Cell
culture-grown influenza vaccines have certain advantages over the traditional
egg-based vaccines, such as higher virus yields, the potential for more rapid
scale-up of vaccine manufacture, the ability to bank and thoroughly
characterize cells used in vaccine production, and the potential for adaptation
to serum free growth. One cell
substrate that is being proposed for use in the US for influenza vaccines is
the Madin Darby canine kidney (MDCK) cell line.
Manufacturers
of influenza vaccines have taken note of the potential advantages of using MDCK
cells in vaccine production. MDCK cells
are a continuous (immortal), neoplastic cell line with the potential to become
highly tumorigenic. Because no vaccine
licensed in the U.S. has been produced in a tumorigenic cell substrate, OVRR
would like to update the VRBPAC on the approach it has taken to the regulation
of neoplastic cell substrates in general and to review with the Committee its
approach to addressing issues associated with cell-substrate tumorigenicity.
This
meeting is part of a process that OVRR has undertaken to keep the Committee
informed of the regulation of novel cell substrates for vaccine
production. Specifically, this meeting
will focus on OVRR's approach to the regulation of cell substrates that are
highly tumorigenic.
In
1998, OVRR initiated discussion with the Committee on the potential use of
neoplastic cell substrates for vaccine manufacture. At that time, OVRR proposed using a Defined Risks Approach (DRA)
to address issues posed by vaccine manufacture in neoplastic cells, and
presented it to the Committee for their consideration. The DRA involves:
1. Identifying the possible risk event; 2. 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; 3. Estimating the possible frequency of the risk event
per dose of vaccine; 4. Developing and determining the sensitivity (with
respect to lower limits of the assay’s ability to detect the risk event) of one
or more assays that can be use to detect the risk event; 5. Developing and
validating one or more processes that can be used to establish a
product-specific safety factor at a defined level of risk. The recommendations from that discussion
were: 1. OVRR should undertake research to provide the experimental basis for
the estimations of risks; 2. Develop a document that describes the approach; 3.
Organize an international workshop at which the issues are presented and
discussed in public; and 4. Continue the dialogue with the Committee.
In
September 1999, the CBER-sponsored international workshop (Evolving Scientific and Regulatory Perspectives on Cell Substrates for
Vaccine Development) took place in Rockville, MD. The proceedings were published in Development in Biologicals,
volume 106, 2001. The goals of the
workshop were to identify concerns and issues associated with neoplastic cells
and identify possible approaches to implementing the DRA by ascertaining the
levels of risk associated with those issues. Several conclusions were drawn
from the presentations and discussions.
1. Because of the multi-factor nature of human carcinogenesis, the risk
that cellular components (other than oncogenic viruses) could induce tumor
formation was considered low. 2. The
risk from adventitious agents was considered greater than from cellular
components such as DNA. In this regard,
primary cells were considered more of a risk than cell lines. 3. The risk from residual cell-substrate DNA
was considered low, although more data were needed on this topic. 4. The possibility that adverse consequences
could arise through interactions of the vaccine virus with the cell substrate
would be specific to the particular vaccine virus and cell substrate. 5. The potential value of designing cell
substrates for the production of specific vaccines was discussed. In general, the approach of immortalizing
primary cells by specific mechanisms to produce "designer" cell
substrates was met favorably. 6. The
issue of whether neoplastic cells had a higher risk of containing a mutation in
the PrP gene such that the cells had an increased capacity to propagate the
agent of transmissible spongiform encephalopathy (TSE) was discussed. As there was no clear mechanism that could
be invoked that would generate de novo
mutations in the PrP gene, and as there was no obvious selection for mutant PrP
proteins, the likelihood of a neoplastic cell developing a mutant PrP was
considered to be extremely low.
Immediately following this international meeting, OVRR summarized these
conclusions to the VRBPAC.
In
May 2000, OVRR discussed with the Committee the use of Vero cells for the
production of live attenuated virus vaccines.
Vero cells are a continuous cell line derived from the kidney of an
African green monkey. Like cells from
most species (except humans), passage of African green monkey cells in culture
can become immortal. The mechanism by
which cells "spontaneously" become immortal is unknown but likely
involves a number of genetic changes that accumulate over time. Vero cells have the advantage in being
permissive to a number of viruses; also, extensive experience with them has
failed to reveal the presence of any adventitious agents. These characteristics have resulted in the
extensive use of Vero cells both for the testing for adventitious agents and
for vaccine production. Early passage
Vero cells are not tumorigenic when assessed in immune incompetent rodents,
such as the nude mouse or newborn rats treated with anti-thymocyte globulin. However, later passage Vero cells can become
tumorigenic, and thus only low passage, non-tumorigenic Vero cells are used for
vaccine production. The recommendations
from the Committee were: 1. That the removal of whole cells be assured; and 2.
That the level of residual Vero DNA be kept to 10 ng per dose or below. In addition, the Committee concluded there
was more of a concern with the use of Vero cells for parenteral vaccines than
for vaccines administered by the oral or nasal routes. The Committee also expressed some concern
that Vero cells have the capacity to become tumorigenic and asked OVRR to
establish a research program to investigate the mechanism by which this occurs
to determine if there could be a safety concern.
In
July 2001, OVRR asked the Committee to consider the use of adenovirus type 5
(Ad5)-transformed cells for vaccine production. These cells are necessary to manufacture defective Ad5 vectors,
since they contain the E1 genes (E1A and E1B) necessary to complement the
E1-defective virus vectors. Because Ad5
vectors propagated on the original production cell line, 293, were found to
contain replication-competent adenovirus (RCA), which was generated by
recombination with the E1 genes present in the complementing cell, a new cell
line was developed such that the only Ad5 sequences present were those
necessary for complementation, and recombination to generate RCA was
eliminated. The new cell line was the
PER.C6 cell line, which was established in the Netherlands from fetal
retinoblasts immortalized by the Ad5 E1A and E1B genes driven by the cellular
promoter from the phosphoglycerate kinase gene. It was found that PER.C6 cells were weakly tumorigenic (see
below), forming tumors at 107 cells per nude mouse, a level similar
to that found with 293 cells.
Because
the use of PER.C6 cells for vaccine manufacture would be the first time a live
viral vaccine produced in a tumorigenic cell substrate would be permitted by
CBER to be used in humans, OVRR brought this topic to the Committee for their
consideration. While adventitious agents
and residual cell-substrate DNA are potential concerns with all novel cell
substrates, there may be a heightened concern when the cell substrate is
tumorigenic or derived from a tumor.
The potential risk of adventitious agent contamination (including TSE
contamination, since PER.C6 cells are neural derived), residual cell-substrate
DNA, and whole cells were discussed.
The sponsor provided documentation on the adventitious agent testing,
calculations as to the risk of TSE transmission, the level of residual DNA, and
the removal of whole cells. The
committee discussed factors that could mitigate concern about these cells,
including the relevance of the known mechanism of transformation and the
requirement for approximately 107 cells to produce tumors in
immunosuppressed animals. The Committee
also discussed the importance of minimizing steps toward oncogenesis in vaccine
recipients (such as initiating events), even when oncogenesis was not a direct
outcome of vaccine components. The Committee agreed that PER.C6 cells
represented an improvement over 293 cells with respect to RCA generation and
were satisfied that the sponsor had addressed the issues of adventitious agents
and residual cell-substrate DNA. The
Committee also endorsed OVRR’s general approach to evaluating these cells,
including the evaluation of cell lysates and DNA from PER.C6 cells for their
oncogenic potential in animals, even though there were no validated assays
available.
The
consideration of Madin Darby canine kidney (MDCK) cells for the manufacture of
inactivated influenza vaccines has prompted this meeting. While some lines of MDCK cells are not
tumorigenic, others are highly tumorigenic. Thus, one goal of this meeting is
for the Committee to comment on our modified DRA as applied to highly
tumorigenic cell substrates.
As
stated above, the main safety concerns with the use of tumorigenic cells are
the potential presence of adventitious agents, particularly of unrecognized
oncogenic viruses, and the amount and the form of the residual DNA present per
vaccine dose. A key and unresolved
scientific question is whether and how the degree of tumorigenicity of a cell
substrate should influence the testing algorithm for the determination of its
suitability for use in vaccine production.
Tumorigenicity
Testing (Dr Andrew Lewis)
For
over 40 years, there was a proscription on the use of tumorigenic cells for
vaccine production due to concerns that products manufactured in these types of
cells could transfer cancer to a vaccine recipient. Whether or not these concerns are scientifically justified cannot
yet be determined. Nevertheless, there
is a recognized perception that the use of neoplastic cell substrates for
vaccine manufacture poses risks for vaccine safety, and these risks are
enhanced the more tumorigenic the production cells are. Therefore, because it is critical for the
public health to retain trust in vaccine safety, regulatory authorities need to
address these risks and the attendant concerns over safety if vaccines are to
be manufactured in tumorigenic cells.
Tumorigenicity
is the property of a cell that allows it to form a tumor in an experimental
animal, usually the nude mouse, while oncogenicity is an activity of an agent
(usually a virus) or cellular component (such as DNA) that is able to transform
cells of that animal into neoplastic cells that can form a tumor. These definitions have practical
consequences, since the species of the tumor cells that occur in a
tumorigenicity assay will be that of the donor cell, while the species of the
tumor cells that arise in an oncogenicity assay is that of the host. Before 2000, the specific requirements for
tumorigenicity testing were single-dose, relatively short-term assays. Such assays are limited both in their
capacity to determine whether a line of neoplastic cells expresses a
tumorigenic phenotype and the degree of aggressiveness (i.e., weakly versus
highly tumorigenic) of the tumorigenic phenotype. The most comprehensive method of obtaining tumorigenicity data is
to evaluate quantitatively, in a dose-response assay with longer observations
times, the capacity of the cells to form tumors. These types of assays have revealed that different cells have
differing capacities to form tumors: some cells are weakly tumorigenic (i.e., requiring large numbers of cells –
106 or more – to produce a tumor), while others are highly
tumorigenic (i.e., requiring as few
as 1 to 100 cells to produce a tumor).
At present, the mechanisms for the variable expression of the
tumorigenic phenotype have not been delineated. Until they are, it will not possible to determine whether the use
of a cell substrate that is strongly tumorigenic poses more of a risk than one
that is weakly tumorigenic. In
addition, determining the dose-response relationships associated with tumor
formation can be used to enhance vaccine safety by revealing the presence of
unrecognized adventitious agents.
Enhanced
testing and characterization of the cell substrate is recommended for
tumorigenic cells. Therefore, assays
more comprehensive than single-dose, short-term assays are required to
determine the level of tumorigenicity of a neoplastic cell substrate. OVRR is currently recommending testing of
multiple doses of cells (107, 105, 103, and 101)
in adult nude mice, extending the observation period of the test animals up to
4 to 5 months, and identifying the species of the cells forming the tumors.
Adventitious
Agent Testing (Dr Arifa Khan)
Testing
for adventitious agents in cell banks and virus seeds involves extensive and
overlapping in vitro and in vivo testing designed to encompass
the detection of different classes of viruses.
These tests have served the public well over the years, as evidenced by
the good safety record of vaccines. However, as new viruses are identified, the
testing has evolved and expanded over the years to take advantage of new
approaches and novel assays. The most
significant addition to adventitious agent testing is the use of PCR
methods. These include the development
of assays for the detection of specific viruses (DNA and RNA) as well as
broadly reactive PCR-based assays, such as the sensitive product-enhanced
reverse transcriptase (PERT) assay for the detection of all retroviruses and
generic PCR assays for the detection of virus
families. The use of tumorigenic cell
substrates in vaccines has added concerns regarding the possible presence of
novel viruses, especially oncogenic viruses, and therefore broad detection
assays that have the potential to detect unknown viruses are needed.
These include treatment of the cell substrate with virus inducers
followed by general detection methods (e.g.,
PERT assay, TEM, generic PCR) for evaluating the presence of latent or
endogenous viruses and the inoculation of lysates prepared from the cell
substrate into newborn mice, newborn rats, and newborn hamsters for detection
of oncogenic viruses. Although neither
type of assay (virus induction or injection of cell lysate) has been validated,
both approaches have been demonstrated to detect viruses. Another approach that is applicable in the
case of inactivated vaccines is to determine the amount of viral clearance
(removal and/or inactivation) due to the manufacturing process. Spiking studies using viruses with different
properties that are expected to influence their removal and/or inactivation are
generally used to determine the level of removal and/or inactivation during the
various steps in production, with subsequent calculation of the total clearance
during manufacture. For highly
tumorigenic cells with negative adventitious agent test results, showing
clearance of appropriate model viruses should provide additional assurance of
safety.
Regulatory Issues Associated with Residual Cell-Substrate DNA (Dr Keith Peden)
Vaccines and other biological
products manufactured in cells contain contaminating residual DNA derived from
that production cell substrate, with the amount and form of this DNA depending
mainly on the type of vaccine. The
potential risk of this cellular DNA has been debated for over 40 years without
resolution. Opinions on residual DNA
have varied from it being considered an inert contaminant, and thus its
presence should not be deemed to be a risk to vaccine recipients, to it being
considered an important risk factor, particularly for vaccines manufactured in
tumorigenic cell substrates. Because
DNA has demonstrable biological activity, OVRR considers that DNA should not be
considered an inert contaminant. The
activities that DNA can transfer are oncogenic activity and an infectivity
activity. DNA oncogenicity is the
capacity of DNA to induce a tumor in an animal, while DNA infectivity would
result if the cell-substrate DNA contains the genome of an infectious virus and
this genome, when transferred to a human vaccine recipient, could result in the
establishment of an infection. The
major risk for residual cell-substrate DNA has generally been considered to be
due to the oncogenicity of the DNA, although DNA infectivity may be more of a
risk. Our approach is to develop assays to quantify the particular biological
activities of DNA, determine the amount of activity in a given quantity of DNA,
and use these values to estimate risk.
In addition, the efficiency of methods to eliminate the activity, such
as by chemical inactivation or enzymatic digestion, can be quantified using the
same assays and thus the level of safety from the clearance of DNA can be
estimated. An in vitro DNA infectivity assay can detect at least 106-fold
less DNA than an in vivo DNA oncogenicity
assay, and thus we are using the in vitro
infectivity assay to estimate the amount of clearance of DNA that can be
achieved using chemical or enzymatic methods. Because estimates of safety are
based on the sensitive in vitro DNA
infectivity assay, this represents a worst case. Using such assays, clearance levels (or safety factors) of
infectivity of >107 can be achieved by inactivation or digestion
with a DNA level of 10 ng per dose.
With such safety factors, the risk of an infectious event can be reduced
to acceptable levels.
The
introduction of highly tumorigenic cells to influenza vaccine manufacture could
yield significant benefits. Although
there is a perception that highly tumorigenic cells may carry greater risks than
less tumorigenic cells, we are proposing that such risks can be mitigated by
careful testing of the cells, validation of the production process for its
capacity to remove adventitious agents, and limitation of residual DNA in the
final product. OVRR would like the
committee to discuss (1) the use of MDCK cells, including those that are highly
tumorigenic, in manufacture of inactivated influenza vaccines, (2) OVRR's
approach to evaluate the safety of highly tumorigenic cells, and (3) any
additional steps OVRR should take to address issues associated with the use of
MDCK cells or neoplastic cell substrates.