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

BIOLÐMS 0159 --- 26/4 press-set --- MB 21/5/99
318 P. R. Krause and A. M. Lewis, Jr
in the effect of multiple dosing of a product and of
size-fractionation of the residual DNA.
In monkeys, direct intramuscular injection of
200 mg of simian immunodeficiency virus (SIV)
provirus DNA (cloned into a lambda phage cloning
vector) led to SIV infection in three of four (75%)
monkeys.8 The retroviral sequences account for
about 25% of the total DNA in such phage.
Therefore, this quantity of DNA represents approxi-
mately 4⋅6 × 1012 retroviral genomes.³ It is not known
whether the infectivity of SIV provirus DNA is
similar to that of other viruses, or whether the
infectivity of retrovirus DNA is directly pro-
portional to its amount. We are not aware of data on
infectivity of viral genomic DNA delivered via other
routes. In the absence of other data, this number is
used as an initial estimate of the infectivity of
residual retrovirus DNA.
We next consider the number of potentially
``infectious'' virus genomes that could be present in
one microgram of cellular DNA. This quantity
represents the DNA from approximately 150 000
cells.§ The number of potentially infectious viral
genomes which could be present per cell may vary
for different viruses and cell lines, and could
potentially be determined experimentally for any
given situation. The possibility that there may also
be non-integrated virus DNA fragments in the final
product may also need to be considered.
Thus, the likelihood of an infection resulting from
intramuscular injection of 1 mg of cellular DNA that
contains a single viral genome per cell is:
(150 000 viral genomes/1 mg cellular DNA)/(4⋅6
× 1012 viral genomes/75% infection) = 2⋅5 × 10 − 8
or 1/40 000 000 infections per mg of cellular DNA
The population risk assoiated with a given
biological product may also depend on the number
of doses an individual is likely to receive (which can
³SIV provirus consists of approximately 10 000 bp, so
the molecular mass of SIV provirus DNA is approximately
10 000 bp × (660 g/mole/bp) = 6⋅6 × 106 g/mole. Fifty micro-
grams of provirus DNA (about 25% of the injected 200 mg
represented provirus) thus represents (50 × 10 − 6 g)/
(6⋅6 × 106 g/mole) = 7⋅6 × 10 − 12 moles. At 6⋅02 × 1023 molecules
per mole (Avogardro's number), this represents (7⋅6 × 10 − 12
moles) × (6⋅02 × 1023 molecules per mole) = 4⋅6 × 1012
molecules.
§Assuming 6 × 109 base pairs/cell and an average
molecular mass per base pair of 660 g/mole, there are
approximately 6⋅7 pg of DNA per cell. One microgram thus
represents 1 000 000 pg DNA/(6⋅7 pg DNA/cell) = 150 000
cells.
vary from one for a vaccine, to over 10 000 for
individuals who take therapeutic products three or
four times daily for a lifetime) and the actual size of
the DNA in the product (very large DNA pieces may
be less likely to enter cells, while if all DNA is
fragmented to a size smaller than the viral genome,
the risk of infection becomes vanishingly small).
Experimental evidence comparing the relative
likelihood of larger versus smaller injected DNA
fragments entering cells could be used to further
refine this estimate. If it can be demonstrated that
some of the DNA within a product is clearly smaller
than the size of a viral genome, then the risk of
infection is reduced by at least this factor. While
recombination events between non-replicating
retroviral subgenomes could theoretically occur to
yield a larger intact virus, the likelihood of this
occurring in the same cell under these circum-
stances appears to be even more remote.
Thus, for a product that contains viral DNA
sequences that are infectious for humans, we
propose that the risk associated with this poten-
tially infectious DNA may be estimated as the
product of the following variables:
, 2⋅5 × 10 − 8 infections per microgram DNA
, number of micrograms of DNA per dose of
product
, average number of viral genomes/cell
, percentage of DNA shown to be smaller than
viral genome
, number of doses given to an individual
This calculation may be illustrated by an example
of a vaccine produced in cells that contain viral
genomes. If there were 1 mg of DNA per dose of the
product, and 100 viral genomes per cell, the risk per
dose would be 1 in 400 000 if each individual were to
receive one dose of the product and there were no
data regarding the fractionation of DNA. Of course,
if estimates of any of the factors of this calculation
were in error, the actual risk could be different.
Other factors influencing the relevance of this
calculation include the susceptibility of human cells
to the specific virus represnted within the residual
DNA and the mode of administration (with oral
administration almost certainly less risky than
parenteral administration). In addition, this calcu-
lation assumes that the risk of infection increases in
a manner proportional to the number of adminis-
tered viral genomes. For any given situation, it
would be possible to improve on these estimates by
performing laboratory experiments to determine the
BIOLÐMS 0159 --- 26/4 press-set --- MB 21/5/99
Safety of viral DNA in biological products
total number of virus DNA copies (which could
either be integrated or not integrated) per cell.
Discussion
DNA from cells used to produce different biological
products vary in their potential to contain
infectious DNA sequences. Cells used to produce
inactivated retrovirus vaccines and other
retrovirus-based products would almost certainly
contain some quantity of potentially infectious
retroviral DNA. The number of viral genomes which
could be present per cell depends on the specific
product and its method of manufacture. For
example, for HIV infection in tissue culture,9 the
number of viral genomes per infected cell appears to
range between 10 and 100. HeLa cells contain
approximately 10±50 copies of the human papillo-
mavirus genome.10 We suggest that products with
potential to contain DNA sequences derived from
viruses known to infect humans may be considered
the most hazardous with respect to this issue.
Hybridoma cells produce murine retroviruses which
can be infectious for human cells,11 and which are
subsequently removed during manufacture of bio-
logical products. Chinese hamster ovary (CHO) cells
produce type A and type C retrovirus-like particles,
which have never been demonstrated to be
infectious for humans. These products are normally
subjected to purification techniques designed to
eliminate these retrovirus-like particles. If the risk
of the retrovirus-like particles is considered suffi-
cient to warrant purification, it follows that the risk
of DNA encoding those particles should also be
considered in evaluating the safety of those
products. There is no evidence that Vero and other
non-tumorigenic continuous cell lines contain
infectious DNA sequences, although the mechanism
by which these cells remain continuous is unknown.
One difficulty faced in the screening of cell lines for
potential latent viral genomes is that it is very
difficult to develop assays for viruses that have
not yet been discovered. Cell lines derived from
human or animal tumors could potentially contain
sequences derived from transforming viruses,
and their DNA could be viewed with suspicion
equivalent to or greater than that of CHO cells,
depending on their capacity to form tumours.
Human diploid cell lines such as WI-38 and MRC-5
cells have no evidence of containing endogenous
infectious DNA. Moreover, DNA from such cell
lines appears to carry no definable risk of
oncogenicity. These issues have not limited the
319
quantity of DNA from these cell lines in biological
products.
The differences between biological products with
respect to this issue suggest that the potential risk
could be calculated separately for each product,
depending on the factors listed above. In order to
facilitate manufacturing and development, it may be
useful to develop criteria for products produced in
any given cell line, based on worst-case scenarios
regarding the unknowns, with instructions for how
to change the limits based on characterization of the
size distribution of DNA in the product, and the
number of doses which a typical patient would
receive. Following the logic outlined here, it should
also be possible to asess risk of other DNA-related
infectious complications arising from biological
products.
At a potential risk per dose of 1 in 400 000 in one
example, the possibility that residual DNA could be
infectious should not be ignored. This may be a
higher than acceptable risk for some biological
products, especially vaccines that are to be
administered to large numbers of healthy children.
It would be difficult to determine the safety of
products with respect to this issue by laboratory
experiments in which the products are directly
injected into animals. Such experiments would
require millions of animals to provide statistically
significant assurances of safety for many biological
products, in which the number of doses anticipated
to be administered to humans may number in the
millions, and where safety factors exceeding one in
a million may need to be established. However,
appropriate animal studies might better estimate
components of the total risk. Before further
increases in limits of residual DNA in biological
products are considered, such studies could improve
assessments of the risks.
With previous limitations on cellular DNA
quantity in biological products, there has been no
documented transmission of viral agents to a
product recipient via infectious residual DNA. This
reassuring experience is consistent with the analy-
sis presented here, which imply that very low risk is
associated with the cell lines currently in use and
the low quantities of cellular DNA in most of these
products. However, this analysis suggests that as
pressure increases to use tumor cell lines to produce
biologicals, to develop novel products (such as
inactivated retroviral vaccines), and to increase the
permissible quantity of residual DNA in biological
products, regulatory decision-makers will need to
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
This analysis is based on experimental data
implying an ID50 of 038 mg for SIV DNA in monkeys.
We are now aware of additional murine data
consistent with an ID50 of 02.5 mg for a murine
retrovirus (Portis JL, McAtee FJ, Kayman SC.
Infectivity of retroviral DNA in vivo. J. AIDS 1992;
5: 1272) and of 04 ng for linearized polyomavirus
DNA (Israel MA, Chan HW, Hourihan SL, et al.
Biological activity of polyoma virus DNA in mice
and hamsters. J Virol 1979; 29: 990±996), consistent
with proportionally greater infectivity risks.
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Received for publication 3 July 1998;
accepted 26 October 1998