:
 Batch/serial potency tests, required for each batch prior to release, are designed to
correlate with the host animal vaccination–challenge efficacy studies
4. OTHER TESTS:
 Depending on the form of vaccine being produced, certain tests may be indicated.
 These tests may concern:
 The level of moisture contained in desiccated products
 The level of residual inactivate in killed products
 The complete inactivation of killed products
 pH
 The level of preservatives and permitted antibiotics
 Physical stability of adjuvant
 Retention of vacuum in desiccated products
 A general physical examination of the final vaccine
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REFERENCES:
1. https://www.gavi.org/vaccineswork/how-are-vaccines-developed?
gclid=CjwKCAjwm7mEBhBsEiwA_of-
TOmpVu9tz8h3jlMJyygrZhJwbXFwUrcPlT9PP8uzsoZt46YtUQw0-hoCc4IQAvD_BwE
2. http://www.phrma-jp.org/wordpress/wp-content/uploads/old/library/vaccine-
factbook_e/2_Vaccine_Development_and_Implementation.pdf
3. https://core.ac.uk/download/pdf/161424545.pdf
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152262/
5. https://www.vaccineseurope.eu/about-vaccines/how-are-vaccines-produced
6. https://www.news-medical.net/health/Vaccine-History.aspx