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BIOLÐMS 0159 --- 26/4 press-set --- MB 21/5/99

Biologicals (1998) 26, 317±320

Article No. bg980161

Safety of Viral DNA in Biological Products*

Philip R. Krause† and Andrew M. Lewis, Jr
Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD, U.S.A.

Abstract. Data from studies of the infectivity of DNA injected directly into laboratory animals are used
to estimate the potential infectivity risk of residual DNA in biological products. The potential for some
novel products to contain infectious quantities of residual cellular DNA is discussed, and further study
of this subject is suggested.
= 1998 The International Association of Biological Standardization

Introduction viral genome could initiate an infection if it were to

enter cells of a recipient of a biological product. In
Previous evaluation of the safety of residual cellular
this regard, recent developments in plasmid DNA
DNA in biological products has concentrated on the
vaccines showed that injected DNA has a small but
risk of the DNA integrating into the chromosomes
finite probability of entering cells,7 although the
of a product recipient, leading to possible disruption
efficiency of entry for larger DNAs is not
of cellular suppressor genes or activation of
well-defined. Potential sources of such viral DNA in
oncogenes, with tumorigenesis as a possible conse-
vaccine production include genomic DNA produced
quence.1±4 Because the risk of such events is now
during the replication of viruses (for example,
perceived to be very low, the previously accepted5
retroviruses, herpesviruses, papillomaviruses,
limit of 100 pg per dose of DNA from continuous cell
polyomaviruses, or adenoviruses) in tissue culture
lines has been increased to 10 ng and a proposal has
and genomes of viruses infectious to humans
been made to consider DNA as an impurity, rather
that may be present as endogenous integrated
than a risk factor.6
sequences (e.g. retroviruses, papillomaviruses, or
As innovation in the development and manufac-
polyomaviruses) in cell substrates derived from
ture of new biologicals requires the use of
tumors or other sources.
increasingly diverse and sometimes novel cell
For live-attenuated virus vaccines, the presence
substrates, we believe it is necessary and useful to
of vaccine-strain nucleic acids might not present a
consider the possibility that residual cellular DNA
risk greater than that of the live vaccine strain.
could be infectious. In this communication, we
Thus, unintended consequences might be more
introduce this possibility and discuss one approach
likely with inactivated vaccines, for which no
to defining the risks associated with DNA
infection risk is usually assumed. Injected infectious
infectivity. This approach is proposed only to
residual DNA derived from retroviruses could lead
introduce the issue and provide a framework for
to oncogenic or immunosuppressive consequences.
further deliberation and collection of relevant data.
For some viruses, a very low rate of infectious
complications may be accepted in an immunocom-
Infectious DNA: theoretical concerns petent population, but administration of such
products to immunocompromised patients may raise
Residual DNA could become infectious in the
additional concerns.
production of viral vaccines, either for DNA viruses
or RNA viruses that replicate through DNA
intermediates. Residual DNA encoding an entire Infectious DNA: modelling risk
To model the potential risk associated with residual
*The opinions expressed in this article are those of the
authors. No official endorsement by FDA is implied or DNA, we estimate the infectivity of such DNA,
should be inferred. calculate the quantity of such DNA that is likely to
²To whom correspondence should be addressed. be in a single dose of a biological product, and factor

1045±1056/99/040317 + 04 $30.00/0 7 1998 The International Association of Biological Standardization

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318 P. R. Krause and A. M. Lewis, Jr

in the effect of multiple dosing of a product and of vary from one for a vaccine, to over 10 000 for
size-fractionation of the residual DNA. individuals who take therapeutic products three or
In monkeys, direct intramuscular injection of four times daily for a lifetime) and the actual size of
200 mg of simian immunodeficiency virus (SIV) the DNA in the product (very large DNA pieces may
provirus DNA (cloned into a lambda phage cloning be less likely to enter cells, while if all DNA is
vector) led to SIV infection in three of four (75%) fragmented to a size smaller than the viral genome,
monkeys.8 The retroviral sequences account for the risk of infection becomes vanishingly small).
about 25% of the total DNA in such phage. Experimental evidence comparing the relative
Therefore, this quantity of DNA represents approxi- likelihood of larger versus smaller injected DNA
mately 4⋅6 × 1012 retroviral genomes.³ It is not known fragments entering cells could be used to further
whether the infectivity of SIV provirus DNA is refine this estimate. If it can be demonstrated that
similar to that of other viruses, or whether the some of the DNA within a product is clearly smaller
infectivity of retrovirus DNA is directly pro- than the size of a viral genome, then the risk of
portional to its amount. We are not aware of data on infection is reduced by at least this factor. While
infectivity of viral genomic DNA delivered via other recombination events between non-replicating
routes. In the absence of other data, this number is retroviral subgenomes could theoretically occur to
used as an initial estimate of the infectivity of yield a larger intact virus, the likelihood of this
residual retrovirus DNA. occurring in the same cell under these circum-
We next consider the number of potentially stances appears to be even more remote.
``infectious'' virus genomes that could be present in Thus, for a product that contains viral DNA
one microgram of cellular DNA. This quantity sequences that are infectious for humans, we
represents the DNA from approximately 150 000 propose that the risk associated with this poten-
cells.§ The number of potentially infectious viral tially infectious DNA may be estimated as the
genomes which could be present per cell may vary product of the following variables:
for different viruses and cell lines, and could
potentially be determined experimentally for any , 2⋅5 × 10 − 8 infections per microgram DNA
given situation. The possibility that there may also , number of micrograms of DNA per dose of
be non-integrated virus DNA fragments in the final product
product may also need to be considered. , average number of viral genomes/cell
Thus, the likelihood of an infection resulting from , percentage of DNA shown to be smaller than
intramuscular injection of 1 mg of cellular DNA that viral genome
contains a single viral genome per cell is: , number of doses given to an individual
(150 000 viral genomes/1 mg cellular DNA)/(4⋅6
This calculation may be illustrated by an example
× 1012 viral genomes/75% infection) = 2⋅5 × 10 − 8 of a vaccine produced in cells that contain viral
genomes. If there were 1 mg of DNA per dose of the
or 1/40 000 000 infections per mg of cellular DNA
product, and 100 viral genomes per cell, the risk per
The population risk assoiated with a given dose would be 1 in 400 000 if each individual were to
biological product may also depend on the number receive one dose of the product and there were no
of doses an individual is likely to receive (which can data regarding the fractionation of DNA. Of course,
if estimates of any of the factors of this calculation
³SIV provirus consists of approximately 10 000 bp, so were in error, the actual risk could be different.
the molecular mass of SIV provirus DNA is approximately Other factors influencing the relevance of this
10 000 bp × (660 g/mole/bp) = 6⋅6 × 106 g/mole. Fifty micro- calculation include the susceptibility of human cells
grams of provirus DNA (about 25% of the injected 200 mg to the specific virus represnted within the residual
represented provirus) thus represents (50 × 10 − 6 g)/
(6⋅6 × 106 g/mole) = 7⋅6 × 10 − 12 moles. At 6⋅02 × 1023 molecules DNA and the mode of administration (with oral
per mole (Avogardro's number), this represents (7⋅6 × 10 − 12 administration almost certainly less risky than
moles) × (6⋅02 × 1023 molecules per mole) = 4⋅6 × 1012 parenteral administration). In addition, this calcu-
molecules. lation assumes that the risk of infection increases in
§Assuming 6 × 109 base pairs/cell and an average a manner proportional to the number of adminis-
molecular mass per base pair of 660 g/mole, there are
approximately 6⋅7 pg of DNA per cell. One microgram thus tered viral genomes. For any given situation, it
represents 1 000 000 pg DNA/(6⋅7 pg DNA/cell) = 150 000 would be possible to improve on these estimates by
cells. performing laboratory experiments to determine the
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Safety of viral DNA in biological products 319

total number of virus DNA copies (which could quantity of DNA from these cell lines in biological
either be integrated or not integrated) per cell. products.
The differences between biological products with
respect to this issue suggest that the potential risk
could be calculated separately for each product,
DNA from cells used to produce different biological depending on the factors listed above. In order to
products vary in their potential to contain facilitate manufacturing and development, it may be
infectious DNA sequences. Cells used to produce useful to develop criteria for products produced in
inactivated retrovirus vaccines and other any given cell line, based on worst-case scenarios
retrovirus-based products would almost certainly regarding the unknowns, with instructions for how
contain some quantity of potentially infectious to change the limits based on characterization of the
retroviral DNA. The number of viral genomes which size distribution of DNA in the product, and the
could be present per cell depends on the specific number of doses which a typical patient would
product and its method of manufacture. For receive. Following the logic outlined here, it should
example, for HIV infection in tissue culture,9 the also be possible to asess risk of other DNA-related
number of viral genomes per infected cell appears to infectious complications arising from biological
range between 10 and 100. HeLa cells contain products.
approximately 10±50 copies of the human papillo- At a potential risk per dose of 1 in 400 000 in one
mavirus genome.10 We suggest that products with example, the possibility that residual DNA could be
potential to contain DNA sequences derived from infectious should not be ignored. This may be a
viruses known to infect humans may be considered higher than acceptable risk for some biological
the most hazardous with respect to this issue. products, especially vaccines that are to be
Hybridoma cells produce murine retroviruses which administered to large numbers of healthy children.
can be infectious for human cells,11 and which are It would be difficult to determine the safety of
subsequently removed during manufacture of bio- products with respect to this issue by laboratory
logical products. Chinese hamster ovary (CHO) cells experiments in which the products are directly
produce type A and type C retrovirus-like particles, injected into animals. Such experiments would
which have never been demonstrated to be require millions of animals to provide statistically
infectious for humans. These products are normally significant assurances of safety for many biological
subjected to purification techniques designed to products, in which the number of doses anticipated
eliminate these retrovirus-like particles. If the risk to be administered to humans may number in the
of the retrovirus-like particles is considered suffi- millions, and where safety factors exceeding one in
cient to warrant purification, it follows that the risk a million may need to be established. However,
of DNA encoding those particles should also be appropriate animal studies might better estimate
considered in evaluating the safety of those components of the total risk. Before further
products. There is no evidence that Vero and other increases in limits of residual DNA in biological
non-tumorigenic continuous cell lines contain products are considered, such studies could improve
infectious DNA sequences, although the mechanism assessments of the risks.
by which these cells remain continuous is unknown. With previous limitations on cellular DNA
One difficulty faced in the screening of cell lines for quantity in biological products, there has been no
potential latent viral genomes is that it is very documented transmission of viral agents to a
difficult to develop assays for viruses that have product recipient via infectious residual DNA. This
not yet been discovered. Cell lines derived from reassuring experience is consistent with the analy-
human or animal tumors could potentially contain sis presented here, which imply that very low risk is
sequences derived from transforming viruses, associated with the cell lines currently in use and
and their DNA could be viewed with suspicion the low quantities of cellular DNA in most of these
equivalent to or greater than that of CHO cells, products. However, this analysis suggests that as
depending on their capacity to form tumours. pressure increases to use tumor cell lines to produce
Human diploid cell lines such as WI-38 and MRC-5 biologicals, to develop novel products (such as
cells have no evidence of containing endogenous inactivated retroviral vaccines), and to increase the
infectious DNA. Moreover, DNA from such cell permissible quantity of residual DNA in biological
lines appears to carry no definable risk of products, regulatory decision-makers will need to
oncogenicity. These issues have not limited the consider the potential for this DNA to be infectious.
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320 P. R. Krause and A. M. Lewis, Jr

Note added in proof 4. Center for Biologics Evaluation and Research. Points
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This analysis is based on experimental data of Infectious Diseases, August 1996.
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Health Organization, Geneva, 1987.
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DNA (Israel MA, Chan HW, Hourihan SL, et al. 1465±1468.
8. Letvin NL, Lord CI, King NW et al. Risks of handling
Biological activity of polyoma virus DNA in mice HIV. Nature 1991; 349: 573.
and hamsters. J Virol 1979; 29: 990±996), consistent 9. Wood R, Dong H, Katzenstein DA et al. Quantifi-
with proportionally greater infectivity risks. cation and comparison of HIV-1 proviral load in
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T Cells. J AIDS 1993; 6: 237±240.
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