VACCINE PRODUCTION

TECHNIQUE
• Standard manufacture uses a bacterial or viral antigen, e.g.
bacterium or virus, which may be killed or may be living
but attenuated.
• To make a live attenuated vaccine, the disease-causing organism
is grown under special laboratory conditions that cause it to lose
its virulence or disease-causing properties.
• The attenuation can be obtained by heat or by passage of the
virus in foreign host such as embryonated eggs or tissue culture
cells.
• Cell cultures are required for viral vaccines since viruses
can replicate only inside the living cells.
• For example To produce the Sabin polio vaccine, attenuation was
only achieved with high inocula and rapid passage in primary
monkey kidney cells.
• Inactivated vaccines are produced by killing the disease-
causing microorganism with chemicals or heat.
 Pathogen(Seed or Clinical isolate)

Inactivation Culture Attenuation Cloning,GMO

Ag Seed(Live
Seed
Purification attenuated)
VACCINE
Purification

wP, Inactivation Culture Culture

HA
V
VACCINE

VACCINE VACCINE VACCINE

Rab, Flu

aP MMR,OPV HBV,HPV
• Vaccines are currently produced by gene techniques, i.e.
instead of using a virus or bacterium, A single gene (usually a
surface glycoprotein of the virus) can be expressed in a
foreign host by Cloning. (Expression vectors are used to
make large amounts of antigen to be used as a vaccine. Most
used vectors for expression are Bacteria: Escherichia coli,
Yeasts
,Baculovirus.)
• This process induces the vector to produce an antigen,
which is then purified.
• The purified antigen, when combined with an adjuvant
results in a safe and very effective vaccine.
• Example: Gardasil, an anti-human papilloma virus
vaccine
that is very effective in preventing cervical cancer.
• The current Hepatitis B vaccine is also this type.
 SELECTING THE STRAINS FOR

PROCESSING
UPSTREAM
VACCINE PRODUCTION

GROWING THE MICRO-

ORGANISMS

ISOLATION & PURIFICATION OF

MICROORGANISM
DOWNSTREAM
PROCESSING

INACTIVATION OF ORGANISM

FORMULATION OF

VACCINE
 SELECTING THE STRAINS FOR
VACCINE PRODUCTION
The Seed(Strain) -
– Manufacturing begins with small amounts of a specific
virus (seed).
– Viruse or Bacteria used in manufacture shall be
derived from a Seed Lot System.
• A record of the origin, passage history (including
purification and characterisation procedures) and storage
conditions should be maintained for each Seed Lot.
• 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.
Selecting the seed(Strain) -

– The choice of the the seed is 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.
– 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.
 GROWING THE
MICROORGANISMS
GROWING BACTERIA

Methods used are :

– BATCH CULTURE
• the microbe is grown in a closed vessel
• typically in a test tube or flask
– CONTINUOUS CULTURE
• the microbe is grown in vessel which has medium
constantly added and spent medium constantly
removed.
• It is performed in a chemostat.
GROWING VIRUSES

– Methods used are :

• CELL (TISSUE) CULTURES – cultured cells grow in sheets
that support viral replication and permit observation
for cytopathic effect.
• BIRD EMBRYOS – incubating egg is an ideal system;
virus is injected through the shell.
• LIVE ANIMAL INOCULATION – occasionally used when
necessary
• TRANSGENIC ANIMALS
 ISOLATION & PURIFICATION OF
MICROORGANISM
• Product isolation is the removal of those components whose
properties vary markedly from that of the desired product.
• Purification selectively separates and retains the
desired product at the highest purity per its pre-
determined specification. (Remove unwanted
compounds)
• The most common method of vaccine production is
based on
an initial fermentation process followed by purification.
• CENTRIFUGATION
• FILTRATION
• CHROMATOGRAPHY
INACTIVATION OF MICROORGANISM
KILLED/INACTIVATED VACCINE:

VIRUS INACTIVATION:
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.
• Solvent/detergent (S/D) inactivation
• Pasteurization
• Acidic pH inactivation(Low pH Treatment)
• Ultraviolet (UV) inactivation
Solvent/detergent (S/D) inactivation -
• Effective with lipid-coated viruses.
• The detergents used in this method, Disrupts the interactions
between molecules in the lipid coat , rendering the coat
dysfunctional and impeding replication
• Most enveloped viruses cannot live without their lipid
coating,
so they die when exposed to these detergents.
• Other viruses may still live, but they are unable to
reproduce, rendering them non-infective.
• The detergent typically used is Triton-X 100.

Pasteurization -
• Effective for both non-lipid and lipid-coated viruses.
• Because pasteurization involves increasing the temperature of
solution to a value that will sufficiently denature the virus, it
does not matter whether the virus has an envelope or not
because the envelope alone cannot protect the virus from
such high temperatures.(at 60 C for 10 hours )
Acidic pH inactivation (Low pH Treatment) –
• Most effective with lipid-coated viruses
• Acidic conditions deactivate virus.
•Incubation typically occurs at a pH of 4 and lasts
anywhere between 6 hours and 21 days.

Ultraviolet (UV) inactivation -

• UV rays can be used to inactivate viruses since virus
particles are small and the UV rays can reach the genetic
material, inducing the dimerisation of nucleic acids.
• Once the DNA dimerised, the virus particles cannot replicate
their genetic material.
 FORMULATION OF
VACCINE
Other than microorganism or its part a vaccine contain
the following substance:-

Suspending fluids - The liquid which contains the chemicals

used during production which kill or weaken the organism for
use in vaccines.
- Sterile water, saline or fluids containing protein,
-Egg proteins are found in influenza and yellow fever
vaccines, which are prepared using chicken eggs
- Yeast Proteins, Hepatitis B vaccines are made by
transfecting cells of Saccharomyces cerevisiae (baker’s yeast)
with the gene that encodes hepatitis B surface antigen, and
residual quantities of yeast proteins are contained in the final
product.
Preservatives and stabilizers (the vaccine remain unchanged)
- Albumin, Phenols, Glycine
- Monosodium glutamate (MSG) and 2-phenoxy-ethanol which
are used as stabilizers in a few vaccines to help the vaccine remain
unchanged when the vaccine is exposed to heat, light, acidity, or
humidity.
-Antibiotics , which are added to some vaccines to prevent the
growth of bacteria during production and storage of the vaccine.
Antibiotics that are used during vaccine manufacture include
neomycin, streptomycin, polymyxin B, chlortetracyline, and
amphotericin B.
-Thimerosal is a mercury-containing preservative that is added to
vials of vaccine that contain more than one dose to prevent
contamination and growth of potentially harmful bacteria. Eg.
diphtheria-tetanus-acellular pertussis (DTaP), hepatitis
B, and Haemophilus influenza type B (Hib).
Inactivating Agents-
- Formaldehyde is used to inactivate bacterial products for
toxoid vaccines, (these are vaccines that use an inactive
bacterial toxin to produce immunity.)
• It is also used to kill unwanted viruses and bacteria that might
contaminate the vaccine during production.
• Most formaldehyde is removed from the vaccine before it
is packaged.
• It is used to inactivate influenza virus, poliovirus, and
diphtheria and tetanus toxins.
- β-propiolactone, which is used to inactivate rabies virus

- Glutaraldehyde, which is used to inactivate toxins

contained in
acellular pertussis vaccines.
 Adjuvants enhance vaccine immunogenicity

Adjuvants or enhancers –

- aluminum gels or salts (Alum)

Alum is used in several licensed vaccines
including:
• diphtheria-pertussis-tetanus
• diphtheria-tetanus(DT)
• DT combined with Hepatitis B (HBV)
• Haemophilus influenza B
• Inactivated polio virus
• Hepatitis A (HAV)
• Streptoccucus pneumonia vaccine
• Meningococccal vaccine
• Human papilloma virus (HPV)
 How to produce
Vaccine? centrifugation

virus
cell (production
seed)

   filtering

Cell culture Inoculation Harvest Bulk Purification


Add

Bulking agent

Adjuvant Stabilizer
   
Preservative

Packaging Labeling Inspection Filling Formulation

45
 Vaccine
Vaccine
virus
virus
multiplied

Virus is spun to
separate it from
the egg white

After incubation the egg

The vaccine virus is
white contains millions of
injected into a 9 to 12
vaccine viruses which
day old fertilized egg and
are harvested and then
incubated for 2 to 3
separated from the egg
days.(during this time the
white.
virus multiplies)
RECOMBINANT VACCINES:
• The vaccines are produced using recombinant DNA
technology or genetic engineering.
• Recombinant vaccines are those in which genes for
desired
antigens of a microbe are inserted into a vector.
Different strategies are:
• Using the engineered vector (e.g., Vaccinia virus) that is
expressing desired antigen as a vaccine
• Introduction of a mutation by deleting a portion of
DNA such that they are unlikely to revert can create an
attenuated live vaccine.
Live attenuated vaccines can also be produced by
reassortment
of genomes of virulent and avirulent strains.
Genetic approaches to Unique antige
vaccine development:

One or more genes

encoding pathogen-
specific antigens
Isolated gene coding for
are isolated and significant antigen
-recombined with a Nonpathogenic
harmless or disabled bacterial plasmid
DNA
vector for delivery
by injection,
- or incorporated
into food plants for
ingestion,
-or modified for
injection as naked Vector
Recombinent
DNA. vaccine
-subunit antigens can
be produced by genetic
engineering.
 GROWING THE
MICROORGANISMS
Growing the microorganism are mainly classified in two
category :

GROWING BACTERIA :
Bacteria are grown in bioreactors e.g.Haemophilus
influenzae type b.

- BATCH CULTURE
- CONTINUOUS CULTURE

GROWING VIRUSES :
- CELL (TISSUE) CULTURES
- BIRD EMBRYOS
- LIVE ANIMAL INOCULATION
- TRANSGENIC ANIMALS
 Batch culture
•Culture incubated in a Air
closed vessel with a single in
Air filter
batch of medium. Air Syringe for
•The fermenter shown here is adding buffer,
out Air
nutrients etc.
set up for a batch culture. filter

Syringe
for
withdrawi
ng samples

Sparger to increase Culture

medium
efficiency
• Batch processing is a way of providing the best conditions
for a micro-organism.
• All the raw materials are put in the fermenter at the start and
then the micro-organism is added.
• The system is then left for a long time – possibly a week –
until all the raw materials have been used up and there is
loads of the product.
• The fermenter is then emptied and other processes are used
to separate the product from the micro-organism.
• The fermenter is then cleaned out and the whole
process begins again.
 Continuous culture

• Growth in an open system

– continual provision of nutrients
– continual removal of wastes
• Continuous culture aims to keep a culture growing
indefinitely. This can be done if:
• fresh nutrients are continually supplied
• Accumulated cells and waste products are removed at
the same rate
• Conditions such as temperature and pH are kept at their
optimum values.
Here the raw materials are
trickled in at the top of a
column in which there are
immobilised micro-organisms .
• The product flows out
the bottom in a pure
state.
• It does not need to be
separated from the catalyst.
• However this process can only
be used for reactions that are
fast – possibly taking 10
minutes.
• A culture vessel designed for
continuous culture is called
a chemostat.