1. Preparation of Reference Influenza Viruses in Mammalian Cells: FDA Perspective Prepared for the Vaccines and Related Biological Products Advisory Committee February 18-19, 2004 Zhiping Ye, M. D., Ph.D. Center For Biologics Evaluation and Research, Division of Viral Products

2. Objectives To review the issues associated with the source of reference influenza viruses To discuss possible strategies for using mammalian cell lines to prepare influenza reference viruses

3. Background Since the 1940s, inactivated influenza virus vaccines have been prepared in embryonated eggs, which provide reasonable virus yields The candidate vaccine viruses are originally isolated in surveillance labs using eggs as opposed to mammalian cells (a global consensus within WHO, national regulatory authorities and manufacturers) Over time, many surveillance labs have come to rely on cell cultures for virus isolation, for reasons of robustness and convenience, even though such isolates are not used as reference viruses

4. Background (cont.) Isolation of circulating viruses from clinical samples in National Influenza Centers (surveillance labs ) Cell culture (Not qualified) - MDCK cells (majority of surveillance labs) - Vero cells (some labs) - Chicken embryo kidney cells (few labs) Embryonated eggs (Few labs)

5. Isolation Rates of Influenza Viruses From Clinical Samples (Govorkova, et al., 1996, J. Virol., 70:5519)

6. Background (cont.) Yearly strain selection can be negatively affected by the requirement for an egg isolate as seed virus –Delayed recommendation for vaccine composition –Altered recommendation for vaccine strain component because of availability A delay in the availability of strain selection during a pandemic situation could have disastrous consequences.

7. Influenza Virus Vaccines (licensed in USA) Inactivated Influenza Virus Vaccines –Commercially available for more than 50 Years in USA –Produced in eggs –Parenteral injection Live Influenza Virus Vaccine –Recently licensed - 2003 –Produced in SPF-eggs –Intranasal inoculation

8. Influenza Virus Vaccines (in development) Cell based- inactivated vaccines –MDCK –VERO Others: –Subunit DNA (Plasmid/bacteria) –Subunit Protein (Insect cells)

9. Reference and Seed Virus Development WHO global surveillance systems (National Influenza Centers) –Provide reference viruses isolated from clinical samples using eggs WHO collaborating centers (Atlanta, London, Melbourne and Tokyo) –Characterize and provide reference viruses (high growth reassortants for type A) to manufactures

10. Reference and Seed Virus Development (cont.) Manufacturers –Develop proprietary seed viruses from egg-isolates recommended by WHO and local authorities Regulatory agencies –Accept reference viruses and approve seed viruses for use in licensed vaccine preparation

11. Reference and Seed Virus Development (Summary) NIC (Egg-isolate) WHOCC (Vaccine candidate) RegulatoryManufactures Ref. Seed Ref. Seed Ref.

12. Use of Eggs for Isolation of Influenza Seed Strains A safety track record of more than 50 years Possible exclusion of some adventitious agents from clinical specimens Leads to high yields in vaccine manufacturing Validated for inactivation of common chicken viruses in vaccine process

13. Pros and Cons for the Consideration of Mammalian Cell Lines for Isolation of Seed Strains Pros –More robust than eggs for primary isolation of influenza A viruses from clinical specimens –Less selective pressure than in eggs for isolation of influenza virus with alternative receptor specificity –Ready availability in many WHO global surveillance centers (MDCK cell lines)

14. Pros and Cons for the Consideration of Mammalian Cell Lines for Isolation of Seed Strains Cons –Limited experience in influenza vaccine development and production (e.g. isolates may not necessarily grow well in eggs) –Issues related to the use of mammalian cells for isolation of seed strains (e.g. the possible contamination of adventitious agents)

15. Regulatory Requirements and Guidance Related to the Use of Mammalian Cell Lines 21 CFR 610.18 (c) Cell lines used for manufacturing biological products (1) General requirements –Identified by history –Described with respect to cytogenic characteristics and tumorigenicity –Characterized with respect to in vitro growth characteristics and life potential –Tested for the presence of detectable microbial agents

16. Regulatory Requirements and Guidance Related to the Use of Mammalian Cell Lines (Cont.) Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals –Published in 1993; currently under revision by Office of Vaccines Research and Review –Provides additional guidance on appropriate standards for characterization and qualification of cell substrates and viral seeds

17. Additional Regulatory Requirements and Guidance Related to the Use of Mammalian Cell Lines WHO Technical Report Series, #878 (Requirements for Biological Substances N o 50-1998) ICH Guidance, Q5A (Viral Safety Evaluation of Biotechnology Products) EMEA Guidance, CPMP/BWP/2490/00 (Cell Culture Inactivated influenza Vaccine-2000)

18. Strategies to Consider for the Use of Mammalian Cell-Isolates as Reference Viruses 1.Retain the clinical samples after initial isolation in NIC and forward the samples to select WHOCC for re-isolation in a qualified cell line –Similar to the current procedure except for the use of cell lines for re-isolation –An appropriate cell bank would need to be produced and qualified for distribution to select centers –Further purification or adaptation of cell-isolates for vaccine production in eggs could be considered if necessary

19. Strategies to Consider for the Use of Mammalian Cell-Isolates as Reference Viruses (Cont.) 2.Use reference viruses directly isolated on cell lines not characterized or qualified –Would require purification of reference viruses to be acceptable (e.g., plaque purification on SPF-CEK cells) –Resources and infrastructure for purification would be necessary

20. Strategies to Consider for the Use of Mammalian Cell-Isolates as Reference Viruses (Cont.) 3.Use reference viruses isolated on characterized and qualified cell lines –Would require production and qualification of an appropriate cell bank for distribution to NIC choice of cell lines (MDCK, Vero, others?) maintenance of quality control after distribution –Further purification or adaptation of cell-isolates for vaccine production in eggs could be considered if necessary

21. Strategies to Consider for the Use of Mammalian Cell-Isolates as Reference Viruses (Cont.) 4.Use reverse genetics methodology and a qualified cell line to generate new reference viruses –Would require production and qualification of an appropriate cell bank for reverse genetics –May eliminate concerns regarding contaminates regardless of the source of the sample –Acceptance of technology and intellectual property issues may not permit near-term implementation

22. Conclusions The use of influenza seeds obtained from non-egg sources may be acceptable if concerns regarding the presence of adventitious agents in the original isolate or in the cell lines used for isolation can be addressed An investment in resources and infrastructure will be necessary A global consensus (WHO, national regulatory authorities, manufacturers) regarding the issues to be addressed would facilitate implementation

23. Acknowledgments Jerry Weir Roland Levandowski Andrew Lewis Keith Peden Arifa Khan Philip Krause