Title: Use of Madin-Darby Canine Kidney (MDCK) Cells for Manufacture of Inactivated Influenza Vaccines: Introduction
1Use of Madin-Darby Canine Kidney (MDCK) Cells for
Manufacture of Inactivated Influenza
VaccinesIntroduction
2Outline
- 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
3Definitions
- 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
4Cell 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 replication-defective recombinant
vaccines (early 2000s)
5History 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
1961 MDCK-NIH
From Gaush, PSEBM, 1996 122931
6Why 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
7Issues 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
8Introducing 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
9Why 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
10The next slides will describe the past 10 or so
years of CBER thinking about introduction of
neoplastic cell substrates
11Neoplastic 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
12VRBPAC 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
1311/19/1998 VRBPACInitial 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
14September 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 as a
conceptual framework for considering the issues
15Defined 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
161999 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
17VRBPAC 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
18VRBPAC 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
19VRBPAC 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
20VRBPAC 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)
21VRBPAC 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.
22The next slides will summarize the concerns that
may be associated with introducing new neoplastic
cell substrates
23Concerns 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
24Concerns 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
25Concerns 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
26Concerns 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
27Concerns 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
28Concerns 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
29Goals 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
30Todays 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