Transcript Use of MDCK Cells for Manufacture of Inactivated Influenza

Use of Madin-Darby Canine Kidney
(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
• Summary of scientific concerns regarding
use of neoplastic cell substrates
• Brief description of the plan for today’s
meeting
Definitions
• Neoplastic cells (continuous cell lines)
– 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
– Highly tumorigenic cells- form tumors at low cell
doses, or form metastatic tumors
– Weakly tumorigenic cells- form tumors only at higher
cell doses
Cell substrates used for vaccine
production
• Primary cells & Tissues (1954)
– Calf lymph for smallpox vaccines
– Monkey kidney 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 replicationdefective recombinant vaccines (early 2000s)
History of MDCK cells
• Multiple, relatively independent derivatives of
the MDCK cell line have been described
1958, MDCK established from
healthy female cocker spaniel
MDCK, ATCC
1963 MDCK-USD
From Gaush, PSEBM, 1996 122:931
1961 MDCK-NIH
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
Issues with MDCK Cell Substrates
• Original line of MDCK cells was non tumorigenic
• Some MDCK derivatives have been found to be
highly tumorigenic
• Highly tumorigenic cell substrates have never
been used to manufacture viral vaccines
• Highly tumorigenic cell substrates pose significant
regulatory challenges
Introducing tumorigenic cell lines
for vaccine development
• Expands the repertoire of neoplastic cells
that can be used in development of new
vaccines, including:
– genetically engineered viral vectored vaccines
– HIV vaccines
– Influenza vaccines
• annual
• pandemic
Why are there concerns about
tumorigenic cells?
• Potential for increased risk of adventitious
agent contamination
• Potential for increased risk associated with
residual DNA
• Potential for increased risk associated with
virus/cell interactions
• Potential for other increased risks?
• Perception of increased risk
The next slides will describe the
past 10 or so years of CBER
thinking about introduction of
neoplastic cell substrates
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 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 inactivated poliovirus vaccines
– Stringent limitations on DNA content
– Used only for inactivated vaccines
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
11/19/1998 VRBPAC:
Initial Discussion with Committee
• Committee recommended:
– 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
– Research to provide scientific foundation for decision-making
regarding use of neoplastic cells in vaccine manufacture
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” as a conceptual
framework for considering the issues
Defined risks approach
• Represents an attempt to establish, where possible, a
quantitative conceptual framework for estimating
upper bounds on potential risks
– 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
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
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
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
VRBPAC 2001: Issues/Topics on in vitrotransformed 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
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
• Discussed importance of minimizing steps (i.e.
initiation events) toward oncogenesis in vaccine
recipients (even if oncogenic outcome is not directly
correlated with use of neoplastic cells, it is important
to assure that vaccine recipients are not “primed”)
VRBPAC 2001: PER.C6 and 293 cells for
vaccine manufacture (continued)
• Discussed adenovirus E1 gene:
– low likelihood of uptake in a significant number of cells
– involvement in apoptosis
– unlikelihood of reaching tumor cell threshold dose
necessary for clinical impact
• Discussed whether degree of tumorigenicity was
important
– varying opinions
• Discussed approach to TSE issues in neoplastic or
retinal cells
• Conclusion that these cells could be used for
manufacture of replication defective adenovirus
vaccines, with appropriate limitation on residual
DNA.
The next slides will
summarize the concerns that
may be associated with
introducing new neoplastic
cell substrates
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 xenograft rejection mechanisms
– Addressed by assuring (via validated methods)
absence of intact cells in final product
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
• Some viruses are known carcinogens in animals and humans
– 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-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
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
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
• In vivo testing of cell lysates
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
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
Today’s talks
• 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
• Manufacturers
– Chiron
– Solvay