1. Use of MDCK Cells for Manufacture of Inactivated Influenza Vaccines: Introduction Philip R. Krause, M.D.

2. Outline Definitions and brief introduction to the issues Recent history of thinking about neoplastic cell substrates Summary of scientific concerns regarding use of neoplastic cell substrates Brief description of the plan for today’s meeting

3. Definitions Neoplastic cells –Cells that are immortalized and can progress along the pathway to tumor formation Tumorigenic cells –neoplastic cells that form tumors when injected into susceptible animals

4. Cell substrates used for vaccine production Primary cells & Tissues (1954) –Calf lymph for smallpox vaccines –AGMK cells for polio vaccines –Embryonated hens’ eggs for influenza, yellow fever vaccines –Chicken embryo cell culture for measles, mumps vaccines –Mouse brain for inactivated JEV vaccine Human diploid cells (introduced in 1960s) –MRC-5, WI-38 for rubella, varicella vaccines CHO cells for highly purified, subunit investigational vaccines (1980s) Vero cells at non-tumorigenic passages for highly purified, inactivated vaccine (IPV) (1980s) Vero cells at non-tumorigenic passages for live-attenuated vaccines (late 1990s) In vitro transformed human cells (e.g., 293, PER.C6) for defective vaccines (early 2000s)

5. Why are MDCK Cells being considered for use in manufacture of inactivated influenza vaccines? Virus growth advantage More rapid scale-up Ability to bank & thoroughly characterize cells Adaptation to serum-free growth Manufacturers will provide more detail

6. History of MDCK cells Multiple, relatively independent MDCK cell lines have been described From Gaush, PSEBM, 1996 122:931 1958, MDCK established from healthy female cocker spaniel MDCK, ATCC 1963 MDCK-USD1961 MDCK-NIH

7. Issues with MDCK Cell Substrates Original line of MDCK cells was non tumorigenic Some MDCK sub lines are highly tumorigenic (1- 100 cells form tumors) Highly tumorigenic cell substrates have never been used to manufacture viiral vaccines Highly tumorigenic cell substrates pose significant regulatory challenges

8. Using tumorigenic cells for vaccine development Permits the development of new vaccines, including: –genetically engineered viral vectored vaccines –HIV vaccines –Influenza vaccines routine pandemic

9. General evaluation of risks associated with producing vaccines in tumorigenic cells includes: Consideration of possible transmissibility of tumorigenic or oncogenic potential from cell substrate residues present in vaccines –Requires consideration of mechanisms by which cells may become tumorigenic and whether this phenotype can be transmitted Consideration of possible activation or transmissibility of tumorigenic or oncogenic potential by vaccine viruses produced in tumorigenic cells

10. The next slides will describe the past 10 or so years of CBER thinking about introduction of neoplastic cell substrates

11. Neoplastic cells in use in 1995 for production of biologicals Namalwa cells for interferon Rodent cells for monoclonal antibodies (hybridomas), therapeutic proteins (CHO and BHK), including CHO cells for investigational protein subunit vaccines –These cells are generally weakly tumorigenic –Non-infectious retroviruses are present –High amounts of viral elimination/inactivation (clearance) are required at least 6 logs clearance in excess of known retrovirus burden; can generally only be demonstrated with multiple “orthogonal steps” Vero cells, at non-tumorigenic passages, used for production of IPV –Stringent limitations on DNA content –Used only for inactivated vaccines

12. VRBPAC discussions regarding neoplastic cell substrates Based on the premise that full public discussion of transition to use of neoplastic cell substrates is important 1998: Initial discussion with committee 1999: International cell substrate meeting & report to VRBPAC 2000: Discussion of the use of Vero cells 2001: Discussion of 293 and PER.C6 cells

13. 11/19/1998 VRBPAC: Initial Discussion with Committee Committee recommended: –Research to provide scientific foundation for decision-making regarding use of neoplastic cells in vaccine manufacture –Development of a document describing a proposed approach to addressing use of neoplastic cells in vaccine manufacture –A workshop to obtain public discussion of this document and additional scientific input on these issues –Continued dialogue with the advisory committee

14. September 1999: International Meeting: Evolving Scientific and Regulatory Perspectives on Cell Substrates for Vaccine Development Sponsored by: CBER, IABS, NIAID, NVPO, WHO Summarized at 9/14/99 VRBPAC Goals: –Identify concerns & issues –Identify approaches to determine levels of risk associated with those issues Discussion of CBER document prepared in response to 11/98 VRBPAC –Presentation of Defined-Risks Approach

15. Defined risks approach Identifying the possible risk event Estimating or determining the frequency with which the risk event might occur or has been observed to occur, either in nature or under experimental conditions Estimating the possible frequency of the risk event per dose of vaccine Developing and determining the sensitivity of one or more assays that can be used to detect the risk event Developing and validating one or more processes that can be used to establish a product-specific safety factor

16. 1999 meeting: Scientific conclusions Multi-factor nature of carcinogenesis suggests very low risk of oncogenicity from cellular components other than oncogenic viruses Unrecognized adventitious agents may be the major concern with neoplastic cell substrates –Primary cells present greater risks for adventitious agents than neoplastic cells Risks from residual DNA were perceived to be low, although there is a need for more scientific data to verify this perception Virus cell interactions: –Risk must be considered based on specific virus/cell substrate combinations, and any selective pressures in the cell culture system Concern was raised that neoplastic cells could contain abnormal PrP genes, of unclear significance Designing cell substrates using defined mechanisms of transformation should be considered

17. VRBPAC 2000: Issues/Topics regarding Vero cell use for vaccine manufacture Vero cells –Non-tumorigenic, but have capacity to become tumorigenic upon repeated passage –Mechanism of transformation is unknown –Substantial experience exists using Vero cells in research and diagnostics –High level of testing detected no evidence for the presence of adventitious agents

18. VRBPAC 2000: Conclusions/Recommendations on Vero cell use for vaccine manufacture Importance of assuring removal of intact cells from vaccine More concern about parenteral than mucosal vaccines produced in Vero cells Significant concern expressed about use of Vero cells at tumorigenic passage levels Some members expressed concern about using cells with the potential to become tumorigenic Limit DNA to 10 ng for vaccines produced in Vero cells at non-tumorigenic passages

19. 293, PER.C6 cells for gene therapy products, investigational live adenovirus vectored vaccines –These cells allow replication of defective adenovirus vectors (PER.C6 designed to minimize RCA formation) –Defined mechanism of transformation (Ad5 E1) –These cells are weakly tumorigenic –Extensive testing detected no evidence of the presence of adventitious agents VRBPAC 2001: Issues/Topics on in vitro- transformed neoplastic cells to produce replication-defective vaccines

20. VRBPAC 2000: PER.C6 and 293 cells for vaccine manufacture Discussed value of these cells for manufacturing vectored viral vaccines Discussed role of known mechanism of transformation- including some skepticism that this provided a clear safety margin Importance of minimizing steps toward oncogenesis in vaccine recipients (i.e, initiation events; even if oncogenic outcome is not directly correlated with use of neoplastic cells, it is important to assure that vaccine recipients are not “primed”) For E1: very low likelihood of uptake in a significant number of cells; effect of apoptosis; unlikelihood of reaching tumor cell threshold dose of more than 1 million cells

21. VRBPAC 2001: PER.C6 and 293 cells for vaccine manufacture (continued) Discussed whether degree of tumorigenicity was important –varying opinions Discussed approach to TSE issues in neoplastic or retinal cells

22. The next slides will summarize the concerns that may be associated with introducing new neoplastic cell substrates

23. Tumorigenic cells may form tumors if transferred to a recipient –Has been reported with human cells –Unlikely if cells are non-human, due to immunological tumor-rejection mechanisms –Addressed by assuring (via validated methods) absence of intact cells in final product Concerns regarding use of neoplastic or tumorigenic cells-1

24. Special considerations regarding adventitious agents –Adventitious agents that may have induced the original neoplastic or tumorigenic phenotype may be present in the cells Viruses are well known to cause cancer in animals –Neoplastic or tumorigenic cells may have expanded capacity to support viral replication, and thus be more likely to contain agents –Addressed to date by: Limiting use of tumorigenic cells to investigational inactivated vaccines for which high levels of purification is performed Expanded testing for oncogenic and other agents Concerns regarding use of neoplastic or tumorigenic cells-2

25. Residual DNA from tumorigenic cells may be infectious or oncogenic –Addressed to date by: In vivo oncogenicity testing on cell substrate DNA Limitation on quantity of DNA Limitation on biological function (i.e., size, other properties) of any residual DNA Concerns regarding use of neoplastic or tumorigenic cells-3

26. Virus-host and Virus-cell interactions: Vaccine virus may package cell DNA or incorporate cell elements that could be oncogenic, thus limiting ability to eliminate these theoretically oncogenic agents from vaccines –Addressed to date by: Demonstration that final vaccine preparations do not contain transforming DNA Not an issue for cytoplasmic RNA viruses like influenza In some cases, inactivation of viral vaccine Concerns regarding use of neoplastic or tumorigenic cells-4

27. Some other mechanism (e.g., oncogenic proteins, RNAs, or other factors that could induce heritable epigenetic changes) associated with immortalization or tumorigenicity could present a risk to the recipient of a vaccine manufactured in tumorigenic cells –Addressed to date by scientific consensus that other such mechanisms are very unlikely use only of weakly tumorigenic cells Concerns regarding use of neoplastic or tumorigenic cells-5

28. Concerns regarding the tumorigenicity testing of neoplastic cells Previously used tumorigenicity assays may not adequately define the tumorigenic phenotype or the risk associated with use of tumorigenic cells

29. Goals for this meeting Discussion of the use of MDCK cells, including those that are highly tumorigenic, in manufacture of inactivated influenza vaccines Discussion of OVRR approach to evaluate the safety of tumorigenic cells for use in vaccine production Discussion of any additional steps CBER should take to address issues associated with the use of neoplastic cell substrates

30. Today’s talks Manufacturers –Chiron –Solvay Andrew Lewis: Regulatory implications of neoplastic cell substrate tumorigenicity Arifa Khan: Adventitious agent testing of novel cell substrates for vaccine manufacture Keith Peden: Issues associated with residual cell substrate DNA