Use of mdck cells for manufacture of inactivated influenza vaccines introduction
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Use of MDCK Cells for Manufacture of Inactivated Influenza Vaccines: Introduction. Philip R. Krause, M.D. Outline. Definitions and brief introduction to the issues Recent history of thinking about neoplastic cell substrates

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Use of mdck cells for manufacture of inactivated influenza vaccines introduction l.jpg

Use of MDCK Cells for Manufacture of Inactivated Influenza Vaccines:Introduction

Philip R. Krause, M.D.


Outline l.jpg
Outline Vaccines:

  • 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


Definitions l.jpg
Definitions Vaccines:

  • 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


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Cell substrates used for vaccine production Vaccines:

  • 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)


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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


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History of MDCK cells of inactivated influenza vaccines?

  • Multiple, relatively independent MDCK cell lines have been described

1958, MDCK established from healthy female cocker spaniel

MDCK, ATCC

1963 MDCK-USD

1961 MDCK-NIH

From Gaush, PSEBM, 1996 122:931


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Issues with MDCK Cell Substrates of inactivated influenza vaccines?

  • 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


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Using tumorigenic cells for vaccine development of inactivated influenza vaccines?

  • Permits the development of new vaccines, including:

    • genetically engineered viral vectored vaccines

    • HIV vaccines

    • Influenza vaccines

      • routine

      • pandemic


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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


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


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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


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VRBPAC discussions regarding neoplastic cell substrates biologicals

  • 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


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11/19/1998 VRBPAC: biologicalsInitial 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


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September 1999: International Meeting: Evolving biologicalsScientific and Regulatory Perspectives on CellSubstrates 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


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Defined risks approach biologicals

  • 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


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1999 meeting: Scientific conclusions biologicals

  • 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


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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


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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


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VRBPAC 2001: Issues/Topics on for vaccine manufacturein vitro-transformed neoplastic cells to produce replication-defective vaccines

  • 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


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VRBPAC 2000: PER.C6 and 293 cells for vaccine manufacture 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


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VRBPAC 2001: PER.C6 and 293 cells for vaccine manufacture for vaccine manufacture(continued)

  • Discussed whether degree of tumorigenicity was important

    • varying opinions

  • Discussed approach to TSE issues in neoplastic or retinal cells


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


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Concerns regarding use of neoplastic or tumorigenic cells-1 associated with introducing new neoplastic cell substrates

  • 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


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Concerns regarding use of neoplastic or tumorigenic cells-2 associated with introducing new neoplastic cell substrates

  • 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


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Concerns regarding use of neoplastic or tumorigenic cells-3 associated with introducing new neoplastic cell substrates

  • 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


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Concerns regarding use of neoplastic or tumorigenic cells-4 associated with introducing new neoplastic cell substrates

  • 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


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Concerns regarding use of neoplastic or tumorigenic cells-5 associated with introducing new neoplastic cell substrates

  • 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


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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


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Goals for this meeting cells

  • 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


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Today’s talks cells

  • 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


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