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

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Use of MDCK Cells for Manufacture of Inactivated
Influenza VaccinesIntroduction

• Philip R. Krause, M.D.

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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 todays meeting

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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

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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)

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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

• 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 122931
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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

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Using tumorigenic cells for vaccine development

• 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

• 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 VRBPACInitial 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
EvolvingScientific 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

• 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

• 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 in
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

• 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(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

• 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

• 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

• 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

• 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

• 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

• 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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Todays 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