conventional methods of animal vaccine production

1

Conventional methods of animal vaccine

production

Submitted by: Dr. Vijayata([email protected])

Pathogen

Culture

AttenuationAg

Purification

Inactivation

VACCINE

Seed (Live attenuated)

Culture

VACCINE

Inactivation

VACCINE Purification

VACCINE

wP,HAV

Rab,FluMMR,OPV

aP

Veterinary Immunology by Tizard

Ada G. The immunology of vaccination. In: Plotkin SA, Orenstein WA. Vaccines. 4th ed. Philadelphia, PA: Saunders, 2003:31-45. Principles of Vaccination

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Types of Vaccine Manufacturing Methods

Egg Based Vaccine Production

Cell Culture Based Vaccine Production

Recommendations for production and control of influenza vaccine ,WHO

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Vaccine production methods

Diseases Vaccines Methods of Production

Dipthera DTaP, DTaP/Hib, Tdap Cell based

Hemophilus infl uenzatype B (Hib)

Hib, DTaP/Hib, Hib/Hepatitis Cell based

Pertussis (whoopingcough)

DTaP, DTaP/Hib, Tdap Cell based

Pneumococcal Pneumococcal conjugate(Heptavalent)

Cell based

Tetanus DTaP, DTaP/Hib, Tdap Cell based

Meningococcal Meningococcal conjugate (quadravalent)

Cell based

Meningococcal Meningococcal polysaccharide(quadravalent)

Egg based

Source : Center for Disease Control and Prevention. VFC: Approved Vaccines and Biologicals. www.cdc.gov/vaccines/programs/vfc/parents/apprvd - vaccs.htm [accessed September 8, 2007].

Bacterial Vaccine:

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Viral Vaccine:

Diseases Vaccines Methods of Production

Hepatitis A Hepatitis A (pediatric) Cell based

Hepatitis B Hepatitis B (pediatric/adolescent) Cell based

Hepatitis B Hib/Hepatitis B Cell based

Poliomyelitis (polio) IPV (Inactivated poliovirus vaccine) OPV (Oral polio vaccine)

Cell based

Rotavirus Rotavirus Cell based

Influenza (Flu) Influenza Egg based

Measles MMR, measles Egg based

Mumps MMR, Mumps Egg based

Rubella (German measles) MMR, rubella Egg based

Varicella (chicken pox) Varicella Egg based

Source : Center for Disease Control and Prevention. VFC: Approved Vaccines and Biologicals. www.cdc.gov/vaccines/programs/vfc/parents/apprvd - vaccs.htm [accessed September 8, 2007].

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Selecting the strains

for vaccine

production

Growing the micro-organisms

Harvesting &

purification of

microorganisms

Inactivation and

splitting of

organism

Formulation of

vaccine

Quality control and lot release

Upstream processing

Downstream processing

Steps in vaccine production

Novel Techniques in the Production of Industrially Imperative Products, Sameera V* Biotechnology Department, REVA Institute of Science and Technology, Bangalore University, India

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SELECTING THE STRAINS FOR VACCINE PRODUCTION

The Seed (Virus/Bacteria)• Manufacturing begins with small amounts of a specific

Virus/Bacteria (seed).• Viruses/Bacteria used in manufacture shall be derived

from a Seed Lot System.• The virus/Bacteria must be free of impurities, including

other similar viruses/ bacteria and even variations of the same type of virus/Bacteria.

• The seed shall pass the tests for sterility and freedom from mycoplasma.

Eurropean medical agency guide line for General requirements for the production and control of live mammalian bacterial and viral vaccines for veterinary use (7BIm1a)

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• A record of the origin, passage history (including purification and characterisation procedures) and storage conditions should be maintained for each Seed Lot.

• The seed must be kept under "ideal" conditions, usually frozen, that prevent the virus from becoming either stronger or weaker than desired.

Selecting the seed (Strain) used for vaccine production

• The choice of strain 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.

The annual production cycle for influenza vaccine:Catherine Gerdil Vaccine 21 (2003) 1776–1779∗

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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.

Standardizing the seed strains for vaccine production

• Once the candidate seed strains for vaccine production have been prepared, their antigens undergo identity testings and to evaluate their suitability for vaccine production.

• This includes satisfactory yields when grown in embryonated eggs/ cell culture and antigenic stability throughout serial passage in eggs as well as the inactivation and purification processes.

The annual production cycle for influenza vaccine: Catherine Gerdil Vaccine 21 (2003) 1776–1779∗

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GROWING THE MICROORGANISMS

• Once the production strain for each vaccine component has been selected, bulk vaccine production can begin.

• Bulk production begins with the cultivation of the virus or Bacteria in a fermenter equipped with numerous process parameters to control temperature, pH, dissolved oxygen, and other factors.

Growing Bacteria

• Batch culture• Continuous culture

Growing Viruses

• Cell (tissue) cultures• Embryonated Eggs

Growing the microorganisms in maximum titre are mainly classified in two category :

The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27,

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Growing Bacteria

• Bacteria are grown in bioreactors e.g. Haemophilus influenza type b.• Batch culture• Continuous culture

Production of Freeze-dried Brucella abortus Strain 19 Vaccine using Cells produced by Continuous Culture† K. J. Boyce, A. W. Edgar, Journal of Applied Microbiology, Volume 29, Issue 2, pages 401–408, August 1966

VACCINE PRODUCTION AS A UNIT PROCESS,PROF.DR.T.O. WIKE'N,1971 Chapter 3.3,pp65-71

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.1966.tb03490.x/abstract

http://onlinelibrary.wiley.com/doi/10.1111/jam.1966.29.issue-2/issuetoc

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• The micro-organism are grown in a closed vessel.

• 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 tularaemia vaccine: review. Sandstrom, G. Journal of Chemical Technology & Biotechnology. Vol. 59, no. 4, pp. 315-20. Apr. 1994


Batch culture

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Continuous culture

• The micro-organism are grown in an open system.

• 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.The tularaemia vaccine: review. Sandstrom, G. Journal of Chemical Technology & Biotechnology. Vol. 59, no. 4, pp. 315-20. Apr. 1994


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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.

• However this process can only be used for reactions that are fast – possibly taking 10 minutes.

• E.g. - in the cultivation of Corynebacterium diphtheriae


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Growing Viruses

• Viruses are grown either on primary cells e.g.for influenza, or on continuous cell lines,e.g. for hepatitis A.

• Cell culture• Embryonated Eggs

OIE Terrestrial Manual 2010 C H A P T E R 2 . 8 . 8 . SWINE INFLUENZA, Recommendations for production and control of influenza vaccine ,WHO

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Cell culture

• Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment.

• Cell cultures are separated into 3 types:-- Primary cell culture- Secondary cell culture- Continuous cell line

Mammalian Cell Culture Technology: An Emerging Field ,D. Eibl, R. Eibl, and R. Pörtner

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Primary cell culture-• Cells that are cultured directly from animal or human tissues and

can be subcultured only once or twice . e.g. Primary monkey kidney cell

• For most of the mammalian vaccines the use of primary cells is not acceptable for the manufacture of vaccines.

• If a vaccine has to be produced on primary cells, they should be obtained from a SPF herd or flock with complete protection from introduction of diseases.

Secondary cell culture- • are derived from human fetal tissue and can be subcultured 20 to

50 times e.g. Human diploid fibroblasts such as MRC-5

Mammalian Cell Culture Technology: An Emerging Field ,D. Eibl, R. Eibl, and R. Pörtner

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Continuous cell lines –• a single cell type that can propagated indefinitely in culture.• e.g. Vero cells, Hep2

Cell Line Selection :• If a cell line is used for the manufacture of vaccines it shall

normally be produced according to a Seed Lot System.• The history of the cell line must be known in detail (e.g. origin,

number of passages and media used for their multiplication, storage conditions).

• The cell line used to cultivate the virus must be able to propagate the virus in large quantities , rapid, efficient in expressing the desired virus, and suitable for a wide variety of virus strains.

• The cells must be checked for their appearance , rate of growth and for contamination with bacteria, virus, fungi, mycoplasma.

The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27 ,

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• Preparation of a cell line for propagation begins with the thawing of the cell line seed lot (e.g., EBx™, VERO, or MDCK).

• Cell line propagation begins with the small scale pre-culture propagation of seed cells after thawing.

• The cells are then introduced to the fermenter vessel with the selected nutrient medium.

• When the cell line reaches a predetermined cell density, the virus is introduced and begins to propagate in the cell line.

• Two methods of mass cultivation of cells are recognized in the industry today, microcarrier cultures and free-cell suspension cultures.

• Both systems begin cultivation of the cell line in a fermenter, which can be scaled up to thousands of liters.


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In microcarrier systems-• Cells are anchorage dependent and grow on solid or

macroporous microcarriers. (microbeads.)• Cell expansion often occurs in roller-flasks.• In the presence of nutrient media, the cells grow and proliferate

covering the beads uniformly.• Microbeads provide a high surface-area-to-volume ratio, which

can lead to high cell densities.• Once a bead is covered, the cells are dislodged, dispersed, and

allowed to reattach to achieve another round of cell growth on the surface of the bead.


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Suspension culture -• Suspension cultures are derived from cells that can survive and

proliferate without attachment (on-adherent)• These cells are maintained by continuous stirring with a magnet

and can multiply while suspended in a suitable medium.• Larger volumes of media are required because the cell line

proliferates while growing freely suspended in the nutrient medium.

• However, the scaling up of the system is easier, and there is no limit to the volume.


Embryonated Eggs

• If the vaccine is to be produced in embryonated eggs, the eggs to be

used should be from closed, specific-pathogen-free, healthy flocks.

• This flock shall be monitored at regular intervals for Bacteria, Virus

and Mycoplasma.

• Many viruses can be propagated in embryonated chicken eggs but

the method is now only used for Influenza viruses.

• At 5 to 14 days after fertilization, a hole is drilled in the shell and

virus injected into the site appropriate for its replication (yolk sac,

chorioallantoic membrane, amniotic cavity, allantoic cavity).• The eggs are incubated at about 33 ° C for 2 to 3 days, candled for

viability and lack of contamination from the inoculation, and then the allantoic fluid is harvested.

22RECOMMENDATIONS FOR PRODUCTION AND CONTROL OF INFLUENZA VACCINE (INACTIVATED) WORLD HEALTH ORGANIZATION)

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Herpes simplex virusPox virusRous sarcoma virus

Influenza virusMumps virus

Herpes simplex virus

Influenza virusMumps virusNew castle disease virusAvian adenovirus

GROWTH OF VIRUSES IN EMBRYONATED EGG –An embryonated chicken egg showing the different compartments in which viruses may

grow. The different routes by which viruses are inoculated into eggs are indicated.

http://www.virology.ws/2009/12/10/influenza-virus-growth-in-eggs/

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Harvesting & Purification of microorganism

• After propagation, the virus is harvested.• Harvesting of virus is largely a manual process that requires

extracting infected cells, breaking down cell walls, and then collecting the virus.

• After treatment of the infected cell line, the virus is released into the supernatant, and the cellular debris is centrifuged away by use of appropriate Centrifugation method.

• Purification selectively separates and retains the desired product at the highest purity per its pre-determined specification. (Remove unwanted compounds)

An international technology platform for influenza vaccines: Jan Hendriksa, , Marit Hollemanb, Otto de Boerb, Patrick de Jongc, Willem ∗Luytjesb

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Centrifugation:• Centrifugation is a process by which solid particles are

sedimented and separated from a liquid using centrifugal force as a driving force.

• Centrifugation is used to separation and purification of pathogenic virus antigens and other agents used in the production of vaccine.

• Centrifugation is also used to remove dead cells, cell debris etc.

Example : Influenza vaccine, rabies vaccine , Hepatitis B vaccine, and Japanese encephalitis vaccine production.

Filtration Technique inVaccine Manufacturing

An international technology platform for influenza vaccinesJan Hendriksa, , Marit Hollemanb, Otto de Boerb, Patrick de Jongc, Willem Luytjesb∗

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Chromatography

• A group of physical separation techniques, which are characterized by the separation of mixtures due to differences in the distribution coefficient of sample components between two phases, one stationary and the other mobile phase.

• Example : Modified Vaccinia Ankara virus (Small pox vaccine)

Filtration• Filtration is a process for separating two substances of two

different physical states. It is used for separating solids from turbid liquids (filtrate), pure gases or solids.

• Separation of particles from liquid by applying a pressure to the solution to force the solution through a filter.

Filtration Technique inVaccine Manufacturing

An international technology platform for influenza vaccinesJan Hendriksa, , Marit Hollemanb, Otto de Boerb, Patrick de Jongc, Willem Luytjesb∗

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• Following purification, the virus is inactivated through a chemical process.

• After inactivation, the whole virus can be purified, split, and ultra purified as a “subunit.”

• Virus splitting follows because only fractions of specific viral surface proteins are required for the subsequent vaccine. (influenza )

• Further purification procedures are then performed.• At this point, the development phase of vaccine is complete.

Inactivation And splitting of microorganism


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KILLED / INACTIVATED VACCINE

• The term killed generally refers to bacterial vaccines, whereas inactivated relates to viral vaccines (Levine et al., 1997).

• For viruses, the outer virion coat should be left intact but the replicative function should be destroyed.

• 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.• Killed vaccines may take the route of heat or chemicals (Turner

et al., 1970).

The Biomedical Engineering Handbook: Second Edition. Ed. Joseph D. Bronzino ,Aunins, J. G., Lee. A. L.,Volkin, D. B. “Vaccine Production.”

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Chemical treatment -• The chemicals used for killed vaccines include formaldehyde

or beta-propiolactone (Lo Grippo, 1960; Gard, 1960).• The traditional agent for inactivation of the virus is formalin

(Weil & Gall, 1940; Kim & Sharp, 1967).• The agent is chosen for effectiveness without destruction of

antigenicity.• For whole organisms, the inactivation abolishes infectivity.• For antigens such as the diphtheria and tetanus toxins,

formaldehyde treatment removes the toxicity of the antigen itself as well as killing the organism.

• Such detoxified antigen, called toxoids, are safe for use in vaccines.

Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development, Abid Nabil Ben Salem1, Rouis Zyed1, Buesa Javier2 and Aouni Mahjoub1

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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.


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By Heat -

A physical inactivation method that leaves intact virus, bacteria.

The heat inactivates the infectious agents, by destroying there

surface antigen.

Heat inactivation usually done at 56°C for 30 minutes.

e.g. A normal suspension of pertussis cells contains a thermolabile toxin that is inactivated by heating at 56°C for 10 minutes.

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.


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LIVE WHOLE VACCINES:

Several methods have been used to attenuate viruses for vaccine

production.

a) Use of a related microorganism from another animal -

The earliest example was the use of cowpox to prevent smallpox.

b) Administration of pathogenic or partially attenuated

microorganism by an unnatural route -

The virulence of the virus is often reduced when administered by an

unnatural route.Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development, Abid Nabil Ben Salem1, Rouis Zyed1, Buesa Javier2 and Aouni Mahjoub1

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c) Passage of the microorganism in an "unnatural host" or host cell

• The major vaccines used in man and animals have all been derived this way.

• Example :- the 17D strain of yellow fever was developed by passage in

mice and then in chick embryos

(Norrby, 2007).- Polioviruses were passaged in monkey kidney cells

(Chezzi et al., 1998).- Measles in chick embryo fibroblasts

(Katz, 1958).

Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development, Abid Nabil Ben Salem1, Rouis Zyed1, Buesa Javier2 and Aouni Mahjoub1

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Formulation of vaccine

Finally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives as

needed.Other than microorganism or its part (antigen), a vaccine contain the following

components:

Component Purpose Example Example of Vaccines

Adjuvants Enhance the immune response to a vaccine

Aluminium salts (Alum)

Diphtheria-pertussis-tetanusDiphtheria tetanus(DT)DT combined with Hepatitis B (HBV)Haemophilus influenza BInactivated polio virus(IPV)Hepatitis A (HAV)

Preservatives Prevent bacterial or fungal contamination of vaccine

Thimerosal Diphtheria-tetanus-acellular pertussis (DTaP)Hepatitis B,Haemophilus influenza type B (Hib).

Component

Purpose ExampleExample of Vaccines

Stabilizers Protects vaccines from adverse conditions such as freeze-drying or heat, thereby maintaining a vaccine’s potency

Gelatine, Monosodium glutamate (MSG)

17D Yellow Fever virus vaccine, Rabies,Varicella

Residuals from manufacturing process

Inactivating agents - used to inactivate bacterial products for toxoid vaccines, to kill unwanted viruses and bacteria that might contaminate the vaccine during production

Antibiotics - prevent bacterial contamination during manufacturing process

Suspending fluids –

kill or weaken the organism for use in vaccines.

Formaldehyde

β-propiolactone

Glutaraldehyde

Neomycin, Streptomycin, Polymyxin B

Egg proteins

Yeast proteins

Influenza virus, Poliovirus, Diphtheria and Tetanus toxins.

Rabies virus

Acellular pertussis

DTaP-IPV/Hib

Influenza, MMR

Influenza and yellow fever vaccines

Hepatitis B vaccines

QUALITY CONTROL AND LOT RELEASE

Test Purpose of Test

Sterility Demonstrates that no live microorganisms are present in product

Safety Demonstrates that overdose of the product causes no harm

Residual toxicity Demonstrates that the product contains no material that can cause harm

Efficacy Demonstrates that each antigen in the product meets the recommended guideline level in internationally recognised tests.

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.

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.

Schedule of final product testing for a veterinary vaccine

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PRINCIPLES OF VETERINARY VACCINE PRODUCTION, OIE Terrestrial Manual 2008

37

1. Batch 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), and each batch of final product prior to release.

BATCH/SERIAL RELEASE FOR DISTRIBUTION :

Prior to release, the manufacturer must test each batch/serial for purity, safety, and potency.

Lot release


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2. Batch 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 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.


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Sampling :

Samples should be selected from each batch/serial of product.

The selector should pick representative sample.

Field tests (safety and efficacy)

Performance monitoring

Filling , Packaging and Labelling

Once all procedures of vaccine production are completed, the

vaccine is blended, filled the doses into vials and packaged.

Which are then sealed and carefully inspected before labels are

applied to show the vaccine batch,lot numbers, and expiration

date.

Standards for labelling products will vary from country to country.


The upstream process, begins

with embryonated eggs brought in on a daily basis from biosecure

flocks.

A seed ampoule is used to

inoculate the chick eggs during the inoculation

phase.

This is followed by a 3 day incubation period during which the virus grows to ensure that sufficient quantities can support further

manufacturing.

Embryonated chicken eggs Inoculation Incubation

Candling

The allantoic fluid is then harvested; a low-

speed clarification process follows.

Harvest (poolallantoic fluid)

After 3 days, all of the eggs are candled to make sure there are no cracks or contamination; the eggs

are then chilled to 2° to 8°C to constrict vessels and make

harvesting easier.

Clarification

Processes of Egg-Based Vaccine Production

40The BRIDGE, Egg-Based Production ofInfluenza Vaccine:James T. Matthews page 21

http://knol.google.com/k/-/-/3fy5eowy8suq3/uhjh6p/10759lores1.jpg

1st inactivation

The first step in the downstream process is

inactivation, which involves the

addition of formalin to inactivate the

virus.

Filtration

Extensive filtration and concentration steps yield a concentrate, which is

then loaded onto zonal centrifugation equipment.

Concentration

Zonal centrifugation

The first purified bulk virus, which is recovered from the

centrifugation process, is split in a fragmentation

step by treatment with Triton detergent.

Triton fragmentationCentrifugation2nd inactivation

Ultrafiltration The material is then clarified by

centrifugation to remove large

particulates and treated with formalin in a

second inactivation step.

An ultrafiltration (UF) step is followed by terminal sterile filtration to generate one monovalent bulk concentrate.

Sterile filtration

Final product 41The BRIDGE, Egg-Based Production of Influenza Vaccine:James T. Matthews page 21

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Cell Culture Based Vaccine Production

virus(production

seed)

Cell culture Harvest Bulk Purification

FormulationFillingLabelingPackaging

Add

Inoculation

cell

Adjuvant Stabilizer

Preservative

Inspection

centrifugation

filtering

Influenza Vaccines (Tina Kröber, Thomas Jarosch, and Laura Fischer) Max-Planck-Gesellschaft, MünchenVaccines / [edited by] Stanley A. Plotkin, Edward A. Mortimer, Jr.

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Thank you

45

• Production of Freeze-dried Brucella abortus Strain 19 Vaccine using Cells produced by Continuous Culture† K. J. Boyce, A. W. Edgar ,Journal of Applied Microbiology, Volume 29, Issue 2, pages 401–408, August 1966

• The tularaemia vaccine: review. Sandstrom, G.Journal of Chemical Technology & Biotechnology. Vol. 59, no. 4, pp. 315-20. Apr. 1994 The live vaccine can be produced in fermenter cultures and the bacteria have to undergo quality controls for each batch of vaccine. It is important to estimate the amount of non-immunogenic bacteria in each batch and to disclose the immunogenicity of the vaccine in man.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.1966.tb03490.x/abstract

http://onlinelibrary.wiley.com/doi/10.1111/jam.1966.29.issue-2/issuetoc

conventional methods of animal vaccine production

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