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REVIEW
Residual DNA Analysis in Biologics Development:
Review of Measurement and Quantitation
Technologies and Future Directions
Xing Wang,
1
Donna M. Morgan,
1
Gan Wang,
2
Ned M. Mozier
1
1
Pfizer, Inc., Global Biologics, 700 Chesterfield Parkway West Chesterfield, Missouri 63017;
telephone: 636-247-6349; fax: 636-247-5712; e-mail: ned.m.mozier@pfizer.com
2
Institute of Environmental Health Sciences, 259 Mack Avenue, Wayne State University,
Detroit, Minnesota
Received 21 June 2011; revision received 9 August 2011; accepted 19 September 2011
Published online 28 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23343
ABSTRACT: Residual DNA (rDNA) is comprised of deox-
yribonucleic acid (DNA) fragments and longer length mole-
cules originating from the host organism that may be present
in samples from recombinant biological processes. Although
similar in basic structural base pair units, rDNA may exist in
different sizes and physical forms. Interest in measuring
rDNA in recombinant products is based primarily on dem-
onstration of effective purification during manufacturing,
but also on some hypothetical concerns that, in rare cases,
depending on the host expression system, some DNA
sequences may be potentially infectious or oncogenic
(e.g., HIV virus and the Ras oncogene, respectively). Recent
studies suggest that a sequence known as long interspersed
nucleotide element-1 (LINE-1), widely distributed in the
mammalian genome, is active as a retrotransposon that can
be transcribed to RNA, reverse-transcribed into DNA and
inserts into a new site in genome. This integration process
could potentially disrupt critical gene functions or induce
tumorigenesis in mammals. Genomic DNA from microbial
sources, on the other hand, could add to risk of immuno-
genicity to the target recombinant protein being expressed,
due to the high CpG content and unmethylated DNA
sequence. For these and other reasons, it is necessary for
manufacturers to show clearance of DNA throughout pro-
duction processes and to confirm low levels in the final drug
substance using an appropriately specific and quantitative
analytical method. The heterogeneity of potential rDNA
sequences that might be makes the testing of all potential
analytes challenging. The most common methodology for
rDNA quantitation used currently is real-time polymerase
chain reaction (RT-PCR), a robust and proven technology.
Like most rDNA quantitation methods, the specificity of
RT-PCR is limited by the sequences to which the primers are
directed. To address this, primase-based whole genome
amplification is introduced herein. This paper will review
the recent advancement in rDNA quantitation and recent
findings regarding potential risks of immunogenicity, infec-
tivity, and oncogenicity of rDNA.
Biotechnol. Bioeng. 2012;109: 307–317.
ß2011 Wiley Periodicals, Inc.
KEYWORDS: residual DNA; real-time polymerase chain
reaction (RT-PCR); whole genome amplification (WGA);
immunogenicity; infectivity; oncogenicity
Introduction
Residual DNA (rDNA) is defined as the sum total of
deoxyribonucleic acid (DNA) and fragments present in
biological samples derived from recombinant host cells
during expression, sometimes it is also referred to as residual
host cell DNA. The potential risks associated with rDNA
are infectivity (through virus such as HIV), oncogenicity
(through oncogenes such as Ras), immunogenicity (through
CpG rich sequences from bacteria), and mutagenesis
(through transposons, retrotransposons, and DNA recom-
bination). The potential risks associated with the presence
of rDNA in products developed for human use, and a
regulatory requirement to confirm its clearance in biopro-
cesses, is the primary reason for rDNA levels in final drug
substances are closely monitored. Test methods need to be
accurate, sensitive, and quantitative to assure DNA is cleared
to specified levels. Throughout the history of the develop-
ment of biotechnological products, methods of analysis for
rDNA have evolved. Many different types of methods have
been developed and are still in use by manufacturers. A
review of these methods and current knowledge on the safety
of rDNA is described.
Regulatory Aspects
In a 1987 World Health Organization (WHO) guideline,
it was suggested that the total rDNA should not exceed
100 pg/dose (WHO, 1987). Later on, the limit was modified
Correspondence to: N.M. Mozier
ß2011 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 109, No. 2, February, 2012 307
to 10 ng per dose (Lebron et al., 2006; WHO, 1998), which is
commonly required by regulatory agencies. This differs from
host cell protein (HCP) impurities, which are handled case
by case (Champion et al., 2005; Wang et al., 2009). For low
dosage biologics (<1 mg/kg), a number of rDNA quantita-
tion methods have sufficient sensitivity. However, when
the dosage of biologics reaches 20–30 mg/kg or higher, it
becomes a challenge to develop a robust method even with
the most sensitive methods. Recent publications from both
FDA and WHO provide more updated guidelines and the
desire for a risk-based assessment of host cell impurities
(FDA, 2010; WHO, 2010).
Risk Assessment
Since rDNA could come from a wide variety of hosts, the
potential risks associated with them are also different.
Furthermore, rDNA may be present in a complex array of
size and composition, so risks can be difficult to generalize.
Nonetheless, many aspects of DNA risk have been studied,
including infectivity, oncogenicity, immunogenicity, and
mutagenesis. The following sections discuss rDNA risks in
more detail.
Virus Infectivity and Oncogenicity
The majority of published studies on rDNA infectivity and
oncogenicity are from Peden et al. (2004) at the Food and
Drug Administration (FDA). In a recent study on rDNA
oncogenicity, they used both H-ras and c-myc to study
tumor induction, and it was found that 12.5 mg each of
plasmid containing H-ras and c-myc were needed to induce
tumor growth in adult or newborn mice (Li et al., 2008). If
this information was put into the context of the whole
human genome, it translates to genomic DNA in the range
of 1–10 g to have similar cell transformation effect because
this test was using the specified gene only whereas in a
genomic DNA preparation this gene is only a very small
proportion of the whole genome. According to this model,
which suggests very high quantities of genomic DNA for
effect, the risk of rDNA for tumor induction appears to be
low. Future studies using different oncogenes and animal
models may expand our knowledge in this area. In a related
publication, for rDNA infectivity, HIV-1 (human immu-
nodeficiency virus type-1) was used as the transfection
agent, and two separate cell lines evaluated to test infectivity
(Li et al., 2009). It was found that linear DNA is more active
than supercoiled DNA, and rDNA at 2 mg was infectious.
These two studies suggested that virus infectivity is more
likely than oncogene transformation from rDNA. However,
direct comparison is confounded by the fact that the
infectivity test was carried out in cell lines whereas the
oncogenesis study was conducted in animals. However,
taken together these studies suggest that the suggested limit
of 10 ng per dose provides a significant safety factor (>10
5
)
for both infectivity and oncogenicity when taking into
consideration that the tests were performed using pure
oncogenes and HIV virus, respectively. An interesting model
was proposed recently to address the risks of residual host
cell DNA (Yang et al., 2010), it is expected that more studies
will be conducted in the future to address this important
issue in biologics development.
Immunogenicity of rDNA
When biologics are administered therapeutically, the
response of the human immune system may be unpredict-
able. The ‘‘cytokine storm’’ induced in the clinic by
TGN1412 indicated the serious consequences of unexpected
mammalian responses in response to a particular biother-
apeutic (Suntharalingam et al., 2006). This is believed to
be target/mechanism based, but underscores the interest in
de-risking biological drug candidates prior to clinical use.
Protein induced immunogenicity has been published
(Cavagnaro, 1995; Cohen et al., 2008; Dayan, 1995; Ryff
and Schellekens, 2002; Sgro, 1995; Thomas, 1995), but much
less is available regarding the capacity of rDNA to induce
immunogenicity. Immune responses have been reported to
high doses of specific nucleic acid sequences, for example,
CpG ODNs (oligodeoxynucleotides) when present as DNA
vaccine or adjuvants in preclinical and clinical studies
(Barouch et al., 2003; Klinman et al., 1997; Kojima et al.,
2002; Shoda et al., 2001; Stan et al., 2001; Tang et al., 2009;
Verfaillie et al., 2005). The induction of anti-DNA
antibodies has also been reported (Arfaj et al., 2007;
Bastian et al., 1985; Gilkeson et al., 1989; Karounos et al.,
1988; Pisetsky, 1997; Stollar, 1987). At some concentration,
all types of DNA are expected to elicit an immune response,
which depends on the source and type of DNA. These
studies have demonstrated that bacterial DNA is more
immunogenic than that from mammalian cells (Gilkeson
et al., 1989), in part due to the high ratio of CpG content in
bacteria and the fact that the CpG from bacteria is less
methylated than mammalian DNA (Klinman et al., 1997).
The extent of immune response in animals has been shown
to be proportional to the amount of CpG in a plasmid and
the ratio of CpG to all DNA present (Klinman et al., 1997;
Kojima et al., 2002). Currently, high CpG content plasmids
are used as adjuvant in DNA vaccine development to
enhance immune response from the host (Daftarian et al.,
2005; Ratanamart et al., 2007). In addition to earlier
evidence showing that CpG ODNs are recognized by TLR9
(Kojima et al., 2002); recognition of bacterial DNA is still a
matter of study and can occur by several pathways. TLR, a
family of pattern recognition receptors essential for innate
immunity (Kumar et al., 2009), recognizes CpG ODN by
TLR9 binding. Bacterial DNA can be recognized by its sugar
backbone especially the 20-deoxyribose, through an un-
known mechanism. In addition, study by Chiu et al. (2009)
suggested that DNA-dependent RNA polymerase III can act
as a DNA sensor linking bacterial or viral DNA to IFN-b
308 Biotechnology and Bioengineering, Vol. 109, No. 2, February, 2012
production and immune response, thus providing an
alternative route to the TLR9 pathway for DNA-induced
immune response.
To further evaluate the potential of rDNA to provoke
a primary innate immune response, we have employed a
human whole blood assay to test interleukin-6 (IL-6)
induction in response to exposure to DNA from Chinese
hamster ovary (CHO), NS0 (a myeloma cell line) and
Escherichia coli. Production of this pro-inflammatory
cytokine evaluates innate immunity, which may be a
precursor to acquired immunity leading to immunogenici-
ty. Cytokine release from the whole human blood has been
used extensively to evaluate immunotoxicity of drug
candidates (Groote et al., 1992; Langezaal et al., 2001;
Meager, 2006; Prabhakar et al., 2002; Vial and Descotes,
1995). These types of tests have also proved useful in
biologics development to evaluate the presence or confirm
the absence of host impurities (Wang et al., 2009). During
the evaluation of the method, it was found that the positive
control material, lipopolysaccharide (LPS), needs to be at
0.02 EU/mL or lower to get a negative IL-6 response in this
whole human blood assay (data not shown). Therefore,
rDNA from CHO, NS0, and E. coli were purified and cleared
of LPS with extensive extraction using LPS-removing
reagent. As shown in Figure 1, for all three different DNA
samples tested at 10 mg/mL, no IL-6 induction activities
were detected either in the presence or absence of
therapeutic proteins. In all the tests, the LPS levels were
<0.02 EU/mL by the limulus amebocyte lysate assay.
It is noted that although a rapid response in a whole
human blood assay mimics the innate response of the
human immune system, it does not necessarily predict
immunogenicity. IL-6 was monitored in these studies and
it is likely that other cytokine responses could be induced
at the levels of DNA tested; further testing with a wider
cytokine panel may be warranted. Another possible
explanation for the inability of fairly high levels of DNA
to elicit IL-6 production in our system is the fact that nucleic
acids are recognized in the endosome or cytosol instead of
on the cell surface where only the receptors for lipids
or protein immunogens exist (Kumar et al., 2009). It is
possible that during the relatively short incubation time, an
insufficient level of DNA was taken up by the cytokine-
producing cells. The evidence to support this argument is in
DNA vaccine development, where gene-bearing plasmids
delivered with a gene gun were required to elicit an immune
response in both preclinical and clinical settings (Leitner
et al., 2009). In contrast, this same human whole blood assay
system has been used successfully to monitor some HCPs at
low levels (data not shown).
High Density Transposable Elements From
Mammalian Genome
Sequencing of both the human and mouse genome indicated
that mammalians have a high percentage of their genome as
repetitive elements, and these repeats exist in a very high
copy number and widely distributed through the mammal
genome (Gibbs et al., 2004; Venter et al., 2001; Waterston
et al., 2002). All mammalians have essentially the same four
classes of transposable elements (Smit, 1993, 1999; Smit and
Riggs, 1996; Smit et al., 1995): class I. autonomous long
interspersed nucleotide element (LINE); class II. Short
interspersed nucleotide element; class III. Retrovirus-like
elements with terminal repeats (LTRs) and class IV. DNA
transposons. The first three classes are produced by reverse
transcription of an RNA intermediate (retroposition),
whereas DNA transposons move by a cut-and-paste
mechanism of DNA sequence. Repetitive sequences are
the single most prevalent feature of mammalian genomes
(Waterston et al., 2002). It is believed that these repetitive
sequences are the remains of transposable elements and
these transposable elements are a product of the evolution of
the genome. Sequencing has revealed that at least 50% and
probably more of the human genome is composed of repeat
sequences and those sequences share considerable homology
with the high copy repeats determined from both the rat and
mouse genome (Allen et al., 2003; Boyle et al., 1990). It is
recognized that as active elements, those repeats have
reshaped the genome by causing ectopic rearrangements,
creating entirely new genes, modifying, and reshuffling
existing genes and modulating overall GC content (Venter
et al., 2001). It is also known that most of the human and
mouse repeat sequences are derived from transposable
elements.
The risks to humans of exposure to these highly abundant
repeat elements remain unknown, but if there is rDNA
present in biological products, a relatively large proportion
of the DNA fragments would contain repetitive sequences.
Figure 1. IL-6 release testing of genomic DNA from E. coli, NS0, and CHO in the
human whole blood assay. Genomic DNA was added at 0.1, 1, and 10 mg/mL in the
absence and presence of monoclonal antibodies. In samples where monoclonal
antibodies we re added, the protein concentration was at 1 mg/mL. Whole blood
samples from three healthy donors (labeled 901F, 829M, and 918F, respectively) were
used for the testing. The IL-6 ELISA kit from R&D System was used for the assay.
Wang et al.: Residual DNA Analysis in Biologics 309
Biotechnology and Bioengineering
For example, if we follow the specification of 10 ng rDNA
per dose proposed by WHO, and also assume that the
average fragment of DNA in the final drug product is 5,000
base pairs and the average nucleotide pair has molecular
weight of 660 Dalton, then 10 ng of rDNA would have
910
8
copies of the repetitive elements. In addition, a
recent study in human neural progenitor cells indicated that
de novo L1 retrotransposition events may occur in the
human brain (Coufal et al., 2009), and it was suggested that
this process may contribute to individual somatic mosai-
cism where cells show different genetic composition in the
same organism. Many rDNA quantitation methods have
been developed and are capable of recognizing highly
repetitive sequences which have been confirmed to be
present in samples derived from bioprocesses (Lovatt, 2002;
Mehta and Keer, 2007; Venable et al., 2007). However, the
integration of repetitive sequences in mouse DNA has not
been shown to be integrated into human cell lines, so the risk
remains hypothetical.
Traditional rDNA Analysis
There are several methods that have been historically
used for the quantitation of rDNA in bioprocesses from
recombinant DNA technology. The first one is the
PicoGreen method. In this approach, a dye called
PicoGreen
1
binds to double stranded DNA and generates
fluorescence, whereas unbound dye emits little fluorescence
(DiPaolo et al., 1999). The advantage of the PicoGreen
1
method is its ease of use and low cost, and has some utility
for samples with relatively high levels of DNA (e.g.,
upstream bioprocess samples). However, PicoGreen
1
has
relatively poor sensitivity and can detect only about 0.2 ng of
a pure solution of DNA. Like all methods for rDNA, it is also
prone to interference and from other components (includ-
ing high recombinant protein) and does not give reliable
results unless DNA is extracted. The lack of sensitivity is the
major limitation, however, given the guideline from WHO
for not more than 10 ng rDNA per dose of drug. For this
reason alone the method is rarely suitable for final product
release, especially high dose proteins. However, low dose
proteins (e.g., vaccines), may be suitable if properly
validated and PicoGreen
1
offers the advantage of high
throughput and low cost.
Another technique used for rDNA quantitation is
hybridization. In this method, probes designed to recognize
specific target single stranded DNA sequences are labeled
with either radioactive tags or fluorescence dyes and
hybridized with denatured DNA samples immobilized on
a membrane. The amount of DNA is estimated from the
signal intensity relative to a DNA standard, which is
generally the genomic DNA from the host (Mehta and Keer,
2007). The advantage of the technology is that it is similar to
the Southern Blot widely used in molecular biology, no
special training is needed, and the cost is relatively low.
Another advantage is the size of the DNA can be estimated
since the DNA can be electrophoresed prior to blotting
alongside DNA standards of various molecular weights. Like
PicoGreen
1
, the major drawback of this method is the lack
of sensitivity (usually about 10 ng/mL). It also suffers from
relatively long testing time (48 h). Because of these
disadvantages, the method is no longer widely used in the
biotechnology industry.
Another common method used in the biotechnology
industry is Threshold
1
technology. In this method, a single-
stranded DNA binding protein (Chase and Williams, 1986)
is used to bind and capture denatured single stranded rDNA
and a monoclonal anti-DNA antibody (Kung et al., 1990)
conjugated to urease is used to detect the bound DNA. The
hydrolysis of urea by the enzyme produces a change in pH in
a small reaction chamber. The rate of change in pH is
therefore proportional to the amount of DNA in samples,
which are then calculated relative to signals from DNA
standards. The advantage of this method is its sensitivity (1–
3 pg) and standardized protocol; the disadvantage is the
relative high cost and low throughput. Another point to
consider for this assay is the limitation of the detectable size
of DNA fragments from biologics. Because this method
depends on the binding of the denatured DNA to the single-
stranded DNA binding protein and the monoclonal anti-
DNA molecule, DNA fragments <600 bp will be missed.
During bioprocessing it is possible that a DNA will be
sheared to smaller sizes during the treatment through filters
and chromatography columns.
Real-Time Polymerase Chain Reaction (RT-PCR)
for rDNA Analysis
Overview of RT-PCR Technology
Given the importance of rDNA testing in biologics
development, a sensitive, fast, and cost-efficient method
is needed. During the past several years, PCR-based
technology has been used for the determination of rDNA
and now becomes the most widely used method in the
biotechnology industry. During the PCR reaction, a
sequence-specific DNA template is amplified to produce
billions of copies within 1–2 h (Fig. 2); this technology thus
allows the detection of extremely low levels of DNA. The
earliest PCR methods were based on an end product
approach where the final PCR products were analyzed with
an agarose gel. This additional processing limited the
quantitative aspects of the method. A major advance was
realized with technologies capable of monitoring DNA
amplification in real time. (Gijsbers et al., 2005; Gregory
et al., 2001; Lahijani et al., 1998; Lovatt, 2002; Lovatt et al.,
2002; Smith et al., 1999; Wang et al., 2006). PCR products
are measured in the early stage of amplification in RT-PCR,
allowing the accurate quantitation of the rDNA in biologics
products because there is a high correlation between the
level of starting DNA and the signals detected in this early
exponential phase. If the DNA is quantified in the linear
310 Biotechnology and Bioengineering, Vol. 109, No. 2, February, 2012
phase or in the plateau phase, the results will be highly
variable (Fig. 3).
Several different chemistries are used in real-time PCR,
three of them are more commonly used and they are all
based on the use of fluorescent dyes. The first one is based on
double-strand DNA binding dyes, such as SYBR
1
Green
(Life Technologies, Foster City, CA). The background
fluorescence from SYBR
1
Green as a free dye is very low
when stimulated by an appropriate light wavelength. In
contrast, as SYBR
1
Green binds to the minor groove of the
double-stranded DNA generated by the PCR reaction, there
is a dramatic increase in fluorescence output, about 2,000
times stronger than the free dyes, therefore this assay has a
very good signal to noise ratio. The second assay chemistry is
based on the dye-primer based signaling systems. In this
case, the fluorescence of the dye is quenched in the primer
because of the special design like a hairpin. During real-time
PCR, this hairpin structure is converted to single-stranded
form and annealed to DNA template for extension. Ten-fold
increases in fluorescence could be observed from the hairpin
form to the linear form of the primer. The advantage of this
method is its multiplexing capability; several genes could be
monitored simultaneously. The third assay chemistry is
based on TaqMan
1
technology. In this case, a fluorescence-
labeled probe with a quencher molecule in the same
fragment is designed between two sequence-specific
primers. During PCR, the exonuclease activity of the
polymerase will cleave the probe, release the fluorescence
dye from the quencher, thus result in significant increase in
fluorescence, similar to the SYBR
1
Green technology,
TaqMan
1
has a signal to noise ratio above 1,000, making it a
very sensitive detection method. Because our effort in
developing the quantitative rDNA analysis was using the
TaqMan
1
technology, a widely used quantitative PCR in the
industry, more details were provided in the following
sections.
Selection of DNA Sequences for PCR Amplification
Since the TaqMan
1
technology depends on the amplifica-
tion of a defined DNA sequence, the first task in developing a
real-time PCR method for rDNA quantitation is to
determine which sequence to be used for the amplification.
Therefore, methods that can sensitively detect and quantify
rDNA need to be evaluated. In regards to the real-time PCR
technology, it is known that the initial copy number of target
DNA sequences is proportional to shorter times to achieve a
threshold signal. Therefore, it is advantageous to select DNA
sequences with as many copies as possible in the genome for
amplification. In the following section, the discussion of the
selection of specific DNA sequences is confined to E. coli,
NS0, and CHO, three of the most commonly used hosts for
production of biological products.
For E. coli rDNA quantitation, the most common DNA
sequences are the ribosomal RNA genes because each E. coli
genome contains seven copies each of the 5S, 16S, and 23S
ribosomal RNA genes (Gregory et al., 2001; Smith et al.,
1999; Wang et al., 2006) and they share very similar
sequences (Fig. 4). In our initial evaluation studies, both the
5S and 16S ribosomal RNA gene sequences were tested for
DNA amplification, and eventually we selected a DNA
sequence of the 16S gene as a target for the RT-PCR
amplification (Fig. 5). In our laboratory, 0.1 pg/mL of E. coli
DNA could be quantified using real-time PCR (Fig. 5),
this is at least 10 times more sensitive than immune assay
or hybridization described earlier. It should be pointed
out that the successful development of a sensitive real-time
PCR for E. coli rDNA quantitation depends on the
purification of high quality DNA as standards (Marmur,
1961).
Figure 2. Diagram of the PCR and its amplification capability.
Figure 3. Amplification curve of RT-PCR on CHO genomic DNA.
Wang et al.: Residual DNA Analysis in Biologics 311
Biotechnology and Bioengineering
For mammalian cell rDNA analysis, the selection of DNA
sequence for amplification was based on an analysis of rat
and mouse genomes. Several families of interspersed repeats
were found to be widely distributed in the mouse genome
and nearly 500,000 copies (Gibbs et al., 2004; Waterston
et al., 2002). Five families of those high copy elements
were evaluated: LINE-1, LINE-2, B1, B2, and LTR. After
alignment of the top 10 most homologous sequences in each
of the aforementioned family, it becomes apparent that one
family of repeats was most conserved in the mouse genome
among these five families. Primers and probes were designed
to recognize these sequences. It is noted that these repetitive
sequences are not as highly conserved as those for the E. coli
ribosomal RNA genes. To address these differences, two
probes were designed to increase the coverage of this
repetitive element family in the mouse genome. Using this
design, it was found that as low as 0.01 pg/mL of genomic
DNA could be quantified with real-time PCR (Fig. 6).
Overall, four amplicons (the fragment that is amplified by
Q-PCR) were designed and one of them showed the highest
sensitivity in the real-time PCR detection. Both NS0 and
CHO-derived genomic DNA were used to test the range of
detection, and it was found that the same amplicon provides
the highest sensitivity for both species of genomic DNA
(Table I). In our further robust testing, it was shown that
both the primers and probes were stable for more than 2
years without noticeable decrease of sensitivity if stored at
208C, and the genomic DNA prepared were also stable if
kept at 808C.
Comparison of RT-PCR With Traditional DNA
Quantitation Methods
Since the threshold method was used for the testing of
internal biologics under development, the transition of
testing method needs a careful bridging study. It was shown
from multiple bioprocesses that these two methods provide
parallel results at the upstream of biologics purification
when the rDNA level is relatively high. However, in
samples from several different stages of purification during
bioprocessing, the rDNA levels is not detectable using
Threshold
TM
yet can be quantified using RT-PCR (Fig. 7).
This is due to the 10improvement in sensitivity. These
data suggest that RT-PCR has comparable coverage of
rDNA species as compared to the traditional method
but with increased sensitivity. In light of the regulatory
agency’s guideline on the rDNA levels from biologics
(10 ng/dose), the sensitivity of RT-PCR provides greater
opportunity to measure at this level and achieve this target
for high dose therapeutics. RT-PCR has been validated
Figure 4. Sequence alignment of the seven E. coli 16S ribosomal RNA gene
sequence and the design of the amplicon for RT-PCR.
Figure 5. Standard curve of E. coli genomic DNA amplified with RT-PCR. Figure 6. Standard curve for CHO genomic DNA amplified with RT-PCR.
312 Biotechnology and Bioengineering, Vol. 109, No. 2, February, 2012
successfully according to the ICH guidelines for many late
stage projects.
Whole Genome Amplification for rDNA Analysis
Overview of pWGA Technology
As previously described, detection of DNA can be achieved
by hybridization-based DNA detection, immune-detection
or real-time PCR-based quantification, but all have draw-
backs. PCR-based detection is based on specificity for
unique representative sequences that do not represent the
entire genome; hence other sequences may not be detected if
not inclusive of the designated target sequences. This may be
important considering that bioprocesses can cause shearing
of DNA to smaller fragments and the PCR can only detect
rDNA if the target DNA sequences are present. Although
hybridization-based and antibody/affinity DNA detection is
designed to detect all potential DNA sequences that might be
present, the sensitivity of this technology is limited since,
unlike PCR, the DNA is not amplified. There are sequence
size restrictions for the antibody/affinity technology offered
by Threshold
1
. Alternative technologies have been investi-
gated, one of which is the whole genome amplification
(WGA) technology (Barker et al., 2004; Gribble et al., 2004;
Hosono et al., 2003; Thorstenson et al., 1998). The WGA
technologies use a different mode of amplification with
unique primers designed to limit the specificity. Like PCR,
all WGA approaches amplify small amounts of DNA to
achieve the required sensitivity. Although several WGA
systems have been marketed, we are not aware of any that is
currently in use for rDNA testing in biologics.
The Mechanism of WGA Technologies
The first WGA system developed was a PCR-based DNA
amplification system in which specific DNA sequences were
added to the ends of all DNA fragments by DNA ligase and
used as the origins for amplification by PCR (Barker et al.,
2004; Gribble et al., 2004; Thorstenson et al., 1998) By these
methods, all DNA fragments are amplified by PCR using
specific primers that bind to the added DNA sequences,
when amplification was performed in real time quantifica-
tion setting, the rDNA can be detected without sequence
preference or selectivity. Therefore, this WGA system
provides higher selectivity and sensitivity in detecting
rDNA from biologics than standard PCR technology. The
limitation of this technology is the effectiveness in adding
specific DNA sequences to all DNA fragments since the DNA
fragments without these sequences will not be amplified by
PCR protocol (and therefore not detected). The Sigma–
Aldrich’s GenomePlex
1
(Sigma-Aldrich, St. Louis, MO)
Complete WGA Kit is one example of this system that is
commercially available.
The second type of WGA system is designed to overcome
the DNA sequence requirement of the PCR technology using
degenerated (lower stringency) DNA primers (Barker et al.,
2004; Hosono et al., 2003). Since degenerated primers
can theoretically bind to a greater number of total DNA
sequences than higher stringency primers, it provides
relatively high uniform DNA amplification across the entire
genome with minimal sequence bias. In addition, this
technology may also benefit the rDNA detection through
overcoming DNA sequence preference encountered some-
times in the standard PCR protocol. In this system,
degenerated DNA primers, usually a hexamer, are used to
bind to DNA target(s), and therefore, broader coverage will
be achieved in comparison to that with specific primers. The
REPLI-g kit system developed by Qiagen Inc. is one example
of this system. The REPLI-g kit contains a degenerated
hexamer and a f29 polymerase for amplification. The
presence of hexamer in the amplification system theoreti-
cally enables any random DNA sequence in the reaction to
be amplified and the strand displacement activity of f29
DNA polymerase also allows amplification at isothermal
condition such as 308C, which minimizes DNA fragmenta-
tion relative to the much higher temperatures required for
standard PCR. In addition, the f29 DNA polymerase also
carries a 30!50exonuclease activity that enables proof-
reading capacity for more accurate amplification of target
DNA. The drawback of this system is that some sequence
binding preference may exist for the degenerate primer
that could result in amplification of certain DNA sequence
over other sequences. However, the ability to provide more
uniform DNA amplification from the entire genome with
Table I. Evaluation of different primer and probe pairs for Q-PCR of NS0
and CHO DNA.
Elements Q-PCR sensitivity, NS0 Q-PCR sensitivity, CHO
B1 mm þþþþ þþþþ
B2 mm þþþ þþþ
L1 mm þþþ
LTR þþþ
Figure 7. Comparison between threshold and RT-PCR for the determination of
rDNA from typical in-process samples.
Wang et al.: Residual DNA Analysis in Biologics 313
Biotechnology and Bioengineering
minimal sequence bias still makes this a very attractive
technology for rDNA detection.
The third type of the WGA system is based on the E. coli
DNA replication system. In this system, primase is used to
replace the Taq DNA polymerase. A DNA helicase is also
utilized. The presence of the primase eliminates any primer
requirement for initiation of DNA amplification. The
presence of a DNA helicase enables double-stranded DNA
targets to unwind at 378C so that the DNA amplification can
also take place at the 378C. In addition, the presence of
primase also allows multiple initiation sites for more
effective amplification of target DNA. The Rapisome
TM
pWGA, developed by BioHelix Inc. (Beverly, MA) is one
example of this type of WGA system (Fig. 8). The
Rapisome
TM
pWGA system, in combination with the real
time DNA quantification technology, provides an attractive
methodology for real time detection of rDNA with high
sensitivity and efficiency.
Evaluation of the pWGA System for Detection of Low
Level rDNA in Biologics
Because of the potential of the Rapisome
TM
pWGA system
in amplifying DNA with high efficiency, we have evaluated
the feasibility of using the Rapisome
TM
pWGA system for
detecting rDNA in low levels in biologics samples. First, we
tested the capability of the pWGA system to amplify low
levels of DNA. The human genomic DNA was diluted from
1mg/mL to 1 pg/mL and 5 mL of the diluted DNA was
incubated with 20 mL of the Rapisome
TM
pWGA mix at
378C for 2 h. The amplified DNA samples were analyzed by
agarose gel electrophoresis using a 0.8% gel (Fig. 9). The
pWGA system effectively detected the human genomic DNA
with the DNA in concentrations as low as 1 pg/mL in 2 h
incubation time. To further test its feasibility for real time
quantification of rDNA in biologics, we further adapted
the Rapisome
TM
pWGA system for real time amplification
of the human DNA using the ABI 7500 RT-PCR system (Life
Technologies, Carlsbad, CA) (Fig. 10). The EvaGreen
TM
dye was used with the Rapisome
TM
pWGA system for
effective detection of amplified DNA in the real time DNA
quantification, the reason to use this dye instead of SYBR is
that EvaGreen
TM
could provide a stronger signal thus
increase the sensitivity of the detection. The human DNA
Figure 8. The model of whole genome amplification by the Rapidsome
TM
pWGA
system.
Figure 9. Detection of human genomic DNA amplified by the Rapidsome
TM
pWGA system using agarose gel. The human genomic DNA was diluted in TE buffer.
The indicated amount of genomic DNA was incubated with 20 ml Rapidsome
TM
pWGA
mix at 378C for 2 h. The reactants were analyzed by agarose gel electrophoresis using
a 0.8% gel. The DNA was visualized under UV light and documented by a Kodak DC290
gel documentation system. Lane 1, 2 mg human genomic DNA; lane 2, 1 ng human
genomic DNA amplified by the pWGA system; lane 3, 100 pg human genomic DNA
amplified by the pWGA system; lane 4, 10 pg genomic DNA amplified by the pWGA
system; lane 5, 1 pg human genomic DNA amplified by the pWGA system; lane 6, 100 fg
mouse genomic DNA amplified by the pWGA system; lane 7, 10 fg human genomic DNA
amplified by the pWGA system; and lane 8, 1 fg human genomic DNA amplified by the
pWGA system.
314 Biotechnology and Bioengineering, Vol. 109, No. 2, February, 2012
was diluted into a series of dilution (1 mg/mL 1 pg/mL)
and was incubated with the Rapisome
TM
pWGA master mix
in order to detect the level of DNA by the ABI 7500 Real time
quantification system (Fig. 10). Because the reaction is
processed in an isothermal temperature, the real time
quantification of the DNA was done with 50 cycles of 368C
for 1 min and 378C for 1 min and the threshold was
determined for each DNA samples. The standard curve was
generated from the threshold value of the diluted DNA
samples. The results obtained from our experiments
revealed a linear correlation between the level of DNA
and the Ct value of the DNA in the reaction from the
concentrations ranges of 1–10 pg/mL. Given the 10 ng
rDNA/per dose of biologics recommended by the WHO’s
guideline, the sensitivity of the pWGA system in detecting
rDNA is well below the limits of the suggested rDNA level in
most doses of biologics. Therefore, the pWGA system
provides an attractive system for effective measurement of
low levels of rDNA in the biologics. Although more studies
are needed to determine the feasibility of the pWGA system
in detecting rDNA from biologics, the results of these
preliminary studies provide strong evidence to suggest the
possibility of applying the pWGA system to DNA detection
for effective detection of rDNA from biologics.
Conclusions
The detection and quantitation of rDNA is required for all
biologics in development, and high dose drugs can be
challenging to test with adequate sensitivity. Similar to the
analysis of HCPs, rDNA is treated as a host related impurity,
and demonstration of clearance in bioprocesses to accept-
able and/or consistent levels is a major parameter in
biologics development. Of the theoretical rDNA risks
discussed here, a knowledge gap still exist to evaluate the
potential risk of the insertional mutation induced by the
transposable retrotransposons that are widely present in the
mouse genome. For other risks, there are limited means for
evaluation, and future studies may elucidate these aspects.
Until then, detection and monitoring of rDNA will be
paramount in the recombinant DNA field.
Different from HCPs, where a vast heterogeneity in both
size and charge makes monitoring and clearance highly
challenging, DNA molecules have similar basic structure
and highly negative charge. Exploitation of this property
with chromatographic and charge-based separation allows
clearance of rDNA several orders of magnitude more
efficiently than that of HCPs. However, it is still possible
that certain biologics or a certain isoforms of particular
recombinant proteins, including those with net positive
charge, possess a strong affinity to DNA, similar to the DNA
binding proteins like zinc finger and leucine zipper
(Ellenberger et al., 1992; Theunissen et al., 1992) that could
make the clearance of rDNA relatively difficult. In our
experience with dozens of different biologics processes,
rDNA has been cleared to parts per billion levels or less. The
detection and quantitation of rDNA has evolved signifi-
cantly over the last decade. The DNA hybridization and
immunoaffinity-based Threshold
TM
method provided valu-
able information in the early days of biologics development,
whereas more recently RT-PCR has gained prevalence
because of its higher sensitivity, throughput and lower cost.
Based on the recent studies using whole genome amplifica-
tion, it is now possible that this novel technology may prove
ideal for the analysis of rDNA due to its sensitivity similar
to RT-PCR, improved sequence coverage, however more
studies need to be done in this area before the adaptation of
this technology for rDNA analysis in biologics.
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