Usp vaccines-biological medicines


Published on

12th USP Science & Standards Symposium - New Delhi

Published in: Health & Medicine, Business
  • Be the first to comment

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Usp vaccines-biological medicines

  1. 1. Track II, Session II: Biological Medicines–Vaccines Wednesday, April 17, 2013 (11:30 a.m. to 1:30 p.m.) IPC–USP Science & Standards Symposium Partnering Globally for 21st Century Medicines
  2. 2. Moderator: Mahesh Bhalgat, Ph.D. USP Medicines Compendium Expert Committee
  3. 3. Comparing the Opportunities and Challenges in Thermostable Vaccines with Conventional Vaccines Rajesh Jain, Ph.D. Joint Managing Director Panacea Biotec Limited, New Delhi Panacea Biotec Ltd. All Rights Reserved, Privileged & Confidential April 17, 2013
  4. 4. Contents • Company Overview • Vaccines needed across all age groups • Concept of Vaccines • Cold Chain • Thermostable Vaccines Panacea Biotec Ltd., All Rights Reserved. Priviledged & Confidential
  5. 5. Company Overview Business Lines Ranking Infrastructure • Pharmaceuticals • Vaccines & Biotherapeutics (MABs, Peptides, r-Proteins) • Largest Vaccine Producer in India • 3rd Largest Biotechnology Company in India (ABLE Survey 2011) • Ranked 39th amongst Pharmaceutical Companies in India (ORG-IMS, 2011) • Four Research & Development Centers • Established Sales & Distribution Network in India, 50 branded products • Direct presence in Germany for specialty hospital products segment • Presence in 55 ROW and Emerging Markets • cGMP Manufacturing Facilities Manpower • 3,800 Human Resource • 291 in R&D • 1,200 in Sales and Marketing Financials • Turnover : INR 1,130 Cr. (~USD 250 Mn.), CAGR of 21% • Listed in BSE and NSE
  6. 6. Vaccine Business – Key Highlights Robust Pipeline Established Presence • Innovative vaccines • Universal Flu (Recombinant viral vectored) • Established presence of over 25 years in vaccines • Dengue (Chimeric Recombinant Tetravalent ) • Reliable partner to WHO, UNICEF: largest supplier of • DTwP/DTaP- IPV combination (Fully Liquid combination Vaccines) vaccines to UNICEF from India • First Indian Company to launch innovative branded • Recombinant Anthrax Vaccine • Seasonal Flu (Quadrivalent seasonal Vaccine) combination vaccine – Easyfive (Hep. B+DTP+Hib) and • Pneumococcal Conjugate (13 valent conjugate) other combination vaccines – Easyfour, Ecovac • Japanese Encephalitis (Inactivated cell culture based viral vaccine) • One of the 3 Companies chosen by Govt. of India to develop Flu vaccine (Seasonal & Pandemic Flu) • Joint venture with Chiron Corp. (Novartis) for marketing of Branded combination vaccines in India • Meningococcal (Tetravalent conjugate vaccine) • Adolescent Vaccines • Tdap (AdTdap) • Tdap- IPV (AdTdap -pol)
  7. 7. Innovation Infrastructure Laksh, Mohali Drug Discovery: Small Molecules • Target identification to development of pre-clinical candidate • Focus areas : Metabolic disorders, Anti-infectives, CNS GRAND, Navi Mumbai NDDS Product Development • Platform NDDS technologies : Nanoparticles, Liposomes, Micro-particles, Depot Injections, SPORT, Oral films etc. • High barrier to entry generics OneStream, New Delhi Drug Discovery: Novel Biologicals • Target identification to development of pre-clinical candidate • Focus areas for Novel Peptides: Metabolic Disorders • Biosimilars & Vaccines Sampann, Lalru Generic Product Development • High barrier to entry generics • NDDS technologies: Depot Injections, Oral modified release, SMEDDS in Softgels, MD tablets, Critical dose drugs • Bio-therapeutics & Vaccine Formulation Development
  8. 8. Pipeline Overview Pipeline Generics Generics US: • 2 ANDAs filed • 45 ANDAs in development NDDS NCE NDDS Europe: • 1 MAA granted • 20 products in development • 7 best in class NDDS products in development (Scientific advice from US / European agencies has been taken for 5 products) NCE & Peptides • 4 Best in Class NCE • 1 Atypical NCE • 2 Novel Peptides Biosimilars, Peptides, Vaccines Biosimilars, Peptides, Vaccines • 4 Biosimilars in development • 4 Peptides in development • 10 Innovative vaccines in development
  9. 9. Commitment to make Affordable Vaccines… The Pledge June 2011, London “We at Panacea Biotec wish to express our continued, unstinted & unequivocal commitment & support to further the cause of GAVI Alliance. In this initiative we pledge to bring down cost of pentavalent vaccine (EasyFive) by minimum 15% in the coming years as a tiny contribution to a mammoth cause!”
  10. 10. Vaccine - Manufacturing Capabilities Vaccine Formulation Facility Location Delhi Formulation Facilities 2 lines for Oral vaccines 2 lines. 1 for Vials , 1 for PFS switch able to lyophilized in Vials Baddi One more filling line under construction Capacities (million doses/ annum ) 1600 PFS-16 Single dose -39 10 Dose -350 Formulation Capacity of 2 billion doses p.a (Includes Vials & PFS) Built Up Area > 50,000 sq ft > 129,167 sq ft
  11. 11. Vaccine - Manufacturing Capabilities Indigenous & fully integrated bulk antigen manufacturing facilities Location Bulk Antigen Facilities Built Up Area Recombinant Vaccines Bacterial Vaccines > 18,000 sq. ft Tetanus Vaccine > 20,000 sq. ft Cell Culture Vaccines Lalru > 40,000 sq. ft > 30,000 sq. ft Licensed Bulk Antigens – • Diphtheria Toxoid • Tetanus Toxoid • Whole cell / acellular Pertussis • Haemophilus influenzae type b conjugate • Recombinant Hepatitis B • Inactivated H1N1 split viron influenza vaccine bulk (by traditional egg based technology) Facility has provision for production of Bulk Antigens which are under development Vaccines : Japanese Encephalitis ,Sabin IPV, Dengue Vaccine, egg based seasonal flu vaccine, yellow fever vaccine Bio therapeutics : Viral proteins , non-viral recombinant bio molecules on cell culture in both conventional & disposable formats . (US-FDA / UK-MHRA compliant)
  12. 12. Vaccines needed across all age groups • Need of pre-birth vaccination • Mothers transfer fewer protective antibodies to their infants because of less exposed to infectious agents, breast feeding is less common & of shorter duration. Also, as current schedules starts @ 11/2 – 2 months of age, there is period of vulnerability during 4–6 months of life with significant mortality & morbidity In 21st century vaccines will be needed across all age groups Source: Rappuoli R. Nature reviews. Immunology, Vol 11, Dec 2011, pp. 865-872.
  13. 13. Vaccines • Concept of Vaccine` – Training of immune system to face various existing disease agents – Generate memory cells. • Prophylactic treatment against disease. • Most effective treatment against any disease so far.
  14. 14. Vaccines-success stories so far • Made eradication of many life threatening diseases possible. – Smallpox – Eradicated in1979 – Polio – Significant elimination – Measles – Significant elimination – Rubella – Significant elimination • Still in progress – Hepatitis-B: 260,000 in the 1980s to about 60,000 in 2004. – Diphtheria: No case reported in USA after 2003.
  15. 15. Vaccines - Limitations • Limited production capacities • Failure to protect immuno-compromised patients. • Instability issues – Heat and freeze degradation. – Unfolding of structure. – Hydrolysis, oxidation, deamidation. – Inherent instability of large molecules. • Additional costs of cold chain logistics and storage.
  16. 16. Temperature sensitivity of vaccines • Vaccines lose potency over time and the rate of potency loss is temperature-dependent. • Both, high temperature as well as (freeze) low are detrimental to vaccine quality – Most aluminum salt adjuvant containing vaccines are freeze-sensitive (Require Freeze stabilization) – Live attenuated vaccines are sensitive to heat (Require Heat Stabilization) Cold-chain have been established to ensure that the potency of vaccines is maintained until the point of use.
  17. 17. Cold chain • A cold chain is a time temperature-controlled and supply chain which provides a series of facilities for maintaining ideal storage conditions from the point of origin to immunization site. • Nearly all vaccines require cold chain for proper transportation while maintaining potency.
  18. 18. Cold Chains- Risks associated • Drawback : Costly and complex distribution logistics. – In the developed world, maintaining the cold chain is estimated to cost up to $200 million a year and increases the cost of vaccination by 14–20% (World Health Organization) • 75-100% of vaccines at some stage of transportation experience “temp excursions” . Usually occurs in the tail end of the cold chain. • Risk of vaccine wastage and associated costs. – 50% losses in emerging nations (GAVI report). – 10% losses in established markets (Australian MoH report). • Need more space for storage and transport.
  19. 19. WHO recommended storage temperature
  20. 20. Reality of cold chains in underdeveloped countries
  21. 21. What we are looking for ??? • Thermo-stable vaccine which can be stored at room temperature (Reformulation) • Redefining the thermal stability of existing vaccines for relaxing the cold chain requirements • Developing new strains with inherent thermostability. Vaccine transported or stored at room temperature ??
  22. 22. Thermo stable vaccines • Vaccines neither requiring refrigeration nor affected by freezing. • Thermostable vaccines will help in – Decreasing the cost of vaccine stockpiling. – Improving the efficacy of vaccine. – Energy cost saving (due to cold chain) – Reducing wastage of vaccines. • Will ensure vaccine stability in remote areas of the world with limited or no access to electricity for cold chain. Thermostable vaccines represent a better opportunity to increase the outreach of global immunization program.
  23. 23. Ambient storage condition-WHO Measured mean annual data Long-term stability testing conditions Zone Climate/Definition Temperature (Open air, °C) Partial vapor pressure (hPa) I Temperate climate ≤15 ≤11 21°C/45% II Subtropical and Mediterranean >15-22 >11 to 18 25°C/60% III Hot and dry >22 ≤15 30°C/35% IVa Hot and humid >22 >15 to 27 30°C/65% IVb Hot and vary humid >22 >27 30°C/70%
  24. 24. How to overcome these problems? • Explore genetically modified strains that address specific stability issues. • Apply novel formulation concepts and processing technologies – Spray drying, foam drying, and lyophilization, etc. • Employ computational analysis of protein structure to inform formulation design.
  25. 25. Thermostable vaccines – ongoing research • Use of silk protein biomaterial matrices for stabilization of MMR vaccine (Tufts University, USA). • Use of lipid particle vaccines platform (VBI ). • Use of osmolytes for Hep-B vaccines (PATH, USA). • ThermoVax platform (RiVax™, Velothrax™). • HydRIS technology platform (Oxford University and Nova Bio-Pharma ) • Use of Glycerin, PEG-300, Propylene glycol for freeze sensitive formulation. • Coated microneedle Patches (influenza virus by University of Queensland). • Oral tablets (E-coli by Johns Hopkins Bloomberg School of Public Health & PATH, Polio vaccines). • Flu vaccine by Powderject, USA
  26. 26. Sugar Glass Technology • A joint effort by Durer Chemical corporation USA, CSIR Australia • Utilize stabilizing abilities of sugars is used for stabilizing vaccines. • Measles , DTaP vaccines successfully stabilized. • Pre-clinical investigations have demonstrated the immunogenicity and potency of the trehalose-dried vaccine candidate.
  27. 27. ThermoVax Technology • A proprietary vaccine formulation platform from Soligenix, thermostable properties to aluminum adjuvanted vaccines. • Makes alum vaccines resistant to freeze/thaw and Heat excursions. • Delivers long-term stabilization of labile antigen-adjuvant combinations • Maintains native structure US bestowing – Applicable to many types of commercial vaccines Polysaccharide conjugates, VLPs, recombinant subunit proteins and peptides – Complex vaccines utilizing “secondary adjuvants” – Combination as well as multivalent vaccines • Scientific merit of ThermoVax™ technology validated through $9.4M grant award Grant provides for stabilization of its proprietary ricin vaccine (RiVax™) and anthrax vaccine (Velothrax™)
  28. 28. Preservation by Vaporization (PBV) • Under study for YF-VAX 17D by Universal Stabilization Technologies, US • Higher activity titer after drying and thermostability during subsequent storage (increased shelf-life). • Allows subsequent particle size reduction (micronization). • Allows short-term stability at 60°C to 90°C that could be used for encapsulation of dry powders for buccal and transdermal delivery avoiding a need of reconstitution with water.
  29. 29. Ambient storage condition-WHO POC VeloThrax, rPA, Soligenix Thermostable IM Measles Microneedle patch for flu HepB Formulations, PATH Preclinical Phase I/II Phase III Marketed
  30. 30. Challenges in thermostable vaccine development • Commercial – – Patents rights are in hand of very few companies and individuals. – • High cost and time associated with development and licensure of thermostable vaccines Higher Product Cost would impact the immunization programs in developing world Regulatory – Addition of novel stabilizer/adjuvant/excipient/process require huge data and cost involved. – Choice of excipients/stabilizer to achieve limited Target population (Infants) have extremely low regulatory tolerance for adverse effects. – Stability indicating markers and correlates of protection should be identified. Better clarity on the stability targets from the policy makers is desired. – Thermostable vaccines without cold chain require new regulations, policies, and logistic systems in additional to new formulation technologies.
  31. 31. Thermostable Vaccine – industry Perspective • Cost vs. benefit has to be evaluated by industry. – Funding model for bearing the developmental cost should be identified – Developmental cost vs. saving by cold chain should be evaluated – Priority development of vaccine candidate e.g. Pentavalent, MMR,Polio. • Wider and open view of regulatory agencies – Relaxation of transportation guideline for already stable vaccines (e.g. D, T & Hep-B). – Separate transport and storage conditions for individual vaccines.
  32. 32. Developmental initiatives • Bill & Melinda Gates Foundation (Measles vaccine project to Dr. Paul Duprex , TransForm Pharmaceuticals, Inc., Massachusetts, United States - US • PATH (Freeze and heat stable Liquid HepB Formulation). • BARDA (Awarded US$2.5 million funding to advance the development of thermostable influenza vaccines)
  33. 33. Summary • Integrate stabilization approaches in early vaccine development. • Many vaccines are more stable than we assume e.g. Human rotavirus vaccine, HepB & tetanus toxoid. New supply chain model (combination of cold chain and controlled temperature chain), distribution policies and logistic system need to developed for them • Funding model to take-care the developmental cost and the risk associated with them needs to be developed.
  34. 34. Evolving regulatory framework for vaccine PQ  IPC-USP 12th Science & Standards Symposium  New Delhi India 16-17 April 2013 Carmen Rodriguez Hernandez QSS-EMP-HIS-WHO
  35. 35. Purpose of WHO vaccines prequalification programme  A service provided to UN purchasing agencies.  Provides independent opinion/advice on the quality, safety and efficacy of vaccines for purchase  Ensures that candidate vaccines are suitable for the target population and meet the needs of the programme  Ensures continuing compliance with specifications and established standards of quality 36 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  36. 36. The following displays the count of finalised submission/review processes which are on time (Internal time is less than or equals 12 months) and those which are overdue (Internal time exceeds 12 months), by year of submission 2012: first streamlined submission under US FDA: Timeframe taken for PQ was 198 days . Quality review done by WHO . Two additional submissions received 30 September, 2012. Evaluation ongoing 37 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  37. 37. PQ activity 2009 2010 2011 2012 Jan-Sept Reassessments 11 12 10 12 Annual reviews and variations 6 21 74* 53** Testing (lots) 124 159 183 105 Complaints/ other issues of concern 3 13 16 12 AEFI 7 7 7 5 Meetings with manufacturers 62 80 71 119 Meeting with NRAs and others 33 86 64 109 38 * Refers to changes ** Refers to Vaccines containing 448 total changes
  38. 38. Vaccine PQ activities to facilitate access  Secure the existing supply of vaccines  Explore alternative sources  Work with NRAs from user countries to facilitate registration of vaccines  Sustain functionality and secure risk mitigation strategies (SOP to be published shortly)  Mechanisms to minimize wastage of vaccines, facilitate outreach (VVMs, MDVP, CTC) 39 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  39. 39. Procedure for expedited review of imported prequalified vaccines for use in national immunization programmes  Concept proposed by 6 SEARO countries in 2005.  Meeting with WHO HQ and RO in 2005  Discussed need for registration in all countries  Proposed a "facilitated process" for registration (MA) of imported prequalified vaccines EXPEDITED REVIEW PROCEDURE FOR LICENSING PQ VACCINES Expert Committee on Biological Standardization 40 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  40. 40. Support to NRAs Implementation of Procedure for expedited review of imported prequalified vaccines for use in national immunization programmes (WHO/IVB/07.08) Firstly used for registration of MenAfriVac (16 countries) 41 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  41. 41. Implementation workshops- AFRO July 2011 July 2011 1 2 3 4 5 6 7 8 Uganda Ghana Nigeria Rwanda Tanzania Guinea Bissau Kenya Ethiopia 1 Burkina Faso 2 Benin 9 3 5 Central African Republic Côte d'Ivoire 6 Gabon Cameroon 7 Gambia 8 Sénégal 11 Eritrea 9 Togo 2 Ethiopia 3 Gambia 4 Kenya 5 Malawi 6 7 Namibia Uganda 8 Sierra Leone Tchad 10 Botswana Mauritanie 10 1 Burundi 4 July 2012 42 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  42. 42. Implementation workshops- WPRO November 2012 1. CAMBODIA 2. LAO PEOPLE'S DEMOCRATIC REPUBLIC 3. MONGOLIA 4. PAPUA NEW GUINEA 5. PHILIPPINES 6. SOLOMON ISLANDS VANUATU 7. 43 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  43. 43. 44 |
  44. 44. 45 |
  45. 45. 46 |
  46. 46. Status of agreements with NRAs NRA/Country Status AIFA/ Italy Discussions ongoing ANSM (France) Discussions ongoing ANVISA (Brazil) Signed BELGIUM Discussions ongoing BULGARIA Signed CECMED (Cuba) Signed HC (Canada) Signed CDSCO – DCGI (India) Discussions ongoing BADANPON (Indonesia) Signed KFDA (Korea) Discussions ongoing JAPAN Discussions ongoing SWISSMEDIC (Switzerland) Discussions ongoing THAI FDA Signed US FDA (US) framework for Vaccines PQ Evolving Regulatory Signed 47 | 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  47. 47. Regulatory networks: DCVRN Network of regulators from developing countries: 2004 – NRA Meets criteria of functionality or Government endorsed workplan & Expertise – Local PQ vaccine manufacture / Clinical trials  Members: Brazil, China, Cuba, India, Indonesia, Korea, South Africa, Thailand and Iran  Strengthen capacity of National Regulatory Authorities • both for Members and other Developing Countries • Initial focus on regulatory control of Clinical trials • through exchange of experience and information 48 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  48. 48. Regulatory networks: AVAREF Network of regulators from African region: 2006 – To provide information to countries targeted for clinical trials of vaccines. – To promote and strengthen communication and collaboration between NRAs and Ethics Committees in countries where vaccines are being developed and those targeted for clinical trials in the African Region. – To provide expertise to African NRAs in support of regulation and evaluation of vaccines. Members:one representative each of the National Regulatory Authorities and of the National Ethics Committees of 19 countries in the African region 49 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  49. 49. Regulatory networks: Discussions DCVRN AVAREF Meetings Bi-annual-Annual FtF Annual meetings: 7 meetings + web meeting: 12 meetings held + 2 web meetings Participants regulator from mature NRAs (USFDA, HC, EMA) Other regulators from the region as observer Sessions Scientific session Closed session 50 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO
  50. 50. QC labs- NETWORK OF WHO VPQ CONTRACTED LABORATORIES  ANSM France  NIBSC UK  Cantacuzino Romania  PEI Germany  Department of Medical Sciences (DMSC) Thailand  RIVM  Health Canada  South African National Control Laboratory  Korea Food and Drug Administration  NCE Hungary 51 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO  SIPH Belgium  Swissmedic  TGA Australia
  51. 51. Qualification of WHO contracted laboratories  Phase 1:  Phase 2: Visit to the lab for evaluating  Review of SOPs and related documents  Organisation of the Laboratory  Validation protocol and report  Testing 3 batches in parallel with a contracted laboratory  Quality System  Personnel  Premises and equipment  Handling of samples  Reagents and Reference material  Test methods  52 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO Test reports
  52. 52. VPQ TESTING PROGRAM Challenges • Increased demand for evaluation of vaccines Novel vaccines to be evaluated: Need for new tests • High demand for evaluation of combination vaccines: Increased complexity, results are not consistent, need for standardization and harmonization 53 | Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO Solutions • Identification of new laboratories/additional capacity Transfer of methodologies to WHO contracted labs • Project on stardardization of Hib: WHO-EDQM • Pre-testing phase • Suitability of the method: Collaborative study • ECBS
  53. 53. National cold room during the campaign 54 | Contribution to development of Controlled Temperature Chain Project Optimize: PATH/WHO Mali, polio campaign, Photos: WHO/Olivier Ronveaux Nicaragua, rotavirus delivery, Photo: Gates Foundation Transport to health centre
  54. 54. Allow specific vaccines to be kept and administered at ambient temperatures, up to 40oC For one, limited period of time immediately preceding administration For vaccines meeting a number of stability conditions Current focus: vaccines administered during campaigns and special strategies: eg Meningo conjugate A, Yellow Fever, Pneumo, Hepatitis B, Rota, Cholera Manufacturers Regulators Studies to enable on label use of vaccines under CTC and regulatory submissions Regulatory pathways 55 | Review data for licensing under CTC WHO CTC Guidelines(QSS) Work w/regulators to define Regulatory Pathways and prequalification (QSS) Field studies to show programmatic challenges , opportunities and impact of CTC (EPI)
  55. 55. Capacity building in countries NRAs • NRA Observers in PQ evaluation procedures • Joint reviews of PSF with NRAs of producing countries/NRA networks 56 | Laboratories • Collaboration between targeted testing program and NCL networks globally • Support to NCL for the establishment of critical testing methods relevant to PQ vaccines • Harmonization of test methodologies Evolving Regulatory framework for Vaccines PQ 17 April 2013 Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO PQ Programme to leverage networking approach to strengthen regulatory capacity including regulation of clinical trials (DCVRN, AVAREF)
  56. 56. Alternative to Animal Testing in the Quality Control of Vaccines and Regulatory Acceptance Mahesh Bhalgat, Ph.D. Biological E Limited
  57. 57. Overview  Current approaches to vaccine manufacturing and testing – The use on in vivo testing methods  Regulatory acceptance of in vitro methods  Opportunities for advancement of alternate methods  Demonstrating product quality, safety and efficacy using the consistency approach  Conclusions
  58. 58. Steps In Vaccine Manufacturing Production of the established (inactivated) vaccines Virulent micro-organism/toxin Culture Concentration Detoxification/inactivation Purification Blending (adjuvant, antigens) Inactivated vaccine/toxoid final lot  Safety tests  Potency tests
  59. 59. Current Considerations In Lot Release Of Vaccines  S  Starting point is uniqueness of every lot produced  Focus of lot release testing on final product  Use of a international reference preparation expressed in IU/ml  Reliance on animal models for safety and potency
  60. 60. Limitations Of Current “In Vivo” Based Approaches  Scientific Aspects  Validity aspects : reproducibility tests, questionable relevance.  Use of reference preparation (not like to like)  Science: based on models developed > 50 yrs ago  Ethical Aspects  Practical : Extensive animal use aspects : Costs, time required
  61. 61. The Relevance Of Biological Assays  Ligand/receptor assays (not cell based)  SPR Variability Relevance  Modern cell based bioassays (early read out)  Receptor binding  KIRA  PACE  Reporter gene assay ?  Classical cell based bioassays (late read out)  Proliferation assay  Death of cells as read out  In vivo bioassays  Includes in vivo clearance event 63 Speed
  62. 62. Species Difference Can Be Unpredictable  For humans the circulating leukocyte profile is 50-70% neutrophils but for rodents it is 50-100% lymphocytes. (Haley, 2003) – Relevance of measuring leukocyte related changes (immunogenicity) in alternate species? ***********************************************************************  Mouse spleens are major sites for lifelong hematopoietic activity while humans have little hematopoietic activity in embryonic spleens and virtually none in adult spleens. (Haley,2003) – Relevance of using mouse spleen cells as target cells in immunotoxicity assays? ***********************************************************************  TCDD (a Dioxin derivative) causes a dose-dependent suppression of the T-cell Dependent Antibody Response in adult female B6C3F1 mice, but enhances the TDAR in F344 and Long-Evans rats even at high doses. (Smialowicz et al., 1994) – Relevance to humans?
  63. 63. Animal Usage In Human And Veterinary Vaccines • Used in: • • • Vaccine development (research, validation of efficacy) Production (sometimes in animals, primary cell cultures, eggs) Batch control testing (safety and potency testing) • Routine batch control testing is responsible for 80% of animal use in vaccine industry and regulation • Batch control testing of vaccines accounts for ~10% of all animal use in biomedical research, using 10 million animals every year • Biologicals testing has the highest proportion and number of experiments causing severe pain and distress to animals out of various types of experiments (basic research, toxicity testing, etc.) Sadhana Dhruvakumar, PETA, Feb 2005
  64. 64. The 3R’s Approach  Replacement: – Substitution of insentinent material for conscious living higher animals  Reduction: – Reduction in the number of animals used to obtain information of given amount of precision  Refinement: – Decrease in the incidence of severity of inhumane procedures applied to those animals which still have to be used Russell and Birch
  65. 65. Overview  Current approaches to vaccine manufacturing and testing – The dependency on in vivo methods  Regulatory acceptance of in vitro methods  Opportunities for advancement of alternate methods  Demonstrating product quality, safety and efficacy using the consistency approach  Conclusions
  66. 66. European Center For The Validation Of Alternative Methods (ECVAM) – Selected Examples  Replace: • Validated commercially available ELISA kits for rabies potency testing (1999) • Validated human-blood-based pyrogenicity test (2005) • Validated Vero cell test for specific toxicity testing of diphtheria toxoid  Refine: • Sponsored development of humane endpoints for rabies, pertussis, and erysipelas challenge tests (1999) • Validated ELISA and ToBI test for batch potency testing of human tetanus vaccine (2000) • Validated ELISA test for swine erysipelas vaccine  Reduce: • Validated ELISA and ToBI test for batch potency testing of human tetanus vaccine (2000) • Validated ELISA test for swine erysipelas vaccine
  67. 67. European Directorate for Quality Medicine(EDQM)/European Pharmacopeia (Ph. Eur.) – Selected Examples  Replace: • • • • •  Refine: • •  Accepts vaccination-serology tests for tetanus, diphtheria, and cholera vaccines (in lieu of vaccination-challenge) Recommends use of humane endpoints in vaccination-challenge procedures Reduce: • •  Accepts antigen quantification test for rabies (1998) Deleted Abnormal Toxicity Test (in favor of production consistency approach) Deleted guinea pig test for diphtheria (residual toxin and irreversibility of diphtheria toxoid) Deleted in vivo test for polio (for some manufacturers*) Deleted residual pertussis toxin test for acellular pertussis (for some manufacturers*) Accepts single dilution assays for diphtheria, tetanus, and acellular pertussis vaccines (in lieu of multiple dilution assays) Accepts vaccination-serology tests for tetanus, diphtheria, and cholera vaccines (in lieu of vaccination-challenge) *A licensing authority can waive tests in monographs if it is assured of production consistency
  68. 68. WHO Animal Use Alternatives Example: Polio Neurovirulence Testing  Transgenic mouse test introduced as alternative to monkey NVT (TRS 904, 2002)  Future direction: can transgenic mice be considered as fully equivalent to monkey NVT?  Future challenges: (a) independent testing by National Regulatory Authority in Tg mouse test, (b) maintenance of competence for testing as polio nears / beyond eradication, (c) need for NVT to control Sabin-IPV?; (d) Need for other molecular tests such as nonisotope method, MALDI-TOF, microarray hybridization, Massively Parallel Sequencing –the ultimate solution for monitoring molecular consistency of live viral vaccines? International Workshop on Alternate Methods-J Shin, WHO, Sept 2010
  69. 69. WHO Reduction And Refinement Examples: DTP Vaccines  Reduction of animal testing during the lot release of DT vaccines  Addendum to Recommendations for potency of DT vaccines (TRS 927, 2003)  -Introduces possibility to use (a) serological assays or (b) challenge assay with a single dilution, both involving reduced number of animals, as an approach for lot release.  -Conditionality; consistency in production and quality control has been confirmed on a continuous basis.  Recommendation for the use of validated humane end points in recording results of potency testing  -Revised recommendations for whole cell pertussis vaccines (TRS 941, annex 6, 2007) International Workshop on Alternate Methods-J Shin, WHO, Sept 2010
  70. 70. WHO Reduction And Refinement Examples: T Containing Vaccines “The innocuity test on the final lot may be omitted for routine lot release once consistency of production has been demonstrated, subject to the approval of the NRA.”
  71. 71. WHO Reduction and Refinement Examples: T Containing Vaccines “The innocuity test on the final lot may be omitted for routine lot release once consistency of production has been demonstrated, subject to the approval of the NRA.”
  72. 72. WHO Reduction And Refinement Examples: Rabies Vaccines  Recommendation for the use of validated humane end points in recording results of potency testing  -Revised recommendations for rabies vaccines (TRS 941, annex 2, 2007)  -plus possibility to use single dilution assay for NIH test  Statement that there is no additional value in performing an accelerated stability test for the purpose of lot release.  -Since this test is based on the NIH test for potency after exposure to the elevated temperature, this statement led to discontinuation of this test on a lot-to-lot basis in a number of countries. International Workshop on Alternate Methods-J Shin, WHO, Sept 2010
  73. 73. WHO Reduction And Refinement Examples: Yellow Fever Vaccines  -Amendment of requirements for yellow fever vaccine potency assay (TRS 872, 1998): 2008  -recent establishment of WHO IS for yellow fever potency  * allows possibility to qualify the cell culture assay in place of the mouse potency assay with improved interlaboratory comparison International Workshop on Alternate Methods-J Shin, WHO, Sept 2010
  74. 74. WHO Continues To Evaluate Additional Opportunities  Lot release guidelines  Recommending mutual recognition of animal tests in exporting and importing countries  Mumps vaccine neurovirulence tests  Repository of mumps vaccine seed strains being established to facilitate evaluation of alternatives to monkey NVT and international collaborative study under development International Workshop on Alternate Methods-J Shin, WHO, Sept 2010
  75. 75. US-FDA Efforts on 3Rs
  76. 76. US FDA Perspective on Potency  610.10 Potency  “Tests for potency shall consist of either in vitro or in vivo tests, or both, which have been specifically designed for each product so as to indicate its potency in a manner adequate to satisfy the interpretation of potency given by the definition in 600.3(s)….” International Workshop on Alternate Methods-T. Finn, US-FDA, Sept 2010
  77. 77. US FDA Perspective On Vaccine Safety And Potency Testing  Tests performed on final bulk/container sample(s) to assure safety…. – e.g.: general safety test, histamine sensitization test, endotoxin…etc  ….and potency – e.g.: D-antigen ELISA test for polio types 1, 2 and 3, ELISA for pertussispotency, diphtheria and tetanus potency… etc.  Lot-release testing  610.1: Test prior to release…  “No lot of any licensed product shall be released…prior to the completion of tests for conformity with standards applicable to each product….” International Workshop on Alternate Methods-T. Finn, US-FDA, Sept 2010
  78. 78. US FDA Perspective On Alternative Safety And Potency Tests  CBER encourages alternatives to reduce, refine and replace the use of animals in safety and potency testing  Relevance  Data to support use  Validation  •Goal: Safe, pure and potent vaccines International Workshop on Alternate Methods-T. Finn, US-FDA, Sept 2010
  79. 79. US FDA Process On Changing A Potency Or Safety Test  Supplement to the License  •601.12: Changes to an approved application – Potency: Rationale and data to support proposed alternative – Safety: Rationale and data to support proposed alternative or demonstration of lack of need International Workshop on Alternate Methods-T. Finn, US-FDA, Sept 2010
  80. 80. US FDA Conducts Research On 3Rs Active research – One of CBER’s Research Priorities: Evaluating, developing and integrating novel scientific technologies and preclinical models for use in product regulation, including development and analysis of novel approaches that reduce, refine, or replace (3R’s) the use of animals high resolution novel technologies for their potential to improve product characterization, e.g.: molecular-based assays (microarray and high throughput sequencing) – alternative to lethal test for anthrax vaccine potency – alternative to monkey neurovirulence test for polio vaccine International Workshop on Alternate Methods-T. Finn, US-FDA, Sept 2010
  81. 81. Indian Authorities Continue to Embrace 3R’s - Examples  Hepatitis B vaccine batches can be released without any in vivo testing for potency (1 in 5 or 1 in 10 lots to be tested)  Indian Pharmacopeia considering deletion of abnormal toxicity test  HIB vaccine lot release testing does not require animal immunogenicity testing  Indian Pharmacopeia considering establishing guidelines to utilize in vitro D-antigen test instead of in vivo test
  82. 82. USP Is Implementing 3R’s in USP-MC Monographs  USP-MC monograph for Hepatitis B does not include any “required” in vivo testing.  Haemophilus influenzae B vaccine monograph under development applies same approach.
  83. 83. Status Of Alternatives In Common Vaccines: Bacterial Vaccines Type Examples Animal test Alternatives (accepted by) Toxoids Tetanus • Safety: absence of toxin, irreversibility of toxoid, specific toxicity • Deletion of specific toxicity test (EU) • Combined absence and irreversibility of toxin tests (EU) • In vitro endopeptidase test for toxin detection has been developed but not validated • Potency: multidilution vaccination challenge on guinea pigs or mice • Single dilution test (EU) • Antibody estimation by ELISA or ToBI (EU, WHO) • Safety: absence of toxin (5 guinea pigs for bulk lot), irreversibility of toxoid, specific toxicity • Deletion of specific toxicity test (EU) • Combined absence and irreversibility of toxin tests (EU) • In vitro Vero cell test for toxin detection (WHO) Diphtheria •Potency: multidilution vaccination challenge on guinea pigs with ~ 20 control animals • Single dilution test (EU, WHO) • Antibody estimation by Vero cell test (WHO) – ELISA and ToBI have also been developed Acellular pertussis vaccines (ACPVs) Bacterins • Safety: absence of toxin (5 mice), irreversibility of toxoid (5 mice) • In vitro CHO clustering test can be done on bulk but not final lot • Potency: multidilution vaccination + serology on 6 groups of mice • Single dilution test (EU) Whole cell pertussis vaccines • Safety: mouse weight gain test with 10 mice for testing specific toxicity • Modified to use 5 guinea pigs (EU) • In vitro alternatives include LAL pyrogen test (WHO) • Potency: Kendrick test - multidilution vaccination and intracerebral challenge in 136 mice – large numbers of animals, severe distress, poor precision and reliability • Humane non-lethal endpoints (EU) • Aerosol challenge instead of intracerebral • Antibody estimation by whole cell ELISA (validated in 2000) Cholera • Potency: multidilution vaccination + serology on 6 mice, guinea pigs, or rabbits • This serology test is accepted by the EU Haemophil us type B conjugate • Potency: multidilution vaccination + serology on 16 mice • This serology test is accepted by the EU • Moving testing upstream: if final bulk testing is satisfactory, can omit potency testing of final lot
  84. 84. Status Of Alternatives In Common Vaccines: Viral Vaccines Examples Animal test Alternatives (accepted by) Rabies • Safety: extraneous agent testing • Cell culture test (WHO) + EU for vet vaccines • Humane endpoints (EU) • Single dilution (EU) • Vaccination + antibody estimation using 5 mice (EU) • Antigen quantification (WHO) •Potency: NIH test (multi-dilution vaccination + intra-cerebral challenge test in up to 170 mice per batch) Hep A (inactivated) and Hep B (recombinant) • Vaccination + serological test in mice or guinea pigs • Antigen quantification (EU, WHO) Inactivated Poliovirus (IPV) • Multi-dilution vaccination + serology in at least 60 rats • Molecular analyses, e.g., MAPREC for poliovirus type 3 (WHO) • Neurovirulence in transgenic mice for poliovirus type 3 (WHO) Oral Poliomyelitis (OPV) • Neurovirulence testing in over 80 monkeys by intra-spinal injection • Molecular analyses, e.g., MAPREC for poliovirus type 3 (WHO) • Neurovirulence in transgenic mice for poliovirus type 3 (WHO) Already not tested in animals: Influenza (tested in eggs), Meningococcal and Pneumococcal (only need pyrogen test which can be done in vitro), Oral typhoid, Varicella, Measles, Mumps, Rubella Currently no alternatives available: BCG (2 safety tests on 6 guinea pigs each) Not covered here: Yellow Fever, Smallpox, Japanese Encephalitis, Anthrax
  85. 85. Overview  Current approaches to vaccine manufacturing and testing – The dependency on in vivo methods  Regulatory acceptance of in vitro methods  Opportunities for advancement of alternate methods  Demonstrating product quality, safety and efficacy using the consistency approach  Conclusions
  86. 86. Tetanus Toxoid Influence on IFN-gamma levels
  87. 87. Tetanus Toxoid Influence on IL-4 levels
  88. 88. Biosensor Analysis of Diphtheria Toxoids
  89. 89. Biomarkers for Pertussis Vaccine Toxicity
  90. 90. Overview  Current approaches to vaccine manufacturing and testing – The dependency on in vivo methods  Regulatory acceptance of in vitro methods  Opportunities for advancement of alternate methods  Demonstrating product quality, safety and efficacy using the consistency approach  Conclusions
  91. 91. New Concepts Followed In Vaccine Manufacturing  cGMP  QBD  Real-time fermentation data  In-process monitoring  In-process testing  Environmental monitoring  Testing using newer techniques  QA oversight and release
  92. 92. What Is Process Consistency ‘…… a concept which includes GMP, process validation and in process and final product tests and is aimed at verifying if a manufacturing process produces final lots which are consistent with one that fulfils all the criteria of Quality, Safety and Efficacy as defined in the marketing authorization, with the ultimate goal of replacing animal tests’ (De Mattia et al. 2011)
  93. 93. Consistency Testing In Vaccine Quality Control: Procedure  Test first few lots thoroughly; in non-animal models but also in laboratory animals and in target species (clinical/historical batch).  Based on this information, specify the profile of the vaccine (fingerprint) based on clinical, manufacturing and testing criteria. Set alert and acceptance criteria and criteria for deviations from consistency.  Subsequent vaccine lots produced should have the same profile as the clinical lot. The consistency in profile is monitored by non-animal techniques.  If so, the vaccine lot can be released for use.
  94. 94. Consistency Based Testing Is A Paradigm Shift Traditional concept of vaccine lot release testing New paradigm: Consistency testing  Each lot produced by a  Each lot produced by a manufacturer is considered to be a unique product manufacturer is one of a series and is NOT unique  Use of Reference preparation  Use of clinical lot  Emphasis in quality control of each vaccine lot is on final product  Makes use of : - strict application of quality systems (GMP, QA) - quality by design - extensive in-process testing - new innovative analytical tools  Quality control includes several animal models and is animal demanding
  95. 95. Testing of D and T Vaccines-Potential Parameters For Consistency Based Testing Production parameters • • • • • • • • • • • Optical density pH Flocculation titre Endotoxin Protein Nitrogen Protein Residual formalin In vitro safety test (Vero) Reversion (Vero) Osmolarity etc. Product quality parameters • • • • • Kf (flocculation time) purity various physico-chemical tests Moab binding (biacore) DAFIA (Direct Alhydrogel Formulation Immunoassay) • etc. Parameters can be used to set alert criteria and acceptance criteria
  96. 96. Consistency Approach Offers Benefits  Scientific Benefits: More meaningful batch release as quality is linked to a clinical lot and better understanding of your product  Ethical Benefits: Apart from clinical lot (first few lots) NO animal use is required for lot release testing  Practical Benefits: Quality control will be less time consuming (a few days instead of 2 months)
  97. 97. Consistency Approach Also Has Its Challenges  Tests: what set of tests is needed and will this be the same for every vaccine. Product specific ??  ‘Risk assessment’: what products are ready for implementing the consistency principle. Consistency is NOT a one-for-all strategy! blending: adjuvant and antigen – antigen interaction.  Vaccine  Validation: how to compare fundamentally different approaches.
  98. 98. Conclusions  Current paradigm for lot release testing is based on extensive testing of final antigen/final vaccine  Reliance on in vivo testing has poses challenges  With introduction of cGMP/QA/in-process testing in vaccine production, consistency based release testing can be considered  All major international bodies such as EDQM/EU/WHO/USP/IP/US-FDA have all embraced the transition from in vivo to in vitro methods  Researchers and manufacturers need to pursue development and implementation of alternate methods
  99. 99. Diverse Techniques Can Be Used In-process And Final Lot Testing Physico-chemical Application  circular dichroism secondary & tertiary structure proteins  fluorescence spectrometry protein conformation, protein modifications  colourimetric assays free amino groups in proteins Immuno-chemical  biosensor analysis epitope quality, antigen-antibody kinetics  ELISA (with Mabs) peptide mapping, ag quantification  electrophoresis purity, protein modification, stability In vitro functional  binding assays  Immune cells antigen binding antigen processing, B/T cell responses, cytokine
  100. 100. Novel Vaccine Adjuvants Manish Gautam, Ph.D. Serum Institute of India Limited
  101. 101. Outline of Presentation  Overview of vaccine adjuvants  Novel Vaccine adjuvants: Mechanistic aspects.  Preclinical Evaluation of Novel Adjuvants  Novel adjuvant development at SIIL- A case study of SIIL-3
  102. 102. Vaccine Adjuvant - Definitions and Guidelines 1) Adjuvants are the substances that are intended to enhance relevant immune response and subsequent clinical efficacy of the vaccines (WHO guidelines on nonclinical evaluation of vaccines, WHO Technical Report Series, No. 927, 2005) 2) A vaccine adjuvant is a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune responses. (EMEA guideline on adjuvants in vaccines for human use. 2005) 3) New Draft Guidance on Preclinical Evaluation of Vaccine Adjuvants is currently being developed by WHO aiming towards harmonization of requirements. (Available at WHO website for comments and discussion).
  103. 103. Novel Vaccine Adjuvants • Previously, Adjuvant development was largely based on approaches focusing largely on humoral immune responses. • Advances in basic sciences, immunology and vaccinology per se, have led to better understanding of host immune response against infection. These advances also impacted antigen discovery and adjuvant development. • Vaccines such as HIV. Malaria, HPV, cancer have brought cellular immunity and its induction in focus • Novel adjuvants targeting cellular immunity are currently being sought. Immunomodulation is becoming the central principle of preclinical assessment of adjuvants • Regulatory frameworks to support such adjuvant development are currently in development.
  104. 104. Novel Vaccine Adjuvants: Changing Paradigms Key to effective vaccines Delivery system Reduces booster frequency Immunomodulation (innate and adaptive immunity ;B and T cell Immunity Inducers Reduces vaccine dose Immune directing Vaccine adjuvant Boosts immunogenicity of sub-unit vaccines Rapid seroprotection Boosts immunogenicity in neonates and elderly
  105. 105. Adjuvant Development Europe Alum liposomes (HAV, flu) AS04 (MPL) (HPV, HBV) MF59 AS03, MF59, AF03 (flu elderly) (pan flu) 1900 1920 1940 1960 1980 2000 2020 Alum USA  The  AS04 (MPL) (HPV) slow process of adjuvant discovery. Alum was the first adjuvant to be licensed in the 1920s. in the USA. The squalene-based oil-in-water emulsion MF59 was first licensed in Europe for a flu vaccine (FLUAD) in 1997.  The LPS analog monophosphoryl lipid A (MPL) formulated with alum (AS04) was first approved for an HBV vaccine (Fendrix) in Europe in 2005. The oil-in-water emulsion AS03 was approved for a pandemic flu vaccine (Prepandrix) in 2008. Taken from M.Friede 2011, WHO
  106. 106. Vaccine Adjuvants and Immune System Targets Innate Immunity Adptive Immunity Antigen-specific B and T cell responses “Inflammatory responses’ Hours Days Innate immune receptors Signal 2 = adjuvants Vaccine vehicle (vector) Signal-1 =Antigen Antigen Presenting cells Months-Years Signal 3 Cytokines Co-stimulation T cells MHC-peptide Min TCR Signal 1 + 2 (+3) = Immune response Signal 1 only = Tolerance /Ignorance
  107. 107. Novel Adjuvants are Engineered to Target APCs, the Key Players of the Innate Immunity Stimulated by discovery and better understanding of role of following targets in host immune response  Discovery of toll-like receptors NLRP3 nucleosomes •Th1/Th2 Immunity
  108. 108. Aluminium Salts: New Insights Formation of inflammasome From DeGregorio, 2009 Stimulates Th2 directed cytokines
  109. 109. Alum induces cell death and the release of not only uric acids but also host cell DNA at injection sites Uric acids 70 60 50 40 6.0E+05 4500 4000 5.0E+05 3500 3000 Cell numbers Concentration(ng/mL) Cocentration(nmol/mL) 80 Dead cells dsDNA 2500 2000 30 1500 20 1000 10 500 0h 4h 24h 3.0E+05 2.0E+05 1.0E+05 0 0 4.0E+05 0.0E+00 0h 4h 24h OVA / Alum OVA Marichal T, Ohata K et al Nat Med. 2011 In press 0h 4h 24h 48h
  110. 110. Adjuvants are the Ligand for Innate Immune Receptors Receptors Ligands Adjuvants TLR2/1 or TLR2/6 Lipo-proteins Lipo-peptide Peptide glycans E coli heat-labile enterotoxins MDP MALP2 TLR3 dsRNA Poly I:C TLR4 LPS MDP, MPL TLR5 Flagellin Flagellin TLR7,8 ssRNA Imiquimod, R848, ssRNA TLR9 CpG DNA Hemozoin CpG ODNs RIG-I, MDA5 dsRNA, ssRNA Poly I:C, ssRNA NALP3 (AIM2) DNA,RNA ATP+PAMPs Uric acid cristal (MSU) Alum , particulate adjuvants (Silica) LPS : lipopolysaccharide MDP : muramyl dipeptide MALP2 : macrophage-activating lipopeptide 2 Poly I:C : Polyinosinic–polycytidylic acid MPL : monophosphoryl lipid A
  111. 111. MoA aluminium, Oil-in-water emulsions From: DeGregorio 2009
  112. 112. New adjuvants: immune potentiators and antigen delivery systems Optimize delivery of Ag (or other adjuvants) to APC in lymphoid tissues Delivery systems Alum, MF59 etc Immune potentiators MPL, CpG, Saponins etc Antigens Combo 2+ immune modulators • Synergy through different MOA (immune cell type or pathways) Recombinant proteins Activates innate immune cells by mimicking “danger signals” normally provided by infection to enhance immune responses Combo: Immune modulator + delivery vehicle • Enhance responses through improved delivery of antigen and/or immune modulator • E.g., ISCOMS Long-lived B & T cell memory
  113. 113. Adjuvant in Development Class TLR3 TLR4 TLR5 TLR7 TLR8 TLR9 Saponins O/W emulsion W/O emulsion Polysaccharides Cationic liposomes Virosomes poly-electrolytes component Poly I:C MPL MPL RC530 GLA flagellin Imiquimod Resiquimod CpG, IC41 QS21 QS21 squalene tocopherol squalene mineral oil Inulin DDA Polyoxidonium phase 1 cancer leish phase II phase III licensed herpes pneumonia malaria cancer HPV, HBV Allergy HIV flu influenza influenza TB pneumonia HIV HIV cancer cancer Allergy cancer cancer Alzheimer HBV, CMV HBV malaria Seasonal flu Pandemic flu malaria cancer HBV, flu TB malaria influenza HAV, flu influenza
  114. 114. T cell Immunity Adjuvants
  115. 115. Dendritic Cells (DCs). • DCs of two lineages: lymphoid and myeloid differentially influence maturation of TH1 and TH2. • Immature DCs: phagocytic, express CCR5 & CCR6, low levels of MHC Class II and B7. • Mature DCs: lose phagocytic capacity, increase presentation ability, enhanced expression of MHC Class II and B7. • Maturation influenced by PAMPs. Influence direction of DC maturation. – PAMPs (LPS, CpG, dsRNA) or host cell molecules (CD40L, IL-1, TNF-a) modulate DC maturation and subsequent TH response. e.g. LPS drives DC1 maturation and TH1 response; PC-GP (nematodes) drives DC2 maturation and TH2 response.
  116. 116. IL-4 vs IFN-g • Antigen recognition through TcR in the context of MHC Class I or II molecules. • Co-stimulatory molecular interactions between T-cell and APC:
  117. 117. Helper T-Cell Subsets • TH1: – IFN-g, TNF-b – Cellular immunity vs. intracellular bacteria, small parasites – Induction of neutralizing antibodies of the IgG2a subclass (in mice) • TH2: – IL-4, IL-5, IL-10, and IL13 – Induced by helminthes parasites, allergens, immunization with soluble or alumadsorbed antigens – Immunity to extracellular parasites, bacteria – Helper function in production of IgA, IgE, and neutralizing IgG to bacterial toxins
  118. 118. Adjuvants and Delivery Systems . Influence on Immune Response Adjuvant or Delivery System TH 2 Response (Antibody Production) TH1 Response (Cytotoxicity) Alum Chitosan CT or LT (+/+) CT or LT (mut) PLG Quil A/QS21 QS21 + MPL +++ +++ +++ + + + +++ +++ ++++ +++ +++ ++ ++
  119. 119. Adjuvants and Delivery Systems Influence on Immune Response Adjuvant or Delivery System TH2 Response (Antibody Production) TH1 Response (Cytotoxicity) IL-12 Live vectors Naked DNA CpG-ODN ++ + + + ++++ ++++ +++++ +++++
  120. 120. Preclinical Evaluation of Adjuvants Proof-of-concept testing • • • • Mechanism of action Effects on the different arms of the immune system Distribution. Local vs. Systemic effects Different components/combinations Safety • • • • General toxicity Inflammation/local effect Pregnancy Autoimmunity
  121. 121. Proof of Concept Testing Major areas • Physical presentation of the antigen in the vaccine • Optimisation of antigen uptake • Targetting to specific cells (dendritic cells, Langerhans cells, macrophages, and others) • Immune potentiation and modulation • intracellular transport and processing of antigens • association with MHC class I or II molecules • expansion of T-cells with different profiles of cytokine production
  122. 122. Pre-clinical Pharmacology of Adjuvant • Screening and optimization – Antibody titers alone can be misleading as don’t consider antibody function (i.e., avidity) – T cell responses complex to understand and kinetics brief • Selection of animal model – Testing anti-sera for functional antigen-binding capacity, opsonization etc. – Disease models: infectious challenge, allergy, cancer, • Points considered – Use of antigen and adjuvant doses and routes that can translate to humans – Use of animal species with physiological response to novel adjuvant similar to that known or expected in humans
  123. 123. Safety Testing of Adjuvants • General toxicity • Inflammation/local effect • Pregnancy - Th1/Th2 ratios vary during different stages of pregnancy - Biased Th1 responses during pregnancy have reported autoimmunity related risk disorders - Interference may result in defective placentation and pregnancy loss
  124. 124. Autoimmunity and Immunomodulatory Adjuvants Some animal data have suggested a link between vaccine/adjuvants and autoimmunity •Complete Freund’s adjuvants (mineral oil, mycobacterium) induces Experimental Allergic Encephalitis •Squalene (adjuvant component of AS03, MF59, AF03) can induce arthritis in rats and lupus in mice. •Holm, Lorentzen, 2004. Dark Agouti rats (arthritis-prone); Intradermal injection of 300 μl at the base of the tail. Satoh et al, 2003. Balb/c mice, i.p. squalene 0.5 ml. Induction of autoantibodies to cellular proteins
  125. 125. Formulation Development (2) • Analytics - must support required specifications and stability – Identity – Quantification – Purity – Characterization – Safety – Potency – Pyrogenicity 130
  126. 126. Formulation Development (3) • Formulation - antigen(s) + adjuvant(s) – Stability testing • On individual components and final formulation, different temperatures & durations • Inadequate stability data for complex formulation at time of Phase 1 trial may necessitate a “mix and shoot” or “bedside mixing” approach • Selection of buffers and excipients for DS and DP • Development of methods for freeze-thaw and lyophilization if required – Longer term stability requires compatibility of antigen-adjuvant with each maintaining their integrity • Dependent on solution conditions such as pH, buffer and other excipients – Considerations • Complexity of formulation • Dose and desired volume • Route • Desired presentation (i.e., vial, pre-loaded syringe) 131
  127. 127. Critical Challenges During Development What parameters ? Physicochemical characteristics Functional characteristics (non-clin) Which model ? Functional characteristics (clin) What effects ? Licensed product Safety (very rare and long term)
  128. 128. Development of Botanical Immunomodulators as Adjuvants for Improving Vaccine Efficacy A Collaborative Project under DST Drugs and Pharmaceutical Research Program Serum Institute of India Pvt Ltd (SIIL). & University of Pune(UoP) Research Team SIIL: Dr. S.S.Jadhav (PI), Dr. Sunil Gairola (Co-PI), Dr. K.Suresh, Dr. Yojana Shinde UoP: Dr. Bhushan Patwardhan (PI), Manish Gautam, Sanjay Mishra and Dada Patil
  129. 129. Chemical Adjuvants: Triterpenoid Saponins
  130. 130. Test material extraction and its Chromatographic characterization Safety studies as per OECD guidelines A • Th1/Th2 immune responses (Flowcytometric studies ) • Ag. specific study: Humoral & cellular immune response B Immunoadjuvant Study against diphtheria in host challenge model Immunoadjuvant Study with polysaccharide based vaccine antigen (T cell dependent antigen) (T cell independent antigen) Immunomodulatory Potential using SRBC’s  Th (CD4) and CTL (CD8) percentage  Th1:IFN-γ, IL-2 & Th2: IL-4 cytokines  Lym. proliferation: I) T- cells: CD3+ II) B - cells: CD19+  Humoral and Cellular immune response. C Immunoadjuvant Potential  VCA: Diphtheria toxin neutralizing Abs.  Total IgG level: Ab ELISA  Challenge associated Morbidity/ Mortality  Functional Ab. estimation: SBA  IFN-γ & IL-4 level (Th1/Th2 immunity)  IFN-γ & IL-4 level (Th1 and Th2  Sera cortisol level. immunity)
  131. 131. Immunomodulatory Study Key Trends….
  132. 132. Immunoadjuvant Potential ‘DPT Vaccine’ Trends… Effect of diphtheria challenge on percent survival 100 *** 90 ** V + ISHS-SIIL 3 V + ISHS-SIIL 2 70 V + ISHS-SIIL 1 60 50 Unimmunized Percent survival 80 Vaccine 1:160 40 30 20 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Days post challenge Challenge Sr. No. Humoral Protection Th1/Th2 ISHS-SIIL-1 ++ ++ Th2 (Toxoid based vaccines,viral vaccines) ISHS-SIIL-2 +++ +++ Th1/Th2 (Polysaccharide, recombinant & Toxoid based vaccines) ISHS-SIIL-3 ++ ++++ Th1 (Polysaccharide, recombinant and Tuberculosis, Malaria)
  133. 133. Immunoadjuvant potential ‘Men A vaccine’ Trends  IgG level: ISHS-SIIL-2 and 3 showed a significant increase of IgG levels as compared to Men A alone group. ISHS-SIIL-2 showed higher modulatory effect as compared to SIIL-3. ISHS-SIIL-2>SIIL-3>SIIL-1  SBA titres: ISHS-SIIL-2 and 3 showed significant increased SBA titers in Men. A immunized animals. ISHS-SIIL-3 showed higher SBA titer as compared to SIIL-2. ISHS-SIIL-3>SIIL-2.
  134. 134. SIIL-3 development and T cell independent antigens • Alternative to conjugation technology currently used in vaccine industry for T-cell independent antigens(Polysaccharide based vaccines) in inducing protective bactericidal antibodies. • CMC conforming to EMEA/WHO guidance on adjuvants • Pharmacokinetics of adjuvant was established when administered alone and along with antigen. SIIL-F4 with Standard adjuvants Total IgG levels (IU/ml) 900.00 800.00 700.00 600.00 500.00 400.00 300.00 200.00 100.00 0.00 Men A Poly. 5 ug Men A + AlPO4 Men A + MPL Men A + TMG Men A + QS21; 10 ug Men A + SIIL-F46.25 Men A CJ 1 ug
  135. 135. Vaccine Adjuvants • Vaccinated animals showed a significant increase antibody titers. • Significant reduction in mortality and morbidity was observed • Better efficacy/ safety over QS
  136. 136. Evaluation of SIIL-3 as vaccine adjuvant Comprehensive analysis: Microarray based Profiling Transcriptome, proteome, metabolome, micro RNAs 0 2 4 h Efforts to detect any biological responses at molecular level
  137. 137. Patents and Publications Patents      1247/MUM/2003. Process for manufacturing immunoactive extracts from medicinal plants for making vaccine adjuvants(Granted 2009) 1253/MUM/2003.A kit containing a vaccine and an immunological adjuvant (Granted 09) 1246/MUM/2003: Process for manufacturing immunoactive aqueous extracts (Granted 09) 1184/MUM/2009: Vaccine composition for improved efficacy (Granted 2010) US Patent on vaccine composition for improved efficacy of T cell independent antigens (Grant expected in 2013, two queries responses answered successfully since publication) in Year 2011).
  138. 138. Challenges • SIIL-F-4 needs further purification and chemistry support. • Up-scaling of process and GMP manufacture of adjuvant. • Advanced studies on pharmacodyanmics and immunotoxicity. • Synthetic routes to synthesize withanolides with correct conformations.
  139. 139. Encouraging Global Trends • WHO comprehensive regulatory guidance document on development of newer adjuvants in under publication. (For the first time, immunomodulatory adjuvants are included in ambit of adjuvant definition). • Nanoparticles based delivery systems needs immunomodulation for efficient mucosal delivery especially at distant sites. (examples of alum and QS-21 recently published with tetanus and malaria antigens). • WHO have launched a Global Adjuvant Development Initiative with a focus on identifying newer vaccine adjuvants for vaccines such as HIV, Malaria, tuberculosis, HPV, etc.
  140. 140. Summary  Newer advances in antigen discovery brings newer hopes for adjuvant adjuvant.  Immunomodulation emerging as central mechansim for adjuvant activity and Immunopharmacology based approaches will be important for newer adjuvant development and assessment  Safety assessment of immunomodulatory adjuvants will be challenging especially autoimmunity, preganancy.  Regulatory framework and science needs to be developed in order to cater to newer adjuvant development  WHO have taken an excellent initiative in this direction and guideline will play an important role in harmonization.  Indian Industry look forward to regulatory support on newer adjuvants.
  141. 141. Issues and Challenges for Development of Combination Vaccines: DPT Based Combination Vaccines as a Case Study Sunil Gairola, Ph.D. Serum Institute of India Limited
  142. 142. Guiding Principles for Introduction of Newer Vaccine – Disease burden – Quality of available vaccine – Affordability – Cost effective – Existence of a robust delivery system
  143. 143. Rationality • Practical way to overcome the constrains of multiple injections to infants and less distress to parents • Improve timely vaccination coverage and less visits to health facilities. • Reducing the cost of stocking and administering separate vaccines. • Reducing the cost for extra health care visits. • Facilitating the addition of new vaccines into immunization program. • May simplify transportation and storage problems (logistics)
  144. 144. Vaccine Types
  145. 145. Combination Licensed in Recent Years • • • • • • • • • • DTwPHib DTwPHB DTwPIPV DTwP-IPV-Hib DTwP-Hib-HB DTaP DTaP-IPV DTaP-Hib DTaP-HB-IPV DTaP-Hib-HB-IPV • • • • • • Hib-HB Rotavirus vaccine Hep A-HepB MMR Pandemic influenzae 23 valent Pneumococci vaccine • Quadravalent Men ACYW vaccine • HPV
  146. 146. Considerations for Combination Vaccines During Development • Product should be stable, each component of the combination vaccine should be given at the same age, and the requirement of booster for each component should be similar. • New combinations cannot be less immunogenic, less efficacious, or more reactogenic than the previously licensed uncombined vaccines. • Free from Immunological, physical, and/or chemical interactions between the combined components have the potential to alter the immune response to specific components. • Many advantages of combination vaccines should not be achieved at the cost of reduced product stability. • From supply standpoint, uncommon transport and storage conditions and complicated bedside mixing could hamper the development of a combination vaccine.
  147. 147. Challenges During Product Development • Formulation development • Immunological interferences- case studies • Manufacturing (scalability) • Analytical methods • Clinical • Regulatory
  148. 148. Formulation Development Development of combination vaccine formulation may need consideration of following: - Antigen-Antigen or Antigen-adjuvant interaction antigens Displacement of antigen from adjuvant leading to reduced immunogenicity. - Differential requirement of each antigen with respect to achieving consistent adsorption -- - pH, selection of excipients and stabilizers adjuvants Similarly buffers, stabilizers and similar components may interfere with the components of the other vaccine. - Scalability of the process - pH preservatives excipients stabilizers Scalability Choice of preservatives Any decision on the considerations is based on impact on Quality aspects including stability and shelf life , Safety (reactogenicity), Efficacy, Finally scalability of the process
  149. 149. Immunological Interferences • Candidate Formulations are studied for appropriate immunogenicity in an animal model. • Immunogenicity induced by the combination vaccine was compared with that induced by the separate but simultaneously administered individual vaccines. • If there was an already licensed combination, then the current licensed formulation has to be used in the control group for comparisons of immunogenicity • The immune response to each of the antigens in the vaccine is assessed, including – the quality of response, – the potential interference and – Incompatibilities between combined antigens.
  150. 150. Case study 1: Immune Interference of Hib antigen with Acellular Pertussis and Tetanus Antigens • Reduction in antibody titers to the Hib component of the vaccine polyribosylribitol phosphate antigen. This has been reported for many DTaPbased vaccines, including the hexavalent vaccine DTaP-HBV-IPV/Hib. • The interference has not been reported to the same extent for DTwP-based combination vaccines. • It has been suggested, due to the adjuvant effect of the whole-cell pertussis (wP) component, this effect is masked in DTwP based vaccines. • Studies in a rat model looking at the interference of Hib and different aP antigens - Reduced anti-PRP response with FHA is in line with the finding that it is a potent suppressor of IL-12 and IFN-γ production in vivo and in vitro, suppressing immune responses to co-injected antigens. - Another explanation for the reduced Hib response when combined with DTaP vaccines is incompatibility with the alum adjuvant (aluminum hydroxide). Experiments in the rat model with Hib alone have reported 5- to 11-fold lower levels of anti-PRP antibodies when adsorbed to aluminum hydroxide adjuvant.
  151. 151. Challenges of Combination Vaccines-formulation • When different antigens are combined into one vaccine, chemical incompatibility or immunological interference can be seen. Case study 1: Immune suppression Case study 2: Immune enhancement
  152. 152. Impact of Antigen Concentration on Potency Parameter (Product: DTPHBHIB) Lot Diphtheria (Lf/dose) Diphtheria Potency (IU/Dose) Tetanus (Lf/dose) Tetanus Potency (IU/Dose) A 25 31.185 (18.455 – 45.67) 7.5 High Survival B 25 37.1323 (22.3396 – 53.0642) 7.5 High Survival C 25 75.745 (52.485 – 105.195) 5.0 High Survival D 25 74.140 (48.525 – 116.125) 5.0 High Survival E 25 85.045 (57.45 – 123.0) 2.5 62.425 (43.809 – 99.266) F 25 56.315 (36.675 – 80.115) 2.5 70.46 (44.48- 114.645) G 25 97.365 (64.40 – 143.76) 2.5 68.71 (41.745 – 109.96) H 25 87.21( 61.285 – 123.515) 4.0 78.84 (57.84 – 108.26) I 25 92.575 (65.34 – 130.63) 4.0 96.77 (72.125 – 132.29) J 25 106.065 (65.655 – 163.32) 4.0 98.85 (65.565 – 152.885) Tetanus antigen when added at 5 to 7.5 Lf/dose resulted in high survival leading to absence of end point in potency assay (WHO challenge method for tetanus component). Tetanus antigen when added at 7.5 Lf/dose also resulted in suppression of immune response against diphtheria component resulting in lower potency estimates.  Tetanus antigen at 2.5 Lf/dose showed borderline conformance to 95 % CI Tetanus at 4.0 Lf/dose concentration for tetanus antigen produced optimum results and hence selected.
  153. 153. Effect of HIB Conjugate (TETANUS TOXOID AS CARRIER PROTEIN) on Tetanus Potency in Combination Vaccine Potency (IU/dose) With 95 % CI 24.619 ( 14.7180 -39.0347) 96.77 (72.125-132.29) 98.85 ( 65.565 – 152.885) 4L f DT PH BH IB IB BH /d os e /d os e 4L f /d os e e 4L f DT PH H /d os 4 IB DTPHBHIB BH 4 e DTPHBHIB Lf 85.20 (57.84-108.29) DT PH 4 /d os DTPHBHIB 7. 5 76.255 ( 53.99-106.92) DT P 7.5 e DTP 0 Lf 72.350 ( 46.06 -111.4) 7. 5 7.5 /d os DTP Lf 69.20 ( 45.305-110.45) DT P 7.5 3 DTP 50 IB 18.4641 (9.6346-29.6911) 7. 5 Carrier protein H Hib conjugate 100 DT P 21.0581 (14.5986-31.7399) 2 Carrier protein IB Hib conjugate Profile of tetanus potency H Carrier protein 1 Hib conjugate IB Tetanus Lf/dose Potency T component (IU/SHD) Vaccine 0 Potency of T component in DTPHBHiB formulation is considerably high as compared to DPT formulation even at 4Lf/dose. This might be due to Hib-TT conjugates which contributed up to 18-24 IU/ml of tetanus potency.
  154. 154. Immunogenicity of Hib-Tetanus Conjugate Immunogenicity of Hib-TT conjugate in presence of other antigens (wP) in rats Sr. No 1 2 Formulation Hib monovalent vaccine (Reconstituted with Non-adjuvanted diluent) DTPHBHib liquid vaccine IgG GMT of Anti Hib Titres (ELISA) 1007.9 5971.40  The presence of adjuvant and a stabilized formulation demonstrate a significant difference in IgG response in pentavalent vaccine with Hib-TT component.
  155. 155. Role of Adjuvants • Method of adjuvant preparation - Insitu or preformed • Method of adsorption - Sequential or simultaneous • Adsorption profile -Kinetics and maturation of adsorption process • Studies on desorption: - Stability of adsorbed antigens
  156. 156. Antigen Adjuvant Interactions During Different Stages of Formulation • Adjuvant can be readymade gel or insitu preparation of adjuvant. • Adsorption of antigens can be significantly change during the process of blending. •It is generally noted that every antigen have special requirements for adsorption for instance at lower pH, D and T antigens are tightly adsorbed. •Monovalent Hepatitis B is 99% adsorbed whereas, in combination vaccines, the adsorption is decreased due to competition. •Addition of Hib to combination blend needs to be monitored for stability of conjugate.
  157. 157. Manufacturing Challenges / Vessels • Combination vaccine can be blended in variety of blending vessels such as – Vessels with vibromixer • Amplitude of operation? – Vessels with magnetic stirrers – Vessels with magnetic stirrers and baffles. • RPM? • Careful assessment has to be done so that the antigens are not denatured based on the vessel type due to shear forces.
  158. 158. Challenges-Regulatory • Case example: Suppose a manufacturer has licensed and qualified DTPHepB and also have monovalent Hib vaccine qualified. What will be regulatory implications if manufacturer decides to develop pentavalent vaccine(DTPHibHepB) formulation:  If a manufacturer has monovalent vaccines licensed, the combination is considered as new product.  As a new product, the combination vaccine has to undergo preclinical and clinical trials. Each stage is governed by regulatory submission (preclinical, phase I, II and III tials).  Inspection of manufacturing facilities by national regulatory authorities and license to manufacture is provided.  Separate regulatory pathways if one of the antigen is recombinant  This escalates into huge development costs and big increase in time lines.  If manufacturer decides to keep monovalent, quadra or penta combinations, product efficacy equivalence needs to be proven.
  159. 159. Analytical Challenges • The assays that are developed for determining the potency in monovalent vaccine may not work for combination vaccine. • New assay methods have to be developed and validated. • • • • • • • For eg,In DTPHBHibTetanus Lf cannot be determined by flocculation methods. ELISA has to be developed for same. The free polysaccharide assay in monovalent Hib is done by Orcinol. In DTPHBHib, the same assay cannot be adopted, instead, an alternate instrumental assay is used, making the assay more expensive. WHO is conducting a collaborative study to harmonize Hib assay in pentavalent vaccine, wherein, Serum institute is one of the participant. In meningococcal conjugate vaccines, the individual polysaccharide or conjugates can be analyzed by either phosphate or sialic acid assays (colorimetric methods). In combination of Men ACYW, the colorimetric assay does not help. An alternate instrumental assays are employed making the assays more complicated and expensive (Dionex AEC-PAD).
  160. 160. Challenges - References • • • • • International or National references are not available for combination vaccines, though combinations are available for more than 50 years. For determining potency assays, monovalent standards are used, which may be responsible for enhanced or suppressed results due to interference of antigens. Way to create an in-house reference is to chose the clinical trial vaccine with proven efficacy, can be calibrated as internal reference std. New standard can be generated by calibrating against the clinical std. Criteria of adsorption is informative but adsorption of all subsequent lots should not be less than the clinical trial lot.
  161. 161. Clinical Immunogenicity Regulatory expectations • Immunogenicity induced by the combination vaccine was compared with that induced by the separate but simultaneously administered individual vaccines. • If there was an already licensed combination, then the current licensed formulation has to be used in the control group for comparisons of immunogenicity • For each component of a combination vaccine, non-inferiority had to be demonstrated against the licensed combination. Challenges • Such studies had to have sufficient power to rule out clinically meaningful differences in GMTs and/or seroconversion rates • Intrinsic variability in assays and subjects, the regulators may not take them into account
  162. 162. Case example-Selection of Concentration of Antigens DTP-HB-Hib with 2.5 mcg Hib • Compared for immunogenicity against licensed DTP-HB-Hib vaccine. (A multi-centric, randomized Phase III clinical trial) • 100 % seroconversion was observed with respect to Hib (≥ 0.15 ug/ml of IgG). • The non-inferiority criteria was met for all components except Hib at ≥ 1ug/ml of IgG (87 % versus 93 % in 10 mcg/dose comparison to comparator. Hence long term protection could not be established. • Another clinical trial was taken with new formulation with increased concentration of Hib antigen (10 mcg/dose).
  163. 163. LABELLING: Primary Container REQUIREMENTS • • • • • • • • • • • • • • • Product generic name and brand name if applicable. Total number of ml in container (liquid) and number of doses in container (freeze-dried). Units/dose or per ml or minimum titer. Dose and route of administration. Nature and amount of any preservative present. Storage condition. Warning/instructions if any i.e. Not to be frozen, shake well. Statements for reconstitution, photo sensitivity, etc. Manufacturing licence number. Expiry date. Name and address of manufacturer. Vaccine Vial Monitor (VVM) if applicable on label for liquids. Visual cue if any. Overprinting / additional information if any. Barcodes if any. CHALLENGES  Label size/space constraint.  Font size/text legibility.  VVMs: 7 x 7mm minimum area required, Visual cues: 5 x 5 mm.  Barcodes: 12 x 12 mm (2D barcode). To accommodate all the above is difficult on the primary label
  164. 164. Summary • Combination vaccines offers opportunities, however development path is complex and challenging. • Increased reactogenicity of combination vaccines is not accepted. Suitable preclinical correlates for toxicity assessment is not available for most of the antigens. In other words, prediction of toxicity or reactogenicity during preclinical assessment is challenging. • Immune interference is a phenomenon in combination vaccines. Needs excellent study designs to predict the same. • Analytics of final lot especially detection and quantification of antigens in combination vaccine is challenging. Several technologies such as label free ELISAs, platforms such as Gyros, MSD, Luminex offers oppurtunities. • Reference standard for evaluation of combination vaccines: (monovalent versus multivalent standard) • Clear guidelines on usage of in vivo potency tests during stability testing: (w.r.t to testing intervals, number,etc)