FDA Briefing Document Vaccines and Related Biological Products Advisory Committee Meeting September 19, 2012 Cell Lines Derived from Human Tumors for Vaccine Manufacture

FDA Briefing Document
Vaccines and Related Biological Products Advisory Committee Meeting
September 19, 2012
Cell Lines Derived from Human Tumors for Vaccine Manufacture
http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/VaccinesandRelatedBiologicalProductsAdvisoryCommittee/UCM319573.pdf 3. Considerations Regarding the Use of Tumor-derived Cell Lines
When tumorigenic cell lines were first considered for vaccine manufacture in 1998 and
then proposed for vaccine manufacture in 2001, three major safety concerns were
identified that needed to be addressed: 1) the presence of residual live cells in the vaccine
that might have the potential of being tumorigenic in humans; 2) the presence of residual
DNA from the cell substrate; and 3) the potential presence of adventitious agents,
including adventitious viruses that might have contributed to the tumorigenic phenotype.
These same concerns also apply to tumor–derived cell lines from human tumors. In
addition, with cells derived from human tissues there is also a heightened concern that
unknown oncogenic factors may be present and contribute to the development of an
oncogenic phenotype if they are co-purified with the vaccine virus.
To assist in the discussion of the risks associated with use of human tumor-derived cell
lines for the manufacture of a viral vaccine and the development of strategies to mitigate
these risks, this section of the briefing document summarizes the available information
regarding how normal cells become tumor cells. The use of human tumor-derived cell lines poses added safety concerns regarding the
potential presence of unexpected and unknown viruses. These include viruses that may
be present in the cell line due to their existence in the patient tissue such as oncogenic,
latent DNA viruses (e.g., adenoviruses, hepadnaviruses, herpesviruses, papillomaviruses,
polyomaviruses) and, 2) endogenous retroviruses (ERVs), which exist normally in a
quiescent state in the host cell DNA of all species, but can become activated and produce
virus particles in tumor cell lines (36, 74).
http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiol
www.fda.gov
1. Guidance for Industry (February 2010): “Characterization and Qualification of Cell
Substrates and Other Biological Materials Used in the Production of Viral Vaccines for
Infectious Disease Indications”
2. Review Article (Anal. Chem. 83, 4327-4341; May 25, 2011): “Landscape of NextGeneration Sequencing Technologies” by Thomas P. Niedringhaus, Denitsa Milanova,
Matthew B. Kerby, Michael P. Snyder, and Annelise E. Barron
1. Introduction
This meeting of the Vaccines and Related Biological Products Advisory Committee
(VRBPAC) is being held to discuss the use of cell lines derived from human tumors as
substrates for the production of preventive viral vaccines.
Over the last decade, it has become clear that the current repertoire of cell substrates is
inadequate to manufacture the next generation of viral vaccines (i.e., certain viruses
cannot be propagated or grow poorly in the available cell lines). Therefore,
manufacturers have submitted additional cell lines to the FDA for consideration for use in
the production of viral vaccines. All of the new mammalian cell lines being considered
are immortal, having been transformed by various oncogenes or are spontaneously
immortalized, and some are derived from human tumors. Over the last 15 years, when
new types of cell substrates have been proposed, starting in 1998, CBER has presented its
review approach to the Advisory Committee to address the issues raised first by the use
of immortalized mammalian cell lines and then tumorigenic cell lines. The purpose of
those discussions with the Advisory Committee was to obtain their input and to make
public a discussion of the issues. This current VRBPAC meeting is a continuation of this
process.
To date, only a limited number of preventive vaccines made in tumorigenic cell lines
have entered clinical trials. Some live-vector vaccines made in laboratory-immortalized
human cells have entered clinical trials (VRBPAC 2001), as have some inactivated
vaccines manufactured in non-human tumorigenic cell lines (VRBPAC 2005). However,
tumor-derived cell lines have not been used for production of prophylactic vaccines (see
Section 2 for definitions). The purpose of the present VRBPAC meeting is to discuss the
scientific issues associated with the use of cell lines derived from human tumors for the
production of both live and inactivated prophylactic viral vaccines and to identify safety
concerns and determine how they can be addressed. As background, this briefing
package includes a brief description of the history of cell substrates used for viral vaccine
manufacture in the U.S., information on the potential concerns associated with the use of
tumor-derived cells, and finally, information on the Agency’s current practices on
reviewing new cell substrates with respect to the presence of adventitious agents and
residual cell-substrate DNA.
2. Background
In this document, tumor-derived cell lines refer to lines established from cells isolated
from tumors. Tumorigenic cell lines refer to cells capable of forming tumors in an
immune-compromised rodent. Continuous cell lines are immortal and may be established
from cells isolated from tumors or from cells originally isolated from healthy tissues and
transformed in vitro spontaneously or by infection with an oncogenic virus or by DNA
transfection with oncogenes or known immortalizing genes such as TERT. On the other
hand, diploid cells are mortal (i.e., they have a limited lifespan in culture) and diploid cell
lines are established from cells isolated from healthy tissues. While diploid cell lines are
4not expected to be tumorigenic, continuous cell lines may or may not exhibit a
tumorigenic phenotype.
2.1 History of Cell Substrates for Viral Vaccines Manufactured in the U.S.:
Primary, Diploid and Tumorigenic Cells
The following are selected highlights of the use of cell substrates for growth of virus for
production of preventive vaccines in the U.S. (see Appendix 1 for details on discussions
with the VRBPAC and other meetings on cell substrates):
1950s and 1960s: Primary cell cultures derived from rhesus macaque kidneys were used
to grow virus to manufacture inactivated poliomyelitis vaccine (IPV) and live-attenuated
oral poliovirus vaccine (OPV).
Embryonated hens’ eggs or primary chick embryo fibroblast cultures were used to grow
virus to manufacture vaccines against yellow fever, influenza, measles, and mumps.
These substrates were considered acceptable both for live-virus vaccines (yellow fever,
measles, and mumps) and inactivated-virus vaccines (influenza), since it was believed
that adventitious agents that could infect chickens would not infect and be pathogenic for
humans.
1960s to 1990s: Human diploid cell lines derived from fetal lung tissue were used to
grow virus for the production of a rubella vaccine (WI-38 cells) and for a polio vaccine, a
hepatitis A vaccine, and a rabies vaccine, and the varicella vaccine (MRC-5 cells). A
diploid cell line derived from rhesus monkey lung cells (FRhL-2) was used for a rotavirus
and a rabies vaccine.
1990s to 2000: A continuous, non-tumorigenic cell line derived from the kidney of an
African green monkey (Vero) was first used for the manufacture of an inactivated
poliomyelitis vaccine (IPV) and then a live-attenuated oral poliomyelitis vaccine (OPV).
The use of Vero cells to grow virus for live viral vaccines was the topic of discussion at a
VRBPAC meeting in 2000. The VRBPAC supported the use of Vero cells for production
of inactivated vaccines. However, some members expressed the concern that Vero cells
had the capacity to become tumorigenic with prolonged passage in culture and
recommended that the cells not be used beyond a specific number of passages. In
addition, several advisory committee members expressed reservations regarding the use
of Vero cells to manufacture live virus vaccines; however, following additional
discussion and refinement of manufacturing controls to mitigate potential risks, three live
viral vaccines that were made using low-passage non-tumorigenic Vero cells have been
licensed in the US (Smallpox vaccine in 2007; and two rotavirus vaccines in 2006 and
2008).
1990s: The use of tumorigenic cell lines to grow virus to produce vaccines against
emerging pathogens was discussed by the VRBPAC in 1998. The topic for discussion
was the appropriate approach/method to assess the risks associated with the use of such
cells in the production of these vaccines. The Defined Risks Approach, which
5experimentally estimates the likelihood of a risk event occurring, was presented to the
committee. This approach requires identification of specific risk events and experimental
measurement of their frequency using quantitative assays. The committee generally
concurred with this approach.
2000s: The first tumorigenic cell line to be considered for use in the production of a live
viral vaccine was PER.C6 for the production of a replication-defective adenovirus
vectored HIV-1 vaccine (VRBPAC 2001). Since the adenovirus type 5 (Ad5) vector was
deleted in the early region 1 (E1 region), human cells expressing the E1 genes in trans
were required to complement the defect in the viral vector. The PER.C6 cell line was
generated in a laboratory by immortalizing human fetal retinal cells with the E1 region of
Ad5. This was the first example of a “designer-cell substrate”, since it was developed
specifically to eliminate the production of replication-competent adenovirus during
manufacture. In addition, because the mechanism of immortalization was known, the
level of concern with these cells was reduced. Following evaluation of the
comprehensive testing plan for these cells and consideration of the purification steps
during manufacture, the VRBPAC supported use of PER.C6 cells for manufacture of
replication-defective Ad5-vectored vaccines against HIV-1. The testing plan included
quantification of the tumorigenic phenotype of PER.C6 cells and evaluation of the
oncogenicity of PER.C6 DNA and cell lysates in newborn nude mice, newborn hamsters,
and newborn rats. Although this extensive oncogenicity testing for the characterization
of a vaccine cell substrate was done for the first time and the assays were not
standardized, the VRBPAC considered that, in the absence of other assays, they were
acceptable for this purpose. In addition, testing for adventitious agents was more
extensive than the usual in vivo and in vitro tests recommended at the time and included a
comprehensive list of specific PCR tests for potential virus contaminants.
The next tumorigenic cell line under consideration was the Madin-Darby canine kidney
(MDCK) cell line, which was proposed for the production of inactivated influenza virus
vaccines (VRBPAC 2005). The MDCK cell line is a spontaneously immortalized cell
line established from the kidney of an apparently normal dog. Some variants of MDCK
cells are highly tumorigenic, while others are not [reviewed in Omeir et al. (64)]. The
Committee accepted the use of tumorigenic MDCK cells for inactivated influenza
vaccines and their evaluation in clinical trials.
Additionally, a non-tumorigenic MDCK cell line was discussed for the manufacture of a
live, attenuated influenza virus vaccine (VRBPAC 2008). Since this clone of MDCK
cells was not tumorigenic, the level of residual cell-substrate DNA in the final product
was low and reduced in size, and the route of vaccine administration was intranasal, the
majority of the Committee supported the evaluation of this vaccine in clinical trials.
2.2 Cell Substrates Derived from Human Tumors Proposed for the Manufacture
of Viral Vaccines
Three cell lines derived from human tumors will be discussed during the September 2012
VRBPAC meeting: the CEM leukemia T cell line, the A549 lung adenocarcinoma cell
line, and the HeLa cervical carcinoma cell line. These cell lines serve as the basis for this
discussion because sponsors have proposed their use in the production of vaccines for
evaluation in clinical trials. CBER requests that these cells be viewed as representative
of this type of cell substrate so that the recommendations of the advisory committee will
be applicable to other tumor-derived cell lines (human and non-human) proposed for
vaccine manufacture in the future.
Over the last 20 years, it has been recognized that cell lines derived from tumors may be
the optimal and in some cases the only cell substrate that can be used to propagate certain
vaccine viruses. For example, to produce HIV-1 in sufficient quantities for inactivated
HIV-1 vaccines, cell lines derived from human CD4-positive T cells are preferred over
primary T cells. The first such vaccine submitted to the FDA was an inactivated HIV-1
produced in the T-cell line HuT78, which was derived from a human lymphoma (24).
The vaccine was an inactivated HIV-1 intended as a therapeutic vaccine for the treatment
of HIV-1-infected individuals and was evaluated in clinical trials in the 1990s (59, 60).
The appropriateness of using HuT78 cells to produce an HIV-1 vaccine was discussed by
the VRBPAC in 1998, and while there were questions as to the need to use such cells, the
Committee accepted that alternative cell substrates were not feasible.
The cell lines under consideration for this meeting are:
 The CEM T-cell line established from cells from an individual with leukemia (22)
and is highly permissive for HIV-1 strains that use the co-receptor CXCR4.
 The A549 cell line established from cells excised from the lung of an individual
with adenocarcinoma of the lung (51). The A549 cell line is highly susceptible to
human adenovirus infection and is used both to detect adenoviruses and to
produce replication-competent adenoviruses for certain investigational vaccines
(4).
 The HeLa cell line, which was established from cells of a cervical carcinoma in
1952 (25, 26). These cells contain approximately 50 copies of the HPV-18
genome and constitutively express the viral oncoproteins E6 and E7 (38, 45, 79).
The HeLa cell line is permissive to many human viruses. HeLa cells have been
engineered to be producer lines for adeno-associated virus (AAV) (87). AAV has
been used to produce investigational gene-therapy products (3, 21, 96) and
investigational vaccines (90).
Summary:
Historically, each new type of cell substrate used for viral vaccine production has
presented novel issues that have been addressed by CBER after scientific consultations
with the VRBPAC, first in 1998 and then in 2000, 2001, 2005, and 2008. The present
VRBPAC is a continuation of these discussions. The current repertoire of cell substrates
is inadequate for manufacture of certain types of new vaccines. To address this
limitation, sponsors have proposed the use of several novel cell substrates that are
tumorigenic or derived from human tumors. Because these cell lines that have not
hitherto been accepted by regulatory authorities for the manufacture of prophylactic
vaccines, CBER has entered into discussions with the VRBPAC so that issues associated
with these types of cells will be discussed in an open scientific forum
3. Considerations Regarding the Use of Tumor-derived Cell Lines
When tumorigenic cell lines were first considered for vaccine manufacture in 1998 and
then proposed for vaccine manufacture in 2001, three major safety concerns were
identified that needed to be addressed: 1) the presence of residual live cells in the vaccine
that might have the potential of being tumorigenic in humans; 2) the presence of residual
DNA from the cell substrate; and 3) the potential presence of adventitious agents,
including adventitious viruses that might have contributed to the tumorigenic phenotype.
These same concerns also apply to tumor–derived cell lines from human tumors. In
addition, with cells derived from human tissues there is also a heightened concern that
unknown oncogenic factors may be present and contribute to the development of an
oncogenic phenotype if they are co-purified with the vaccine virus.
To assist in the discussion of the risks associated with use of human tumor-derived cell
lines for the manufacture of a viral vaccine and the development of strategies to mitigate
these risks, this section of the briefing document summarizes the available information
regarding how normal cells become tumor cells.
3.1 Mechanism of Tumorigenesis and Considerations for Characterization of
Cell Substrates
Experimental data on the mechanism of cancer development has demonstrated that the
transformation of a normal human cell to one with a tumorigenic phenotype is a multistage process. The tumorigenic transformation of normal cells can be caused by
infectious agents (i.e., oncogenic viruses), somatic mutations, and epigenetic changes.
The fact that multiple oncogenic events are required to establish a tumor has been
considered to provide an additional safety margin for vaccines manufactured using a
tumor-derived cell substrate for virus growth. However, while it has been reported that
several oncogenic events are required to convert a normal human cell into a cell that can
establish a tumor in an experimentally inoculated mouse (31), the concern remains that a
single oncogenic “hit” can predispose a cell to subsequent oncogenic events that will,
perhaps over many years, lead to a malignant cell. At this time, if such initiating events
occur it is not possible to detect them. Nevertheless, because this is a theoretical
possibility, regulatory authorities have required that multiple methods be used to reduce
potential oncogenic factors in vaccines produced in such cell substrates.
The transformation of a normal cell to one with a tumorigenic phenotype is a multi-stage
process. Below is a brief description of each of the mechanisms of tumorigenesis and
how they can affect the safety profile of human-derived tumor cells as substrates for
vaccine production.
3.1.1 Oncogenic Viruses
The first cancer virus described was the Rous sarcoma virus, a retrovirus that causes
sarcomas in chickens (72, 73). The determination that the gene responsible for the
neoplastic transformation was the src gene, a gene acquired from the chicken genome and
modified during multiple virus infection cycles (41, 56, 57), was instrumental in
establishing the field of oncogenes (85, 91). The subsequent identification of cellular
analogues of many other viral oncogenes followed rapidly (7-9, 15, 92, 93, 100), as did
the discovery of viruses that can cause human tumors (12, 18, 78).
Viruses can be oncogenic in several ways. They can carry dominant oncogenes that
directly induce a transformed phenotype in the infected cell or they can integrate into the
host genome and cause the activation of cellular oncogenes or the inactivation of tumorsuppressor genes. For example, viruses such as papillomaviruses, adenoviruses,
polyomaviruses, gamma herpesviruses (such as EBV), and replication-defective and
replication-competent retroviruses, such as MC29 and RSV, respectively, have been
shown to carry oncogenes. However, in most cases, expression of the viral oncoproteins
alone is insufficient to result in cancer, and subsequent genetic and/or epigenetic changes
are necessary to convert the initially transformed cell into a malignant cell.
The presence of oncogenic adventitious viruses would represent the most likely source of
oncogenic activity in a tumorigenic cell substrate, and these viruses would have the
greatest potential for transmitting cancer. Thus, it is important to address whether there
are any infectious adventitious viruses present in the cell substrate. To accomplish this,
various virus-detection assays and clearance strategies are recommended for evaluating
the safety of a tumorigenic cell substrate for use for vaccine manufacture (see Section
4.1).
3.1.2 Somatic Mutations
The acquisition and accumulation of somatic mutations contributes to the development of
many cancers. For example, point mutations can result in conversion of proto-oncogenes
to oncogenes, and chromosomal rearrangements and insertional mutagenesis, either
through infection with an exogenous retrovirus or the mobilization of endogenous
retroviruses or retrotransposons, can result in activation of proto-oncogenes by elevating
their expression, as can gene amplification. Insertional mutations can also inactivate
tumor-suppressor genes. However, because the two copies of tumor-suppressor genes are
generally expressed, the oncogenic consequences of such an insertional mutation would
require inactivation of the other allele, which would be a rare event. Tumor-suppressor
genes can acquire gain-of-function mutations to become dominant-negative proteins,
which can interfere with the function of the normal protein; only in this case would the
oncogenic risk of DNA be increased.
The main safety issue related to transformation by somatic mutations is the presence of
residual cell-substrate DNA in a vaccine and whether this DNA represents an oncogenic
or an infectious risk. As discussed in Section 4.2, the oncogenic and infectious risk of
DNA can be reduced by lowering the amount of DNA, and/or decreasing the size of the
DNA (by nuclease digestion), and/or by reducing the activity of the DNA (by chemical
treatment or gamma irradiation).
3.1.3 Epigenetic Changes
Epigenetic mechanisms of gene regulation include: chromatin remodeling, histone
modifications, DNA methylation, and altered microRNA (miRNA) expression. Although
such mechanisms have been described to alter gene expression, it was not thought that
the consequences of epigenetic events could be heritable, i.e., passed on from one
somatic cell generation to the next and thus permanently modify the cell’s phenotype.
However, recently it has been recognized that treatment of cells with certain agents or
proteins can induce phenotypic changes through epigenetic processes, and that these
phenotypes can be passed on to subsequent cell generations.
If a vaccine cell substrate becomes tumorigenic through epigenetic mechanisms, the
oncogenic risk associated with using these cells for vaccine production is reduced for the
following reasons:
 If the stimulus that induced the phenotypic change(s) in the cells was endogenous,
either it is no longer present in that cell or, if still present, then its concentration in the
vaccine produced from the cell would likely be too low for it to exert a biological
effect in the vaccine.
 If the stimulus that had induced the phenotypic change in the cells was exogenous, it
would not be present in the cell substrate and thus would not be present in the
vaccine.
 If the outcome of the stimulus on the cells was to change the phenotype through
alterations in gene expression via DNA methylation, the risk would be reduced to one
associated with the DNA or chromatin. However, because changes in gene
expression often involve multiple normal cellular genes, transmission of the
tumorigenic phenotype to cells in the vaccine recipient via DNA or chromatin would
be highly improbable, since it would require the transfer (uptake) and co-expression
of multiple genes. Because these genes would not be expected to be linked, the cotransfer of two or more genes would be improbable. And because these genes are
normal cellular genes, their expression would likely not be transforming. In addition,
since oncogenesis through DNA methylation frequently involves the silencing of
tumor-suppressor genes (6, 14, 19, 30, 39, 40, 43, 70), the uptake of such a silenced
gene by cells would not be expected to alter the cell’s phenotype due to lack of
expression.
 If the outcome of the stimulus on the cells was to change the phenotype through
alterations in gene expression via histone modifications and chromatin remodeling,
the risk would again be reduced to one associated with the chromatin and
transmission of these traits to other cells would be improbable as the factors affecting
these modification requires the transfer and expression of multiple genes
In summary, if the tumorigenic phenotype arose through epigenetic mechanisms, the
capacity to transfer this phenotype to vaccine recipients via residual cellular components
would be improbable or involve hitherto unrecognized mechanisms.
3.2 Metastatic Potential
Several types of cells can form tumors when inoculated into immune-compromised
rodents; however, not all of these tumors can metastasize. The steps involved in
metastasis are: the invasion of the basement membrane (invasion), entry of the blood or
lymphatic systems (intravasation), survival in these tissues and migration to distant sites
through the blood vessels or, more commonly, the lymphatics, traversal of the vessel
walls (extravasation), and colonization of distant tissues (89). The mechanisms whereby
cells become metastatic are unknown, and it is not clear how the fact that a cell forms
metastases in an immune-compromised rodent influences the degree of risk for a vaccine
manufactured in such a cell.

Several proposed mechanisms for the acquisition of a metastatic phenotype are briefly
presented below (17). If a mechanism involving the activation of additional protooncogenes were operating, then compared with a non-metastatic cell, the DNA from a
cell capable of metastasis might represent more of an oncogenic risk. However, if the
generation of a metastatic cell results from the increased expression of proteins involved
in invasion, intravasation, etc., or the reduced expression of proteins that maintain
cell/cell junctions, then any residual DNA from metastatic cells in a vaccine would not
represent more of a risk than DNA from non-metastatic cells.
If the generation of a metastatic phenotype involves single or multiple activated
oncogenes, demonstration of DNA clearance in the vaccine can address this concern.
Furthermore, for mechanisms involving multiple genes, the conversion of a normal cell to
a metastatic cell would require the simultaneous uptake of multiple DNA fragments by a
single cell, which would be an improbable event. Thus, with appropriate manufacturing
controls, the use of tumorigenic cells that can metastasize in immunocompromised
rodents may not represent an additional safety concern for the use of these cells for
vaccine production.
Summary: Although the possibility of unknown oncogenic factors remains as a
theoretical concern with cells derived from human tissues, current knowledge related to
the mechanisms of oncogenesis supports the notion that it is highly improbable that any
whole cell or complex of cellular factors from a tumor-derived cell will be present in
sufficient quantities and lead to the development of tumors in the vaccine recipient.
Thus, cellular DNA and adventitious agents are considered the major safety concerns for
vaccines manufactured in tumorigenic cell lines or cell lines derived from human tumors.
Therefore, extensive characterization for adventitious agents in the cell substrate and
validation of DNA removal through the manufacturing process are required. The current
recommendations for cell-substrate and vaccine testing with respect to adventitious
agents and residual cellular DNA are discussed in Section 4.
4. Recommendations for Testing of Cell Substrate and Vaccines
Current recommendations for testing of mammalian cell substrates and vaccines are
generally based on three main safety concerns: the presence of residual, potentially
tumorigenic, live cells in the vaccine; the presence of residual DNA from the cell
substrate; and the potential presence of adventitious agents.
The characterization of cell substrates includes tumorigenicity testing. This testing is
described in the 2010 guidance document [Guidance for Industry for Characterization
and Qualification of Cell Substrates and Other Biological Starting Materials Used in the
Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases]
(included as Attachment 1). This testing is performed to determine if the cell substrate is
capable of forming tumors after inoculation into animals to characterize the tumorigenic
phenotype of a cell line. In the case of a tumorigenic cell substrate, an extended
observation period of 4 months or more is recommended with additional characterization
of the tumorigenic phenotype by dose-response studies.
Because infectious or oncogenic viruses could be transmitted through whole cells present
in a vaccine, the demonstration of removal of cells is required for vaccines manufactured
in the U.S. in any cell substrate.
4.1 Adventitious Agents

4.1.1 Background
The presence of adventitious agents is a safety concern in the use of any cell substrate
and throughout the manufacture of any biological product. Adventitious agents are
defined as microorganisms that are not intended to be present in a biological product and
include bacteria, fungi, mycoplasma/spiroplasma, mycobacteria, rickettsia, protozoan
parasites, transmissible spongiform encephalopathy agents, and viruses. In addition to
specific regulatory requirements for adventitious agent testing, FDA has issued
regulatory guidance for industry and points to consider documents to help manufacturers
formulate a comprehensive testing regimen for viral contaminants based upon the cell
substrate, raw materials, and manufacturing steps for each vaccine. These documents are
further supplemented by information in relevant International Conference on
Harmonization (ICH) documents and World Health Organization (WHO) documents.
The use of human tumor-derived cell lines poses added safety concerns regarding the
potential presence of unexpected and unknown viruses. These include viruses that may
be present in the cell line due to their existence in the patient tissue such as oncogenic,
latent DNA viruses (e.g., adenoviruses, hepadnaviruses, herpesviruses, papillomaviruses,
polyomaviruses) and, 2) endogenous retroviruses (ERVs), which exist normally in a
quiescent state in the host cell DNA of all species, but can become activated and produce
virus particles in tumor cell lines (36, 74).
Furthermore, many tumor-derived cell lines support infection and replication of a wide
variety of viruses and therefore may be more susceptible to infectious viruses present in
the host (e.g., some RNA viruses such as LCMV) or through contaminated human or
animal-derived reagents used during cell-line derivation and passage history. There may
be additional concerns in the case of virus-like particles (VLPs) such as co-packaging of
“unwanted” oncogenic RNAs or DNAs that may be transferred to the recipient by
vaccination.
All cell substrates need to be assessed for the potential presence of adventitious viruses
during cell-line qualification and demonstrated to be free of infectious virus.
4.1.2 Current Recommendations for Characterization and Testing of Cell
Substrates
CBER’s current recommendations for adventitious virus testing for preventive vaccines
and approaches for development of mitigation strategies to reduce the risk of virus
contamination are describe in the Guidance for Industry for Characterization and
Qualification of Cell Substrates and Other Biological Starting Materials Used in the
Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases
(2010). The recommended adventitious agent testing listed in this guidance is shown in
Appendix 2.
Adventitious agent testing of mammalian cells includes non-viral and viral agents. Nonviral agent testing includes tests for mycoplasma, bacteria and fungal sterility, and
mycobacteria. Adventitious-virus testing includes: general tests for virus detection, i.e.,
in vivo assays generally in adult mice, suckling mice, and embryonated hens’ eggs; in
vitro cell-culture tests for cytopathic and hemadsorbing/hemagglutinating viruses using
three cell lines (same species and tissue type as used in production, human diploid cells,
and monkey kidney cells); and assays for retroviruses, such as transmission electron
microscopy (TEM), PCR-based reverse transcriptase assays (e.g., PERT assay), and
infectivity assays. In certain cases, retrovirus-detection assays may include drug
treatment to induce latent viruses (discussed below).
Assays for species-specific adventitious viruses are also recommended. These are based
not only on the animal species from which the cells were derived, but also on the passage
history and the raw materials of animal origin used to culture the cells from original
isolation to the time of use. Recommended tests include: 1) antibody-production assays
such as MAP for mouse viruses, HAP for hamster viruses, and RAP for rat viruses and
challenge assay for lymphocytic choriomeningitis virus (LCMV), in the case of rodent
cell lines or potential exposure to rodent materials; and 2) tests for bovine and porcine
viruses in the case of cells lines derived from these species or exposure to bovine serum
and porcine trypsin. Virus-specific assays should also be included based upon donor
species and passage history (e.g., PCR and infectivity assays for human pathogens in case
of human cell lines).
While safety concerns related to cell tumorigenicity associated with residual intact cells
or residual cell DNA may be addressed by removal of whole-cells and reduction of DNA
size and amount (as discussed above), demonstrating the absence of latent and occult
viruses in the cell substrate is more challenging. Therefore, additional testing
recommendations include investigating for the presence of latent viruses such as DNA
viruses and endogenous retroviruses using chemical induction assays, and to detect
oncogenic viruses by testing cell lysates in vivo in three newborn rodent species. For
detection of latent viruses, a step-wise strategy was developed at CBER for treating cells
with four chemical inducers that have different mechanisms of action to activate
endogenous retroviruses and DNA viruses, followed by detection of induced viruses
using various general and specific assays (42, 52, 53). Detection of induced viruses may
be performed through the use of transmission electron microscopy (TEM) or PCR, which
is highly sensitive and compared with TEM is more specific, as detection is based on
primer design from known sequences.
The detection of known and novel viruses may be further facilitated by the use of broad
virus detection technologies such as massively parallel sequencing, long range PCR with
mass spectrometry, and virus microarrays, as discussed below.
4.1.3 Emerging Technologies for Viral Detection
Technologies such as massively parallel sequencing (MPS) or deep sequencing, virus
microarrays, and broad-range PCR with electrospray ionization mass spectrometry
(PCR/ESI-MS; commercial name PLEX-ID) have enabled rapid identification of novel
virus strains and novel-virus discovery in research and clinical samples (These
technologies are further detailed in Appendix 3). These emerging methods have also
been used by some researchers and testing labs to investigate cells and source materials
used in manufacture of vaccines as well as to test commercially available vaccines to
demonstrate the potential applications of the technologies. Such studies resulted in the
discovery of porcine circovirus-1 (PCV-1) in a rotavirus vaccine (Rotarix) by virus
microarrays and MPS (95), bluetongue virus in Vero cells by PLEX-ID (76), as well as a
xenotropic murine leukemia virus-related virus designated as XMRV that was initially
discovered in a human clinical sample using virus microarrays (88) and later found to be
a novel recombinant murine retrovirus (66). The use of MPS to screen raw materials
such as bovine serum has resulted in identification of several novel bovine viruses and
the detection of a large number of noninfectious viral genome fragments (65). These
results highlight the need for development of a follow-up strategy for evaluation of a
positive signal with any of these nucleic acid-based technologies to assess the biological
significance and relevance of such findings to product safety. A logical approach is to
determine if any detected sequence is a part of a complete virus genome, if the genome is
particle associated, and whether the particle is infectious. Further evaluation of potential
risk would be to determine if the particle is able to replicate in human cells.
Emerging technologies that can detect both known and novel viruses have generated
great interest and extensive discussions regarding the application of these methods for the
evaluation of biological products. Although the advantages of these methods are p.14
recognized, currently they have not been recommended for use due to technical
challenges that are being addressed. These challenges include standardization and
validation, data analysis, interpretation, and storage, and development of follow-up
strategies for investigation of a positive result. Additionally, more data are needed to
determine when these technologies should be applied, for which product types, and at
what stage of manufacturing.
4.1.4 Viral Clearance by the Manufacturing Process
In addition to the testing of the cell substrates and other raw materials, vaccine safety
may be further assured through assessment of the capacity of the manufacturing steps to
clear potential contaminating viruses. Clearance includes both virus removal during the
vaccine manufacturing process, as well as specific inactivation steps incorporated into the
production process (2). Clearance of potential adventitious agents can be assessed
quantitatively by spiking with model viruses of various types to determine the reduction
in titer afforded by each step in the manufacturing process to derive a log-clearance
value. Inactivation steps (e.g., chemical treatment, gamma irradiation) cannot typically
be used with live-virus vaccines, and consequently, the extent of adventitious agent
clearance is usually higher for an inactivated vaccine than with a live-virus vaccine. The
extent of viral clearance by the manufacturing process is product dependent, and there are
limitations inherent in the studies to assess viral clearance (e.g., number of model viruses
evaluated). Thus, viral clearance studies are a useful contribution to the evaluation of
vaccine safety, but they cannot by themselves ensure the absence of adventitious agents.
Summary: A comprehensive evaluation of vaccine cell substrates for the presence of
adventitious agents has been described in the current FDA guidance document. This
evaluation currently includes recommendations for conventional in vitro and in vivo tests
for unknown adventitious agents, as well as specific tests (e.g., PCR) for agents relevant
to the origin, passage, and use of the cell in vaccine production. Evaluation of
adventitious virus removal and/or inactivation also plays an important role in establishing
the safety of vaccine products. As recent examples illustrate, emerging technologies
(e.g., massively parallel sequencing) may have the potential to be powerful tools for the
detection of known and unknown adventitious agents and may be especially useful for
evaluation of novel cell substrates. Nevertheless, at the present time, these emerging
technologies are not standardized, nor is there a defined path for follow-up of potential
signals generated during their application. Further discussions, including input from the
current September 2012 VRBPAC, is needed to determine the role of such technologies
in evaluating novel cell substrates, including cell lines derived from human tumors.
4.2 Residual Cell-substrate DNA

4.2.1 Background: DNA Oncogenicity, DNA Infectivity, and DNA Integration
Small amounts of residual cell substrate DNA unavoidably occur in all viral vaccines as
well as other biologics produced using cell substrates. There are several potential ways
15
DNA could be a risk factor. DNA can be oncogenic or infectious; in addition, it can
cause insertion mutagenesis through integration into the host genome.
DNA Oncogenicity
A major concern about residual cell-substrate DNA has been the potential for the
induction of cancer, particularly if the DNA was from a tumorigenic cell or from a cell
line established from a human cancer.
Cellular oncogenes were originally identified based on their homology with viral
oncogenes. For example, the viral src gene (v-src), the transforming gene of the Rous
sarcoma virus (RSV), the first tumor virus identified (73), was found to have counterparts
in cellular DNA, first in the chicken genome (85, 94) and then in all mammalian genomes
(27, 67). Additional cellular versions of viral oncogenes, termed proto-oncogenes, were
identified (34, 93). However, proto-oncogenes are normal genes and are involved in
normal cellular processes. It is only when “activated” (most frequently through
mutation) that they become dominant oncogenes. It is the potential presence of such
activated dominant oncogenes in the genomes of certain cell substrates, such as
continuous cell lines and tumorigenic cells, that has raised concern over residual DNA in
vaccines prepared using such cell substrates, since complete removal of DNA from
vaccines is not possible. Therefore, the issue has been whether the low levels of residual
cell-substrate DNA in vaccines could be a risk factor in recipients of these vaccines. This
issue has been debated over many years with the conclusion that residual DNA amount
and size should be controlled. A summary of these discussions is presented in
Appendix 4.
DNA infectivity
If the DNA from the cell substrate contains the genome of an infectious virus, either as an
extrachromosomal element or integrated into the host genome of the cell, uptake of that
DNA could result in expression of the corresponding virus, and if that virus could
establish a productive infection in the human host, the consequences could potentially be
pathogenic.
Although there are publications on the infectivity of genomes of DNA and RNA viruses,
few studies have determined the specific infectivity of virus DNA. Studies at CBER
have shown that cloned HIV-1 DNA was infectious in vitro at 1 pg, and DNA from HIV-
1-infected cells was infectious at a level of 2 µg (83). Apart from determining the
specific infectivity of an HIV-1 genome, this study demonstrated that an integrated copy
of a viral genome has similar infectivity to a non-integrated copy of the same genome. In
addition, this in vitro infectivity assay has been used to quantify the reduction in DNA
activity afforded by various treatments commonly used during vaccine manufacture.
Such treatments as -propiolactone (BPL) for inactivated vaccines and benzonase
digestion for live vaccines can reduce the infectivity of DNA by more than 100,000 fold
(83). Combining the reduction in the biological activity of DNA afforded by
manufacturing steps with lowering the permitted amount of residual DNA to 10 ng per
dose or below provides safety factors in excess of 10
7
(82, 83). DNA integration
While it is theoretically possible that integration of DNA could induce oncogenicity,
either through the activation of a proto-oncogene or the inactivation of a tumorsuppressor gene, this risk depends on the efficiency with which naked DNA integrates.
Integration was a concern when DNA vaccines were developed in the 1990s, and a
VRBPAC meeting was convened in 1997 to discuss the issue. It was decided that it was
unlikely that DNA integration occurred at a high enough frequency to be a concern,
although no data were available at the time. Subsequently, this decision was confirmed
experimentally, when Ledwith and colleagues demonstrated in a rodent model that
integration of a plasmid DNA vaccine occurred at a very low efficiency (46, 47, 97), with
4 integrants being identified following injection of 50 µg plasmid DNA. At this low
level of integration (at the most, 1 integration event per 150 cells), the oncogenic
consequences of DNA integration would not be a significant concern. In addition,
studies at CBER have shown that up to 25 µg of a plasmid containing a strong promoter
failed to induce tumors in sensitive mouse strains, whereas tumors were induced by the
same vector expressing oncogenes (81).
As stated above, the major risk from DNA is considered to be through the introduction of
a dominant activated oncogene or an infectious genome, particularly one from a virus that
can establish a productive infection in the human host.
4.2.2 Current Recommendations for Control of Whole Cells and Host-cell DNA
Content in Vaccines
Potential infectious or oncogenic agents could be transmitted through whole cells present
in the vaccine; therefore, demonstration of removal of cells from vaccines has been
required in the U.S. for several decades.
The current testing recommendations in viral vaccines for the testing of host-cell DNA
and cell lysates from tumorigenic cells was established when the first tumorigenic cell
line for vaccine production was reviewed and presented to the VRBPAC in 2001.
Although there were no standardized assays to test for oncogenic activity of DNA, the
Committee agreed that cell DNA and lysates should be evaluated in three newborn rodent
species. The species were newborn nude mice, newborn hamsters, and newborn rats.
These species were selected based on their broad sensitivity to oncogenic viruses. The
recommendations were to administer by the subcutaneous route in the three newborn
species 100 µg or more of DNA or a lysate prepared from 10
7
cells. Animals should be
monitored for more than four months, and any tumors would need to be assessed for their
species of origin to determine whether they arose spontaneously or were induced by the
test article. Such testing of cell lysates in animal species could also contribute to the
detection of oncogenic viruses capable of withstanding the cell lysis preparation.
The oncogenic and infectious risk of residual DNA in vaccines can be reduced by the
implementation of manufacturing steps designed to lower the amount of DNA, decrease
the size of the DNA, and/or to reduce the activity of residual DNA by chemical treatment
or gamma irradiation. Studies with BPL and benzonase demonstrated that DNA
17infectivity can be reduced by several orders of magnitude (83). Current
recommendations are that the level of residual cell-substrate DNA should be ≤10 ng per
dose and a median DNA size of 200 bp or lower.
Summary: Although current testing recommendations include evaluation of the
oncogenicity of host cell DNA and cell lysates in vivo, the oncogenic and infectious risk
of DNA is primarily addressed by lowering the amount of DNA, decreasing the size of
the DNA (by nuclease digestion), and/or by reducing the activity of the DNA (by
chemical treatment or gamma irradiation).
5. Summary
The current repertoire of cell substrates is inadequate for manufacture of certain types of
new vaccines. To address this issue, sponsors have proposed the use of several novel cell
substrates that are tumorigenic or derived from human tumors. In fact, it has been
recognized that such cell lines derived from tumors may be the optimal and in some cases
the only cell substrate that can be used to propagate certain vaccine viruses. However, at
the present time, only a limited number of vaccines made in tumorigenic cell lines have
entered clinical trials. In each case, the use of a specific tumorigenic cell substrate has
been discussed with the vaccines advisory committee before clinical trial initiation. The
purpose of the present VRBPAC meeting is to discuss the scientific issues associated
with the use of cell lines derived from human tumors for the production of both live and
inactivated prophylactic viral vaccines and to identify safety concerns and determine how
they can be addressed. The use of such cell lines for the manufacture of prophylactic
vaccines has not hitherto been discussed nor have such products entered clinical trials.
In previous discussions regarding the possible use of tumorigenic cell lines for vaccine
manufacture, three major safety concerns were identified that needed to be addressed: 1)
the presence of residual live cells in the vaccine that might have the potential of being
tumorigenic in humans; 2) the presence of residual DNA from the cell substrate; and 3)
the potential presence of adventitious agents, including adventitious viruses that might
have contributed to the tumorigenic phenotype. The current data suggest that these are
also the major issues associated with tumor-derived cells, including cell lines derived
from human tumors. In the current meeting of the VRBPAC, the committee will review
and discuss these issues and the acceptability of the use of human tumor-derived cell
substrates for production of preventive vaccines. In addition, the committee will discuss
the overall approach used to characterize such cell substrates, including the role of new
technologies in evaluating novel cell substrates.
APPENDIX 1
Prior VRBPAC Discussions and other Meetings on Cell Substrates
This appendix expands on information presented in the body of this briefing document.
It includes some additional details on discussions and meetings related to critical
decisions made regarding the use of new cell lines for vaccine manufacturing. Also
included are links to the transcripts of earlier VRBPAC meetings as described in the text.
In the 1970s, the first continuous cell line was used for the production of a vaccine
intended for the general public. Pasteur Mérieux Sérums & Vaccins (now known as
sanofi pasteur) used Vero cells, a continuous cell line derived from the kidneys of the
African green monkey by serial passage in vitro (101), for the manufacture of an
inactivated poliomyelitis vaccine (IPV) and then for the production of a live-attenuated
poliomyelitis oral vaccine (OPV). While both vaccines are licensed in Europe, only the
Vero-cell produced IPV is licensed in the US.
By the 1990s, additional types of cells were being considered for the production of new
vaccines such as those against agents of bioterrorism, novel infections viruses, and
pandemic influenza. The Office of Vaccines Research and Review (OVRR) in CBER
entered into internal discussions to identify what are the issues associated with the use of
cells that are tumorigenic or were derived from human tumors. The discussions resulted
in a Defined Risks Approach (50), which consists of identifying the risk events,
determining the frequency of these events by developing quantitative assays to measure
them experimentally, and then from these results estimating the likelihood of the risk
event. A component of this approach was to enter into a dialogue with the VRBPAC.
OVRR considered that this dialogue would be an ongoing enterprise both to inform the
committee as to OVRR’s evolving thinking on cell-substrate safety issues and to gain
their advice. The Defined Risks Approach was presented to the VRBPAC, in 1998, and
the transcripts of that meeting are available
(www.fda.gov/ohrms/dockets/ac/98/transcpt/3476t1.pdf). A component of the OVRR
approach was that an international meeting was to be organized at which the issues
identified were to be discussed, gaps in knowledge identified, and future studies to
address these gaps considered. The meeting was held in 1999; the proceedings were
published in the Developments in Biologicals in 2001 (11).
In 2000, a VRBPAC meeting was convened to discuss whether the use of the continuous
non-tumorigenic Vero cell line represented a safety concern for the production of live,
attenuated viral vaccines and if so how these concerns could be addressed. The
transcripts from this meeting are available
(www.fda.gov/ohrms/dockets/ac/00/transcripts/3616t2a.pdf
www.fda.gov/ohrms/dockets/ac/00/transcripts/3616t2b.pdf). Members of the VRBPAC
supported the use of the non-tumorigenic Vero cells for production of inactivated
vaccines; however, some members expressed some concern that, because Vero cells have
the capacity to become tumorigenic, they might not be suitable for the production of live,
attenuated vaccines until the mechanism whereby these cells become tumorigenic is
understood. The Committee recommended that OVRR investigate the mechanism
whereby the cells become tumorigenic to ascertain whether this capacity itself
19represented a safety concern. Nevertheless, with additional ttesting and discussions,
several live viral vaccines produced in Vero cells have been licensed in the United States
over the last decade: smallpox vaccine (2007) and two rotavirus vaccines (2006 and
2008). In addition, Vero cells are the substrate for a number of investigational vaccines.
In 2001, OVRR was asked to consider the first tumorigenic cell substrate for the
manufacture of a replication-defective adenovirus-vectored vaccine against HIV-1
infection. Propagation of replication-defective adenovirus type 5 (Ad5) vectors requires
a human cell line that expresses in trans the genes deleted in Ad5 vectors. The Ad5
vector under discussion expressed the transgene in place of the early region 1 (E1
region), and thus a human cell line that expresses E1A and E1B is necessary for vector
production. The first such line constructed for this purpose was the 293 line (29), which
contains a single copy of the left 12% of the Ad5 genome (84) and expresses E1A and
E1B. However, a problem with 293 cells for the production of Ad5-vectored products
was that a measurable level of replication-competent adenovirus (RCA) was generated by
recombination with the integrated Ad5 E1 region (33). This was considered undesirable
for prophylactic vaccines administered parenterally, and new lines were developed that
produced no measurable RCA, such as the PER.C6 line (20) for E1-deleted Ad5 vectors.
PER.C6 cells are tumorigenic, although dose-response tumorigenicity studies (35, 49, 86)
in the nude mouse have shown that they are “weakly” tumorigenic, since 10
7
cells are
required to form a tumor (28, 80).
The PER.C6 line and the Ad5 vectored HIV-1 vaccine were presented to the VRBPAC in
2001. This was the PER.C6 cell line generated by immortalizing human fetal retinal cells
by the early-region 1 (E1) of adenovirus type 5 (Ad5). For this discussion, the
Committee included several experts in tumorigenicity and oncogenicity. Issues
associated with the potential risks from DNA were discussed at the meeting. Based on a
comprehensive testing regimen for these cells, it was decided that these cells could be
used for the safe manufacture of replication-defective Ad5-vectored vaccines against
HIV-1. In addition to quantifying the tumorigenic phenotype of PER.C6 cells, the testing
involved evaluating the oncogenicity of PER.C6 DNA and cell lysates in newborn nude
mice, newborn hamsters, and newborn rats for oncogenic activity. This extensive
oncogenicity testing was done for the first time and, while the assays were not
standardized, the VRBPAC considered they were appropriate at the time. In addition,
testing for adventitious agents was more extensive than the usual in vivo and in vitro tests
recommended at the time and included a comprehensive list of specific PCR tests for
potential virus contaminants. Transcripts from that meeting are available
(www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_01.pdf
www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_02.pdf
www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_03.pdf
www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_04.pdf).
The next meeting to discuss safety issues associated with novel cell substrates occurred in
2004 in Rockville, MD, and was organized by the National Institutes of Health (NIH) and
the International Alliance for Biological Standardization (IABS). The proceedings were
published in 2006 in the Developments in Biologicals series (68). At that meetingfirst data on the oncogenicity of DNA obtained from studies conducted in the OVRR
were presented.
In 2005, tumorigenic Madin-Darby canine kidney (MDCK) cells were proposed for the
production of two inactivated influenza virus vaccines, and OVRR requested that the
VRBPAC evaluate their suitability for this purpose. The MDCK cell line is a
spontaneously immortalized cell line established from dog kidney in 1958 by Madin and
Darby, although there is no publication reporting its derivation. [However, kidney cell
lines were established by the same group from sheep and cattle, and these were described
(54, 55); thus, it is likely that similar methods and reagents were used to establish the
MDCK cell line.]
Over the years, conflicting data have been published as to whether this spontaneously
immortalized cell line is tumorigenic [reviewed in Omeir et al. (64)]. This confusion is
most likely due to the inherent behavior of the MDCK cells themselves, since it is known
that MDCK cells have an unstable phenotype with respect to several characteristics. For
example, their predominantly epithelial morphology can be changed to a fusiform cell
type by treatment with various cytokines such as hepatocyte growth factor (37), and they
can be induced to undergo epithelial to mesenchymal transition (EMT), a process that
occurs during normal development and one that also occurs during metastasis, in
response to growth factors (5, 44, 58, 71), transcription factors (63), or activated
oncogenes (16, 61, 62, 71). Nevertheless, what seems to be clear is that MDCK cells
currently available from the American Type Culture Collection are tumorigenic in both
adult and newborn athymic nude mice (64).
At a VRBPAC meeting in 2005, OVRR presented data on the use of both the infectivity
and oncogenicity of DNA to estimate risks and manufacturers presented data on the
vaccine purification process. Because of the demonstrated low level of residual small
size DNA in the final product and the fact that the vaccines were inactivated, the majority
of the Committee recommended that the use of tumorigenic cells did not represent a
significant risk. The transcripts from that VRBPAC meeting are available.
(www.fda.gov/ohrms/dockets/ac/05/transcripts/2005-4188t1.pdf)
In 2008, the VRBPAC were asked to consider the use of non-tumorigenic MDCK cells
for the manufacture of a live, attenuated influenza virus vaccine. Because this line of
MDCK cells was not tumorigenic, the level of DNA was low and reduced in size, and the
route of vaccine administration was intra nasal, the majority of the Committee supported
the evaluation of the vaccine in clinical trials. Nevertheless, it should be noted that
because some MDCK cell lines were tumorigenic, some members remained concerned
that residual MDCK DNA might escape digestion to a small size. The transcripts from
that VRBPAC meeting are available.
(www.fda.gov/ohrms/dockets/ac/08/transcripts/2008-4384T1_1.htm
www.fda.gov/ohrms/dockets/ac/08/transcripts/2008-4384T1_2.htm)
APPENDIX 2
Cell Substrate Recommended Testing from “Guidance from Industry:
Characterization and Qualification of Cell Substrates and Other Biological
Materials Used in the Production of Viral Vaccines for Infectious Disease
Indications”
Multi-stage testing with a variety of different assays is used to assure product safety and
ensure product quality. This includes testing all the biological materials in vaccine
manufacture, especially the initial components, which are the virus seed and the cell
substrate. Different stages for testing are selected based on the maximum likelihood of
adventitious agent detection and effect on product quality. This table shows a typical
testing scheme. Selected tests may be recommended on a case-by-case basis depending
upon the cell substrate and other biological materials used for virus propagation as well
as production conditions for vaccine manufacture
Table 1: Example of Biosafety Testing Scheme for Manufacture of a Viral Vaccine
1
tests may be different for cells versus intermediate and final products
Cell Testing Testing of Intermediates and Final Product

I watched the entire Oversite Committee meeting, called 3 of the US Reps offices plus the oversite comittee to tell them that Dr.Tenpenny's office said what Dr. Boyle said about"the Mercury has been taken out" "But she and the NIH man said they were not "Vaccines Experts". Dr.Tenenny's , a vaccine researcher and anti-some of the vaccines, due to the health issues she is seeing in her practice, in Cleveland, Ohio. Her office told me that What Dr. Boyle said is not true that Mercury is being put in all vaccines, and thennnnn, they try to take it out after putting it in , all except the Multi-vials.
http://google2.fda.gov/search?q=MERCURY+IN+VACCINES&client=FDAgov&site=FDAgov&lr=&proxystylesheet=FDAgov&output=xml_no_dtd&getfields=*

Common Ingredients in U.S. Licensed Vaccines
July 7, 2011
http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/VaccineSafety/ucm187810.htm
The vast majority of the over one billion doses of vaccines manufactured worldwide each year are given to healthy babies, children and adults. Thus, it is critical that vaccines be demonstrated to be safe and effective. FDA demands that vaccines undergo a rigorous and extensive development program in the laboratory, as well as in animal studies and human clinical trials, to determine their safety and effectiveness. Highly trained FDA scientists and clinicians carefully review all the information in a marketing application before approval and use by the public. Prior to licensure, as part of FDA’s review, FDA takes all the ingredients into account, including the active ingredients as well as other substances. After FDA approves a vaccine, FDA and CDC continuously monitor its safety.

Why is aluminum in some vaccines?
Aluminum salts are incorporated into some vaccine formulations as an adjuvant to enhance the immune response in the vaccinated individual. The aluminum salts in some U.S. licensed vaccines are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixed aluminum salts. For example: aluminum salts are used in DTaP vaccines, the pneumococcal conjugate vaccine, and hepatitis B vaccines.

Aluminum adjuvant containing vaccines have a demonstrated safety profile of over six decades of use and have only uncommonly been associated with severe local reactions. Of note, the most common source of exposure to aluminum is from eating food or drinking water.
The response to formaldehyde being put in vaccines is the sounds like a kindergarten explanation...."pickled frogs in formaldehyde its really water"....
Why is formaldehyde in some vaccines?
Are other adjuvants used in US vaccines?

Yes, there is one other adjuvant used in one U.S. licensed vaccine. Cervarix, a vaccine licensed by FDA on October 16, 2009, to prevent cervical cancer caused by human papillomavirus types 16 and 18, includes AS04 in its formulation. AS04 is a combination of aluminum hydroxide and monophosphoryl lipid A (MPL). MPL is a purified fat-like substance.

How does FDA evaluate adjuvants for safety and efficacy?
When evaluating a vaccine for safety and efficacy, FDA considers adjuvants as a component of the vaccine; they are not licensed separately.

Why are antibiotics in some vaccines?
Certain antibiotics may be used in some vaccine production to help prevent bacterial contamination during manufacturing. As a result, small amounts of antibiotics may be present in some vaccines. Because some antibiotics can cause severe allergic reactions in those children allergic to them (like hives, swelling at the back of the throat, and low blood pressure), some parents are concerned that antibiotics contained in vaccines might be harmful. However, antibiotics most likely to cause severe allergic reactions (e.g., penicillins, cephalosporins and sulfa drugs) are not used in vaccine production, and therefore are not contained in vaccines.

Examples of antibiotics used during vaccine manufacture include neomycin, polymyxin B, streptomycin and gentamicin. Some antibiotics used in vaccine production are present in the vaccine, either in very small amounts or they are undetectable. For example, antibiotics are used in some production methods for making inactivated influenza virus vaccines. They are used to reduce bacterial growth in eggs during processing steps, because eggs are not sterile products. The antibiotics that are used are reduced to very small or undetectable amounts during subsequent purification steps. The very small amounts of antibiotics contained in vaccines have not been clearly associated with severe allergic reactions.

Why is formaldehyde in some vaccines?
Formaldehyde has a long history of safe use in the manufacture of certain viral and bacterial vaccines. It is used to inactivate viruses so that they don’t cause disease (e.g., influenza virus to make influenza vaccine) and to detoxify bacterial toxins, such as the toxin used to make diphtheria vaccine. Formaldehyde is diluted during the vaccine manufacturing process, but residual quantities of formaldehyde may be found in some current vaccines. The average amount of formaldehyde to which a young infant could be exposed to at one time through vaccines is considered to be safe.

Formaldehyde is also produced naturally in the human body as a part of normal functions of the body to produce energy and build the basic materials needed for important life processes. This includes making amino acids, which are the building blocks of proteins that the body needs.

Formaldehyde is also found in the environment and is present in different ways. It is used in building materials, as a preservative in labs and to produce many household products.

The body continuously processes formaldehyde, both from what it makes on its own and from what it has been exposed to in the environment. The amount of formaldehyde in a person’s body depends on their weight; babies have lower amounts than adults. Studies have shown that for a newborn of average weight of 6 -8 pounds, the amount of formaldehyde in their body is 50-70 times higher than the upper amount that they could receive from a single dose of a vaccine or from vaccines administered over time (1,2,3).

Excessive exposure to formaldehyde may cause cancer, but the latest research has shown that the highest risk is from the air when formaldehyde is inhaled from breathing, and occurs more frequently in people who routinely use formaldehyde in their jobs. There is no evidence linking cancer to infrequent exposure to tiny amounts of formaldehyde via injection as occurs with vaccines.

(1) Heck HD, Casanova-Schmitz M, Dodd PB, Schachter EN, Witek TJ, Tosun T. 1985. Formaldehyde (CH2O) concentrations in the blood of humans and Fischer-344 rats exposed to CH2O under controlled conditions. Am Ind Hyg Assoc J 46(1):1-3.

(2) Cascieri TC, Clary JJ. 1992. Formaldehyde -- oral toxicity assessment. Comments Toxicol 4 (4): 295-304.

(3) Dhareshwar SS, Stella VJ. 2008. Your prodrug releases formaldehyde: should you be concerned? No! J Pharm Sci 97(10):4184-93.
Why are sugars, amino acids, and proteins added to some vaccines?
These materials may be added as stabilizers. They help protect the vaccine from adverse conditions such as the freeze-drying process, for those vaccines that are freeze dried. Stabilizers added to vaccines include: sugars such as sucrose and lactose, amino acids such as glycine or the monosodium salt of glutamic acid and proteins such as human serum albumin or gelatin. Sugars, amino acids and proteins are not unique to vaccines and are encountered in everyday life in the diet and are components that are in the body naturally.

Why are there preservatives in some vaccines?
Preservatives are added to vaccine formulations filled into multi-dose vials to prevent the growth of bacteria or fungi that may be introduced into the vaccine during its use, e.g., repeated puncture of the vaccine vial.

Why is fetal calf/bovine serum in some vaccines?

In the manufacture of viral vaccines, the virus may be grown in cells. These cells need a source of nutrition, which in some instances may be provided by fetal bovine serum.
WAIT TIL YOU SEE THAT THEY MAKE VACCINES OUT OF INJECTING THE VIRUS'S INTO HUMAN TUMORS , FETAL LUNG TISSUE, GREEN MONKEY AND CAININE/DOG KIDNEYS....I ASK WHERE ARE THEY GETTING THESES SPECIMENS FROM ? AND THEY ARE CONCERNED IT WILL CAUSE CANCER BY USING THEM BUT THEY STILL DO IT.....

CDC's Dr. Boyle provides valuable info! She writes, "For me the big issue is... clearly there is gross under-reporting." And Boyle gives us stats, 15% or more of the population.

There I agree, i.e., I'm seeing (informally) diseases in the autism spectrum can be indicated by several diagnostic parameters at an early age, as parents and medicos push symptoms under the rug in denial, no one able to handle the hot potatoes they created. Autism spectrum is very under-reported, and of course misnamed. Diagnostic indicators are manic facial expression, cat-like screams, slow head-tracking, little interest in real people, unresponsive to real people, and other signs. Autism parents are being told that "autistic spectrum is 1% of the population". I see it as 50% or much more if you compare ultrasound-vaccine infants with natural infants.

Dr. Boyle writes of statistical differences of 15%, compared to national surveys. Additionally, what about the parents and medicos who don't or can't admit problems to "national surveys"? I've seen those parents commonly, in denial. What about the ultrasound-vaccine dampened "spark of life" that is not measured? Do we have to wait for gross physical signs? All stats are low.

Jim West
NYC

Dr. Yazbek;
It is I that am thankful.

Jim Thompson;
Thanks for the link.
I have a lot of reading to do.
51 people there with impressive titles behind their names were there at that meeting, I am correct on that number?

So far much is about the mercury - all those impressive titles and they seem to be idiots. How can that be?

but there was this too:

"Aluminum salts have a very wide margin of safety.
Aluminum and mercury are often simultaneously administered to infants, both at the same site and at
different sites.
However, we also learned that there is absolutely no data,
including animal data, about the potential for synergy,
additivity or antagonism, all of which can occur in binary
metal mixtures that relate and allow us to draw any
conclusions from the simultaneous -----"

Well that should get to the point right there.

You know if these guys were talking like this back in late 70s or early 80s; I would say well bless their hearts --- but this was in 2000 and I hate them all!

Thank you Benedetta for your kind words; they are deeply appreciated.

John, as usual … you have unearthed valuable information.

Dr. Coleen Boyle remains very busy.

In 2002, after carefully explaining to the House Committee on Government reform all about the MMR/Thimerosal/Autism “Danish studies” Dr. Boyle stated:

“CDC remains committed to collecting accurate data on prevalence of autism and conducting studies to find causes of autism. Research is already underway, and more is planned, to look at the relationship between the MMR vaccine and autism. We want each child to be born healthy and to grow and develop normally, so that they are able to lead productive lives. We are dedicated to finding the answer to what causes autism and how it can be prevented. We will continue our work until the answer is known.”

http://www.hhs.gov/asl/testify/t020418.html

Ten years later, in a press release on March 29, 2012 announcing that the “CDC estimates 1 in 88 children in United States has been identified as having an autism spectrum disorder”, Dr. Boyle was quoted as saying “To understand more, we need to keep accelerating our research into risk factors and causes of autism spectrum disorders,”

http://www.cdc.gov/media/releases/2012/p0329_autism_disorder.html

In her 2002 address to the House Committee on Government Reform (above) Dr Boyle reported that “The CDC spent the following amounts on autism research: $943,000 in FY 1999, $2.6 million in FY 2000, $8.3 million in FY 2001, and plans to spend $11.3 million in FY 2002 and $10.2 million in FY 2003.”

It would be safe to predict that in 2013, the CDC will spend many more millions on autism research and that Dr. Boyle will still not know what causes autism even though she will still be convinced that it is neither a vaccine nor heavy metals … because after all, Danish studies said so.

I could cry. There's nothing like seeing evidence like this to bring home what we already suspected. This letter was from 2000 and my children were born in 2003. Every person who participated in this could have warned of it instead. My children were catastrophically injured because of fraud that Boyle obviously contributed to. I have friends whose children are dead and others whose kids have no future. Did they think we were going to let go of this?

I love Dr. Ed Yazbak; I love the stuff he wrote too! I keep finding such treasures after all these years that surprise me. Really smart -- I mean really intelligent people that should be running the CDC keep showing up with great work!

Thankyou John for reminding us that email.

Translation: We are so excited, after searching the world over we have finally found a scumbag researcher that would sell his soul, his mother's soul down the river and little kids too.
He'll be flying in tonight!

This is of course what they did also with the Madsen/MMR study. Even before the Simpsonwood meeting Marshalyn Yeargin-Allsopp was writing to José Cordero, then deputy head of the National Immunisation Program (email, 2.36pm May30, 2000).

'As we discussed on Friday, we have become aware through Poul Thorsen of an exciting opportunity to study the role of MMR vaccine and autism using several registries/existing studies and the repository of biologic specimens and laboratory capabilities in Denmark. Attached below is a proposal for such a study. Poul will be leaving on Thursday to travel to Denmark where he will be meeting with the PIs for the proposed study on June 6th. We would like to be able to have Poul say whether it is likely that CDC (NIP) can fund the study, if NIP is interested. The proposed budget is included; there may be additional sources of funding (in addition to NIP) but we are not certain at this time. Unfortunately, the DD Branch does not have much (if any) $$ to fund the study, but we do have the expertise that we have developed due to the autism surveillance in Atlanta and the MMR/autism case control study. I will be out of the office tomorrow, but you may contact Diana or Poul if you have questions. Thank you so much for considering this proposal.'

The data of this study was later reviewed by Goldman and Yazbak, and Stott, Blaxill and Wakefield in in Journal of American Physicians and Surgeons:

http://www.jpands.org/vol9no3/goldman.pdf

http://www.jpands.org/vol9no3/stott.pdf

but also in the Cochrane review of MMR in 2005 where it was stated:

"The follow up of diagnostic records ends one year (31 Dec 1999) after the last day of admission to the cohort. Because of the length of time from birth to diagnosis, it becomes increasingly unlikely that those born later in the cohort could have a diagnosis"

http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD004407.pub2/pdf/standard

Of course, as Ed Yazbak pointed out in Red Flags when he first reproduced the Yeargin-Allsopp letter the deputy director of NIP would not be the obvious person to approach if there was any danger that the study might find anything.

"The NIP Mission is described in 145 words that do not include the word autism. (11)
It is evident from the above e-mail that:
.. The “proposed” study had already been planned and discussed in Denmark, at the
DD (Developmental Disabilities) Branch of the CDC and with key NIP people
.. Dr. José Cordero had the authority to promptly approve NIP funding of the
“autism” study
.. Dr. Cordero who was responsible for the “planning, coordination, and conduct of
immunization activities nationwide” could not possibly appropriate funds - or in
any way support a study - that COULD potentially have compromised the MMR
vaccination program
.. Before the study was started, it was guaranteed NOT to find any connection
between MMR and regressive autism.
Drs. Diana Schendel, a CDC epidemiologist (Diana) and Dr. Poul Thorsen (Poul) went
on to co-author the study in question.
It is no wonder at all then that DS 2002 that was
1. Funded by the CDC
2. Co-authored by a CDC epidemiologist
3. And evidently supported by the CDC’s “expertise that we have developed due to the
autism surveillance in Atlanta and the MMR/autism case-control study” did indeed
conclude that MMR did not cause autism in Denmark and did justify the CDC’s financial
investment.
The “additional source of funding” in the US was the National Alliance for Autism
Research (NAAR). The organization has asserted: “To be clear, our unrestricted donation
of $25,000 from Merck in 2000 was not used to fund the Danish study – or any other
vaccine-related studies.” (12)"

http://www.poac-nova.org/news_uploads/332/DanishStudy2005.pdf

Yazbak's article was, of course, written long before we knew anything about Thorsen's alleged financial mis-dealings. But now we know that the method for masking the statistical correlation with the vaccine was also dreamt up at the CDC before the study was commenced. The pretence that the studies themselves were bona fide despite Thorsen's indescretions only becomes more outrageous as the years roll by - far too many years.

She mentions showing data with/without perinatal/congenital conditions such as prematurity....hmm, she's ALMOST saying, "those with perinatal/congenital conditions such as prematurity ARE AT INCREASED RISK OF VACCINE REACTION."

Almost.

Those of us who tried (and failed) to keep them from vaccinating our children who had perinatal/congential conditions can tell them a thing or two...

I have an email address for anyone who wants to write a complaint to Dr Frieden about Colleen Boyle. directors [email protected] She should be held accountable for her lies under oath.

Hera:
Exposure groups - of course -- what is wrong with me?

I guess because they have said so much that such a study could not be done because it was unethical -- that I thought they never considered the question -- I guess.

#4 your take on that was:
"Item 4 apparently relates to soci-economic status (SES) being a factor in early Autism diagnosis. She isn't very clear, so this is probably the most important key point she makes. Not sure if SES is related to Thimerosal"

Take into account the next sentence - the compliance rate of the vaccination rate.

Who would be the ones in our society that would be the most compliant?

Benedetta, The term exposure refers to the kids having received more or less Thimerosal. A nice euphemism; it sounds so much nicer than saying something like "The babies who were injected with more mercury" At the time this study was done, many CDC employees would not have understood what Thimerosal was, so why enlighten them?
Dr. Boyle is interested in the fact that babies who got less or no Thimerosal had low rates of speech delay and autism, so she is looking for "characteristics" of the families with lower rates of autism , - perhaps so that she can find some other explanation for the absence of autism. (It just CANT be the Thimerosal or the lack of it)
Edward, you seem to be correct about the year terms. The situation at the time of this study was that very few babies were diagnosed with autism before age 3 and most were diagnosed at age 4,5. So the correct way to do the study would have been to take , for example, two groups of 7 year olds, one group with high Thimerosal exposure, and one with low or none and then to record the outcomes. If you throw in two and three year old kids into the mix, the autism diagnoses will come down.
I dont think we can make too much out of this letter. Although the intent of the author seems fairly clear, the letter has been written in such a manner, that if questioned, the author can give all sorts of satisfactory answers, illustrating her immense desire to learn the truth about Thimerosal and autism. Remember friends, When it comes to autism, THE TRUTH IS A MOVING TARGET.

Forfeit their pensions? These highly educated people, entrusted with the nation's health, need to be tried for mass aggravated assault and sentenced to life in jail, and for treason, a capital offense !

Obviously, this gal is more than helpful. She's soliciting.

The issue with her second point relates to diluting data to make the association go away. She knows whatever statistical significance lies in the numbers, if they throw in a age group not likely to have a lot cases with Autism diagnoses in it, well..., there goes the statistical significance of what they are looking at for the entire study.

Apart from a few people claiming that their baby was "born with Autism" (maybe 1 in 10,000) the majority of ALL kids under the age of two could be considered lacking in such elementary social graces.

Attempting to throw in 18 month olds?

Experts agree that even though some so called Autism Speaks experts have the ability to diagnose Autism within mommy's womb, typically the signs of Autism do NOT present prior to being distinguishable from neuro-typical behavior.

Remember what the "core" issue with Autism is: problems with cognitive skills, language, speech, etc. The pharmaceutical companies continue to leverage old research against these core symptoms for the simple reason new research will expose the causal role their vaccines play in generating cases of Autism.

Item number three, she points out she already knows the results from years of secret surveillance at pharmaceutical company Autism "outposts" across the country that premmies are the hardest hit.

When the vaccine schedule was increased during the Clinton years in Washington that's when the Autism store fronts opened up, too. The same people that continue to fork out the cash to Thorsen are also the same people that opened up the Autism shops.

Doctors knew that infecting children with neurologically-active diseases such as Measles, Mumps, Rubella, or Varicella, was going to do something. There are "breakthrough" cases of Measles after vaccination all the time. That means the diseases in the vaccine vial were the cause of the outbreak.

It was standard knowledge a generation ago that a child exposed to Measles was a great risk for developmental delay if he or she already immune-compromised, also caught a second infection too soon after the initial infection with a neurologically-active disease virus.

There is enough evidence also to strongly suggest that negative neurological outcomes were also known to be associated with Thimerosal which is why vaccine trials pit a Thimerosal-containing vaccine against a Thimerosal-containing placebo. Poisoning the placebo with mercury to achieve balance in negative neurological outcomes is nothing new.

Item 4 apparently relates to soci-economic status (SES) being a factor in early Autism diagnosis. She isn't very clear, so this is probably the most important key point she makes. Not sure if SES is related to Thimerosal? Hmmm. Maybe vaccines were already being used as a genocidal weapon like Bill Gates talks about?

Item 5 is interesting. The Simpsonwood people thought they could include a case file study to verify their data. The transcripts mentioned that they even started pulling cases up and started examining them. They stopped and decided to not do that when they realized that the birth doses of the Hep B vaccine were mysteriously NOT being entered on neonatal birth records as well as they were when the shots were first on the market. Now why would a hospital not want to put Hep B birth doses into their records jackets? They certainly put it in their insurance billing records!

Item six she wants to know a lot about the zero exposure group.

If you were expected to make a trusty study, a lady like this would give you the creeps. No ifs, ands, or buts about it.

At Cass and Hera:

I'm reading her # 2 differently. She says most DD don't occur until the second or third year of life. A child in her second year of life is between 12 and 24 months. A child in her third year of life is between 24 and 36 months. If one accepts that most diagnosis occur in the second or third year of life, and age cutoff of 18 months or 2 years is perfectly consisten and appropriate. The 18 month old is in her second year of life and the two year old is in her third year of life.

To Edward: Because ASD is not diagnosed until after age 2, including kids younger than 2 (who might later become autistic) will artificially inflate the number of non-ASD kids for the statistical analysis and make the data look better. The translation of what she says is: Go fudge the data to make the statistics look better.

I assume Burton had this email. Wished he asked her about this an about many other emails.

It was nice watching her get whip-sawed around today. And surprising. Who knew we'd actually win a round?

At the next hearing, someone has to bring up Poul Thorsen next time CDC testifies. The CDC heads need to be confronted about Thorsen. Also the blatant conflicts of interest among members of the CDC's Advisory Committee for Immunization Practices (ACIP) and the fact that former head of the CDC, Julie Gerberding, now works for Merck's Gardisil division. CDC Administrators better get their priorities straight and remember who pays their salaries and it's not Merck.

I'm not sure I understand: What is inconsistent about the statement in #2?

Loved it when Posey busted Colleen Boyle for side-stepping his question on a vaccinated vs. unvaccinated study and said "You just wasted 2 minutes of my time!" It was clear how unimpressed many on the panel were with these two.

Boyle said Thimerosal is not in any vaccines except the flu multi shot. Not true. Thimerosal was in the H1N1 flu vaccine that the CDC pushed during the 2009 pandemic. As a matter of fact the CDC had to dispose of unused H1N1 vaccines in a hazardous waste landfill by order of the EPA because of the thimerosal content. What a short memory she has. I hope this significant little factoid is mentioned at the next committee hearing since I expect there will be many more.

Very creepy!

We can only pray that "what goes around, comes around" for her. I watched her squirm and avoid all important questions while pressing on that "better diagnosis" is the answer. My son was born 3 years after Simpsonwood. If the truth had come out then, I may have heard about it before my son's fate was sealed. Thanks to her, many children have suffered and new ones will begin the suffering. I don't know how she can sleep at night. Oh, wait, she doesn't have an autstic child keeping her up all night.