Conventional methods ofanimal vaccine production       Submitted by:        Dr. Vijayata       (dr.viz.vet@gmail.com)     ...
Pathogen                                            Inactivation                               Culture                    ...
Types of Vaccine Manufacturing Methods      Egg Based Vaccine        Production                             Cell Culture B...
Vaccine production methods     Bacterial Vaccine:             Diseases                                          Vaccines  ...
Viral Vaccine:               Diseases                                              Vaccines                               ...
Steps in vaccine production              Upstream                                                                         ...
SELECTING THE STRAINS FOR VACCINE                             PRODUCTION    • Manufacturing begins with small amounts of a...
• A record of the origin, passage history (including purification  and characterisation procedures) and storage conditions...
• Alternatively, if the chosen vaccine strain is an “in house”  clinical isolate, it will be necessary to compile a comple...
GROWING THE MICROORGANISMS   • Once the production strain for each vaccine component has been     selected, bulk vaccine p...
• Bacteria are grown in                                                             bioreactors e.g. Haemophilus          ...
Batch culture   • The micro-organism are grown in a closed     vessel.   • All the raw materials are put in the fermenter ...
Continuous culture       • The micro-organism are grown in an open system.       • Continuous culture aims to keep a cultu...
Here the raw materials are  trickled in at the top of a  column in which there are  immobilised micro-  organisms.• The pr...
• Viruses are grown either on                                                               primary cells e.g.for influenz...
Cell culture• Cell culture is the complex process by which cells are grown  under controlled conditions, generally outside...
Primary cell culture-•   Cells that are cultured directly from animal or human tissues and    can be subcultured only once...
Continuous cell lines –• a single cell type that can propagated indefinitely in culture.• e.g. Vero cells, Hep2Cell Line S...
• Preparation of a cell line for propagation begins with the  thawing of the cell line seed lot (e.g., EBx™, VERO, or  MDC...
In microcarrier systems-• Cells are anchorage dependent and grow on solid or macroporous   microcarriers. (microbeads.)• C...
Suspension culture -• Suspension cultures are derived from cells that can survive and  proliferate without attachment (on-...
Embryonated Eggs     • If the vaccine is to be produced in embryonated eggs, the eggs to       be used should be from clos...
Herpes simplex                                                                              virus                         ...
Harvesting & Purification of                            microorganism       • After propagation, the virus is harvested.  ...
Centrifugation:    • Centrifugation is a process by which solid particles are      sedimented and separated from a liquid ...
Chromatography  • A group of physical separation techniques, which are    characterized by the separation of mixtures due ...
Inactivation And splitting of                microorganism• Following purification, the virus is inactivated through a  ch...
KILLED / INACTIVATED VACCINE• The term killed generally refers to bacterial vaccines, whereas  inactivated relates to vira...
Chemical treatment -       • The chemicals used for killed vaccines include formaldehyde         or beta-propiolactone (Lo...
Solvent/detergent (S/D) inactivation –      • Effective with lipid-coated viruses.      • The detergents used in this meth...
By Heat -       A physical inactivation method that leaves intact virus, bacteria.       The heat inactivates the infectio...
LIVE WHOLE VACCINES:      Several methods have been used to attenuate viruses for vaccine          production.     a) Use ...
c) Passage of the microorganism in an "unnatural host" or        host cell     • The major vaccines used in man and animal...
Formulation of vaccineFinally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives asneeded.Other...
Compone                                                                 Example of                         Purpose        ...
QUALITY CONTROL AND LOT RELEASE                      Schedule of final product testing for a veterinary vaccine           ...
Lot releaseBATCH/SERIAL RELEASE FOR DISTRIBUTION :Prior to release, the manufacturer must test eachbatch/serial for purity...
2. Batch safety test -Batches are considered satisfactory if local andsystemic reactions to vaccination with the batch tob...
Sampling :  Samples should be selected from each batch/serial  of product.  The selector should pick representative sample...
Processes of Egg-Based Vaccine Production   Embryonated   chicken eggs                             Inoculation            ...
1st inactivation                Filtration                 Concentration The first step in the                            ...
Cell Culture Based Vaccine Production                                                                                     ...
Thank you        43
44
• Production of Freeze-dried Brucella abortus Strain 19 Vaccine  using Cells produced by Continuous Culture† K. J. Boyce, ...
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Conventional methods of animal vaccine production

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This ppt provide information about the conventional methods of animal vaccine production..it is somewhat differ from my earlier ppt of vaccine production techniques..

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  • OPV-Oral polio vaccine sabin,MMR-measles,mumps,rubella –live attenuated vaccine.Rab-Rabies,Flu-Influenza (killed viral),Ap- Acellularpertussis (Subunit vaccine,surface protein) Hepatitis A(killed viral vaccine)
  • There are different methods which vaccine manufacturers use to produce vaccines, and they are:
  • preservative free
  • Following cultivation, the antigen is recovered, isolated in crude form, further purified, and/or inactivatedto give the unformulated product; these steps are referred to collectively as thedownstream process
  • Culture incubated in a closed vessel with a single batch of medium.
  • in the cultivation of C.diphtheriaecontinually constantly or frequent occuringMany environmental factors (e.g. pH, oxygen levels, nutrients and temperature) can be controlled very precisely.The culture is stirred continuously - ensuring nutrients and oxygen reach the cells and metabolic products are distributed away from them.
  • Catalyst a substance that increaes the rate of a chemical reaction without itself undergoing any permanent chemical change
  • (e.g. disease barriers, filters on air inlets, no new animals introduced without appropriate quarantine).
  • a homogenous population of single cell types, which can divide up to 100 times before dying. The most common are those established from human embryos e.g. WI 38 from human embryonic lungs.
  • PerC.6 (Crucell)• EBx™: a stem cell line derived from chicken embryos(Sigma-Aldrich Group)• VERO: a kidney cell from the African green monkey(GSK)• MDCK : Madin-Darby canine kidney cells used byChironCell lines. Cultures of cells that have a high capacity for multiplication in vitro.
  • culture cells growing on microcarrier beads, MDCK cells on microcarrier
  • anchorage independent. In contrast to cells that grow in plastic dishes to form a monolayer, other cells are non-adherent and can be maintained as suspension cultures. Larger volumes of media are required to achieve the same results with free-cell suspension because the cell line proliferates while growing freely suspended in the nutrient medium.
  • Newcastle disease virus and other avian parainfluenza viruses, avian reticulo-endotheliosis virusspecific agents: Mycobacterium avium , fowl pox virus, Newcastle disease virus and, avian encephalomyelitis virus, infectious laryngotracheitis virus,, Marek's disease virus, infectious bursal disease virus, Haemophilusparagallinarum , Salmonella gallinarum, Salmonella pullorum, Mycoplasma gallisepticum and Mycoplasma synoviae.
  • This occurs in a clean, closed environment, whereas harvesting of an egg based virus is largely a manual process that requires extracting infected cells, breaking down cell walls, and then collecting the virus.
  • Removal of microbes by passage of a liquid or gas through a screen like material with small pores
  • Initial chromatography with ultrafiltration is often followed by treatment with beta-propiolactone.Since seasonal influenza vaccines contain three viral strains, the production process must be performed for each strain. Initial chromatography with ultra filtration is often followed by treatment with beta-propiolactone, which deactivates the virus; final splitting of the virus is followed by ultracentrifugation. This ultrapurification technology is basically similar to the egg-based vaccine ultrapurification process, and the resulting purified subunit vaccine is identical in composition to egg-based vaccine.
  • The outer virion coat should be left intact but the replicative function should be destroyed. For nonattenuated whole organisms or for toxin antigens, the preparation must be inactivated to eliminate pathogenicity.
  • Chemicals used include formaldehyde(e.g.Polio,Hepatitis), glutaraldehyde, and beta- propiolactone(e.g. Rabies, influenza)
  • Typical incubation lasts for 10 hours and is performed at 60°C
  • But this inactivation of surface antigen results in poor immune response. dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids. It is aquaternary structure of a protein. Inactivation of poliovirus in 10 minutes required an 8% concentration of formalin, but all other viruses tested were inactivated with 2% formalin 72.
  • This principle is used in the immunization of military recruits against adult respiratory distress syndrome using enterically coated live adenovirus type 4, 7 and (21).
  • After repeated passages, the virus is administered to the natural host.The initial passages are made in healthy animals or in primary cell cultures. Human diploid cells are now widely used such as the WI-38 and MRC-5.
  • Formulation is a manual process consisting of the addition of diluent to achieve the required 15 microgram antigenic component per strain (H1, H3, and B strain) for each dose of vaccine. Other than microorganism or its part (antigen), a vaccine contain the following components:
  • toxoid vaccines, (these are vaccines that use an inactive bacterial toxin to produce immunity.) Inactivate toxins contained in acellularpertussis vaccines. Influenza and yellow fever vaccines, which are prepared using chicken eggs
  • Sterile filling of vials or syringes and subsequent inspection of the filled vials or syringes are
  • .influenza seed virus, is added to the cell-containing bioreactor (container) where the virus then infects the cells and multiplies, making more virus particles. After several days the influenza virus has infected and destroyed all the cells in the bioreactor. The virus is then harvested by removing the debris made by the cells and made non-infectious, before being further purified. This purified, non-infectious solution is then blended, concentrated and filled into syringes or vials ready for use.
  • Conventional methods of animal vaccine production

    1. 1. Conventional methods ofanimal vaccine production Submitted by: Dr. Vijayata (dr.viz.vet@gmail.com) 1
    2. 2. Pathogen Inactivation Culture Attenuation Ag Purification Seed (Live VACCINE Purification attenuated) Inactivation wP, HAV Culture VACCINE VACCINE aP VACCINE Rab,Flu MMR,OPVAda G. The immunology of vaccination. In: Plotkin SA, Orenstein WA. Vaccines. 4thed. Philadelphia, PA: Saunders, 2003:31-45. Principles of VaccinationVeterinary Immunology by Tizard
    3. 3. Types of Vaccine Manufacturing Methods Egg Based Vaccine Production Cell Culture Based Vaccine Production 3Recommendations for production and control of influenza vaccine ,WHO
    4. 4. Vaccine production methods Bacterial Vaccine: Diseases Vaccines Methods of Production Dipthera DTaP, DTaP/Hib, Tdap Cell based Hemophilus infl uenza Hib, DTaP/Hib, Hib/Hepatitis Cell based type B (Hib) Pertussis (whooping DTaP, DTaP/Hib, Tdap Cell based cough) Pneumococcal Pneumococcal conjugate Cell based (Heptavalent) Tetanus DTaP, DTaP/Hib, Tdap Cell based Meningococcal Meningococcal conjugate Cell based (quadravalent) Meningococcal Meningococcal polysaccharide Egg based (quadravalent) 4Source : 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].
    5. 5. 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) Cell based OPV (Oral polio vaccine) 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 5Source : 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].
    6. 6. Steps in vaccine production Upstream Downstream processing processingSelecting Harvesti the Growing Inactivati ng & Quality strains the on and Formulat purificati control for micro- splitting ion of on of and lot vaccine organism of vaccine microorg releaseproductio s organism anisms n 6Novel Techniques in the Production of Industrially Imperative Products, Sameera V* Biotechnology Department, REVA Institute of Science and Technology, Bangalore
    7. 7. SELECTING THE STRAINS FOR VACCINE PRODUCTION • 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. 7Eurropean medical agency guide line for General requirements for the production and control of live mammalian bacterial and viral vaccines for veterinary use (7BIm1a)
    8. 8. • 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. 8 The annual production cycle for influenza vaccine:Catherine Gerdil∗Vaccine 21 (2003) 1776–1779
    9. 9. • 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. 9 The annual production cycle for influenza vaccine: Catherine Gerdil∗Vaccine 21 (2003) 1776–1779
    10. 10. 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 • Batch culture Growing the Bacteria • Continuous culture microorganisms in maximum titre are mainly classified in two category : Growing • Cell (tissue) culturesThe BRIDGE,Cell-Culture-Based Viruses • Embryonated Eggs 10Vaccine Production: Rino Rappuolip 26-27,
    11. 11. • Bacteria are grown in bioreactors e.g. Haemophilus influenza type b. Growing Bacteria • Batch culture • Continuous cultureProduction of Freeze-dried Brucella abortus Strain 19 Vaccine using Cells produced by Continuous Culture† K. J. Boyce, A. W. Edgar, Journal ofApplied Microbiology, Volume 29, Issue 2, pages 401–408, August 1966 11 VACCINE PRODUCTION AS A UNIT PROCESS,PROF.DR.T.O. WIKEN,1971 Chapter 3.3,pp65-71
    12. 12. Batch culture • 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. 12 VACCINE PRODUCTION AS A UNIT PROCESS,PROF.DR.T.O.The tularaemia vaccine: review. Sandstrom, G. Journal of Chemical Technology & Biotechnology. WIKEN,1971 Chapter 3.3,pp65-71Vol. 59, no. 4, pp. 315-20. Apr. 1994
    13. 13. 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. 199413 VACCINE PRODUCTION AS A UNIT PROCESS,PROF.DR.T.O. WIKEN,1971 Chapter 3.3,pp65-71
    14. 14. 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 14 VACCINE PRODUCTION AS A UNIT PROCESS,PROF.DR.T.O. WIKEN,1971 Chapter 3.3,pp65-71
    15. 15. • Viruses are grown either on primary cells e.g.for influenza, or Growing on continuous cell lines,e.g. for hepatitis A. Viruses • Cell culture • Embryonated Eggs 15Recommendations for production and control of influenza vaccine ,WHO OIE Terrestrial Manual 2010 C H A P T E R 2 . 8 . 8 . SWINE INFLUENZA,
    16. 16. 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 16 Mammalian Cell Culture Technology: An Emerging Field ,D. Eibl, R. Eibl, and R. Pörtner
    17. 17. 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 17 Mammalian Cell Culture Technology: An Emerging Field ,D. Eibl, R. Eibl, and R. Pörtner
    18. 18. Continuous cell lines –• a single cell type that can propagated indefinitely in culture.• e.g. Vero cells, Hep2Cell 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. 18 The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27,
    19. 19. • 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. 19 The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27,
    20. 20. 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. 20 The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27,
    21. 21. 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. 21 The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27,
    22. 22. 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)
    23. 23. Herpes simplex virus Pox virus Rous sarcoma virus Influenza virus Mumps virus Influenza virus Mumps virus New castle disease virus Avian adenovirus Herpes simplex virus 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. 23 http://www.virology.ws/2009/12/10/influenza-virus-growth-in-eggs/
    24. 24. 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) 24An international technology platform for influenza vaccines: Jan Hendriksa,∗, Marit Hollemanb, Otto de Boerb, Patrick de Jongc, Willem Luytjesb
    25. 25. 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. 25An international technology platform for influenza vaccines Filtration Technique inJan Hendriksa,∗, Marit Hollemanb, Otto de Boerb, Patrick de Jongc, Willem Luytjesb Vaccine Manufacturing
    26. 26. 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 in Vaccine Manufacturing 26An international technology platform for influenza vaccinesJan Hendriksa,∗, Marit Hollemanb, Otto de Boerb, Patrick de Jongc, Willem Luytjesb
    27. 27. Inactivation And splitting of microorganism• 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. 27 The BRIDGE,Cell-Culture-Based Vaccine Production: Rino Rappuoli p 26-27,
    28. 28. 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). 28 The Biomedical Engineering Handbook: Second Edition. Ed. Joseph D. Bronzino ,Aunins, J. G., Lee. A. L.,Volkin, D. B. “Vaccine Production.”
    29. 29. 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. 29Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development, Abid Nabil Ben Salem1, Rouis Zyed1, BuesaJavier2 and Aouni Mahjoub1
    30. 30. 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. 30The Biomedical Engineering Handbook: Second Edition. Ed. Joseph D. Bronzino ,Aunins, J. G., Lee. A. L.,Volkin, D. B. “Vaccine Production.”
    31. 31. 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. 31The Biomedical Engineering Handbook: Second Edition. Ed. Joseph D. Bronzino ,Aunins, J. G., Lee. A. L.,Volkin, D. B. “Vaccine Production.”
    32. 32. 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. 32Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development, Abid Nabil Ben Salem1, Rouis Zyed1, BuesaJavier2 and Aouni Mahjoub1
    33. 33. 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). 33Recombination and Point Mutations in Type G Rotavirus Strains: The Challenges of Vaccine Development, Abid Nabil Ben Salem1, Rouis Zyed1, BuesaJavier2 and Aouni Mahjoub1
    34. 34. Formulation of vaccineFinally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives asneeded.Other than microorganism or its part (antigen), a vaccine contain the followingcomponents:Component Purpose Example Example of VaccinesAdjuvants Enhance the immune Aluminium salts Diphtheria-pertussis-tetanus response to a vaccine (Alum) Diphtheria tetanus(DT) DT combined with Hepatitis B (HBV) Haemophilus influenza B Inactivated polio virus(IPV) Hepatitis A (HAV)Preservatives Prevent bacterial or Thimerosal Diphtheria-tetanus-acellular fungal contamination of pertussis (DTaP) vaccine Hepatitis B, Haemophilus influenza type B (Hib). 34
    35. 35. Compone Example of Purpose Example nt VaccinesStabilizers Protects vaccines from adverse Gelatine, 17D Yellow Fever conditions such as freeze-drying Monosodium virus vaccine, or heat, thereby maintaining a glutamate (MSG) Rabies,Varicella vaccine’s potency Inactivating agents - used to Formaldehyde Influenza virus, inactivate bacterial products for Poliovirus, Diphtheria toxoid vaccines, to kill and Tetanus toxins. unwanted viruses and bacteria β-propiolactone Rabies virus that might contaminate the GlutaraldehydeResiduals vaccine during production Acellular pertussisfrom Antibiotics - prevent bacterial DTaP-IPV/Hib Neomycin,manufactur contamination during Streptomycin, Influenza, MMRing process manufacturing process Polymyxin B Influenza and yellow Suspending fluids – Egg proteins fever vaccines kill or weaken the organism for Hepatitis B vaccines use in vaccines. Yeast proteins
    36. 36. QUALITY CONTROL AND LOT RELEASE Schedule of final product testing for a veterinary vaccine Test Purpose of TestSterility Demonstrates that no live microorganisms are present in productSafety Demonstrates that overdose of the product causes no harmResidual toxicity Demonstrates that the product contains no material that can cause harmEfficacy Demonstrates that each antigen in the product meets the recommended guideline level in internationally recognised tests.Increase in With live vaccines, there is concern that the organism might be shed fromvirulence tests 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, onePRINCIPLES OFVETERINARY VACCINE component causing a decrease in the protective immunological responsePRODUCTION, OIE Terrestrial 36Manual 2008 to another component.
    37. 37. Lot releaseBATCH/SERIAL RELEASE FOR DISTRIBUTION :Prior to release, the manufacturer must test eachbatch/serial for purity, safety, and potency. 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. 37 PRINCIPLES OF VETERINARY VACCINE PRODUCTION, OIE Terrestrial Manual 2008
    38. 38. 2. Batch safety test -Batches are considered satisfactory if local andsystemic reactions to vaccination with the batch tobe released are in line with those described in theregistration 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. 38 PRINCIPLES OF VETERINARY VACCINE PRODUCTION, OIE Terrestrial Manual 2008
    39. 39. Sampling : Samples should be selected from each batch/serial of product. The selector should pick representative sample.Filling , Packaging and LabellingOnce all procedures of vaccine production are completed, thevaccine is blended, filled the doses into vials and packaged.Which are then sealed and carefully inspected before labels areapplied to show the vaccine batch,lot numbers, and expirationdate.Standards for labelling products will vary from country to country. Field tests (safety and efficacy) Performance monitoring 39 PRINCIPLES OF VETERINARY VACCINE PRODUCTION, OIE Terrestrial Manual 2008
    40. 40. Processes of Egg-Based Vaccine Production Embryonated chicken eggs Inoculation Incubation The upstream A seed ampoule This is followed by a 3 process, begins is used to day incubation period with embryonated inoculate the during which the virus eggs brought in chick eggs grows to ensure that on a daily basis during the sufficient quantities from biosecure inoculation can support further flocks. phase. manufacturing. Harvest (pool Candling allantoic fluid) After 3 days, all of the eggs are The allantoic fluid is candled to make sure there are no then harvested; a low- cracks or contamination; the eggsClarification speed clarification are then chilled to 2° to 8°C to process follows. constrict vessels and make harvesting easier. 40The BRIDGE, Egg-Based Production ofInfluenza Vaccine:James T. Matthews page 21
    41. 41. 1st inactivation Filtration Concentration The first step in the Zonal centrifugation downstream Extensive filtration and concentration process is steps yield a concentrate, which is inactivation, which then loaded onto zonal centrifugation involves the equipment. addition of formalin to inactivate the virus. Triton fragmentation 2nd inactivation CentrifugationUltrafiltration The material is then The first purified clarified by bulk virus, which isAn ultrafiltration centrifugation to recovered from the(UF) step is remove large centrifugationfollowed by terminal particulates and process, is split insterile filtration to Sterile filtration treated with a fragmentationgenerate one formalin in a step by treatmentmonovalent bulk second inactivation with Tritonconcentrate. step. detergent. 41 Final product The BRIDGE, Egg-Based Production of Influenza Vaccine:James T.
    42. 42. Cell Culture Based Vaccine Production centrifugation virus cell (production seed)    filtering Cell culture Inoculation Harvest Bulk Purification  Add Adjuvant Stabilizer     Preservative Packaging Labeling Inspection Filling Formulation 42ccines / [edited by] Stanley A. Plotkin, Edward A. Mortimer, Jr. Influenza Vaccines (Tina Kröber, Thomas Jarosch, and Laura Fischer) Max-Planck-Gesellschaft, Münche
    43. 43. Thank you 43
    44. 44. 44
    45. 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. 45
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