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MagNA Pure System Application Note No. 4
Highly Sensitive and Robust
Automated DNA Extraction from
Pharma Manufacturing In-Process
Quality Control (QC) Samples using
the MagNA Pure LC 2.0 System
Dr. Jutta Mayr und Marina Wagner
Roche Diagnostics GmbH, Penzberg, Germany
Pharma Division
September 2011
1 Abstract
To ensure final product safety, clearance of host cell DNA
from the drug substance is essential in the manufacturing
process for therapeutic proteins and monoclonal antibody
(MAB) drugs. We have developed and validated an
automated process for monitoring clearance of Chinese
Hamster Ovary (CHO) DNA impurities during the
capture and polishing steps of downstream processing.
This method, which eliminates manual steps in DNA
extraction and subsequent qPCR analyses, can help overcome
the analytics bottleneck during process development and
routine testing. DNA extraction with Roche’s MagNA Pure LC
System offers excellent DNA recovery rates and robustness
towards matrix effects for subsequent highly sensitive
quantitative PCR. The MagNA Pure LC System eliminates
manual dilution of high protein or DNA loads, manual
neutralization of samples from acidic protein purification,
and manual addition of carrier RNA. Here, we show
validation data from in-process control QC samples
collected during the pharmaceutical manufacturing process
that were spiked with defined amounts of CHO DNA, and
from in-process controls, using Roche’s MagNA Pure LC
System and Qiagen’s QIAcube Instrument.
For general laboratory use.
2 Introduction
Roche’s Penzberg production site is a center of competence
for therapeutic proteins (see Figure 1). The focus of
therapeutic protein production is the manufacturing of
life-sustaining drugs for oncology, anemia and virology.
The removal of host cell protein and host cell DNA during
downstream processing of therapeutic proteins is required
to guarantee high quality and purity of the biopharmaceutical
product (Figure 2). Both process developers and analysts
working in quality control have to demonstrate the clearance
of DNA impurities from the CHO (Chinese Hamster Ovary)
production cell lines.
Figure 1: Fermentation at the Roche plant in Penzberg, Germany.
In-Process-(Quality) Controls (IPC) check for clearance of
residual CHO DNA contamination:
MagNA Pure LC
Instrument
QIAcube
Instrument
180 min
32 Samples
90 min
12 Samples
Proteinase K digestion included/
automated
N o dilution of samples needed
Fermentation
Bulk
Purification
N o carrier RNA needed
No neutralization of pH needed
(~10% acidic samples)
All reagents contained in kit and
ready to use
Increasing sensitivity need
A utomated PCR Setup
Run Duration
Less hands-on-time and reagents
Sensitivity (DL/QL)
Accuracy
Standard protein content
(up to 25mg/ml)
Roche/Qiagen
comparable
Roche/Qiagen
comparable
High protein content
(up to 150 mg/ml)
Roche better
than Qiagen
Roche better
than Qiagen
LightCycler ®Instrument
Loading of standard curves/
only 1 standard per run
Less hands-on-time and reagents
Figure 2: The workflow schematic shows fermentation manufacturing and the chromatographic purification process of a therapeutic antibody.
Repeated testing is necessary to demonstrate removal of CHO DNA during the production, which indicates a high quality and high purity of the final drug
product. Using Roche systems for automated nucleic acid purification and qPCR, we saw striking advantages compared to using QIAcube DNA purification.
In 1996, the WHO defined a standard value of 10 ng CHO
DNA per therapeutic dose.[1] PCR-based DNA quantification
of in-process QC control samples is performed to demonstrate
the effectiveness of certain purification steps in reducing
CHO DNA content. This in-process testing requires
reproducible and sensitive methods for DNA extraction with
high recovery rates.
DNA extraction of in-process controls is not trivial. Samples
can have high protein loads ranging from 8 up to 150 mg/ml
and having a range of 0 to 100 ng/ml DNA.
2
Proteins present can negatively influence recovery of DNA
extraction and sensitivity of subsequent PCR analyses.
Samples which are acidic after purification steps using
chromatography can also interfere with DNA extraction.
In addition, DNA adsorption to surfaces of disposables
(e.g., pipette tips) can impair recovery rates and accuracy.
To prevent this, some DNA extraction kits require addition
of carrier RNA to ensure high recovery rates and high
sensitivity in subsequent PCR analyses.
2 Introduction
In the present study, we provide data for a new integrated
workflow using automated CHO DNA extraction from
pharmaceutical samples and PCR sample preparation using
Roche’s MagNA Pure LC System, followed by sensitive
quantitative real-time PCR analyses. The MagNA Pure LC
Instrument is fully automated and could be programmed
to prepare PCR mixtures. PCR mixes are transferred
to the glass capillaries of the Light Cycler® 2.0 Instrument
or the multiwell plates of the LightCycler® 480 Instrument
(both Roche).
In the present study, we compared extraction of CHO DNA
using the MagNA Pure LC System to a DNA extraction
protocol using spin column technology with Qiagen’s
QIAcube purification platform. Sensitivity, linearity and
reproducibility of both systems were evaluated using
real-time PCR to determine DNA recovery rate in samples
spiked with defined amounts of CHO DNA. The robustness
of both systems with respect to matrix effects was
determined by quantifying CHO DNA for in-process QC
controls from different purification stages of a therapeutic
protein. In addition, both systems were tested with
respect to the neutralization of acidic samples prior to DNA
extraction and their requirement for carrier RNA.
3 Materials and Methods
Samples
Samples containing 1 to 150 mg/ml drug substance from
different downstream processing steps of a manufacturing
process, and different purification stages for therapeutic
proteins using the Chinese Hamster Ovary (CHO)
production cell line, were obtained from Roche Production
and Development at the Penzberg facility in Germany.
Nucleic acid extraction
Nucleic acid extraction from in-process QC controls spiked
with a final concentration of 0.4 to 40,000 pg/ml CHO DNA,
at different protein concentrations (0-150 mg/ml protein),
was carried out using the MagNA Pure LC Total Nucleic
Acid Kit – High Performance (Roche Diagnostics GmbH,
Mannheim, Germany). The MagNA Pure LC 2.0 Instrument
was used with the „High Sensitivity“ purification protocol,
with a sample volume of 100 µl and an elution volume of 100
µl. Each run of 180 min included: 30 samples, a positive
control (~ 90 pg/ml CHO DNA in TE buffer), and a negative
control (TE buffer only).
To identify optimal conditions, samples were purified with a
Proteinase K digestion step, and with and without addition
of carrier RNA. For samples obtained from an acidic column
based purification step (pH 3.5), DNA isolation was carried
out with and without a prior neutralization step. All
extractions were performed in replicates.
Reference method
CHO DNA extraction using the column-based QIAamp
Viral RNA Mini QIAcube Kit on the QIAcube Instrument
(Qiagen, Hilden, Germany) was used as the reference
method. Each run of 90 min included: 10 samples, a positive
control (~ 90 mg/ml CHO DNA in TE buffer), and a
negative control (TE buffer only). Sample and elution volumes
were 140 µl each. Carrier RNA was manually added to
the samples (included in the Qiagen kit). For the Qiagen kit,
a manual Proteinase K digestion step must also be performed
prior to extraction. This Proteinase K digestion was done
at +72°C using Proteinase K and SDS. The purification on
the MagNA Pure LC instrument includes an integrated
Proteinase K step.
Purification was then carried out with and without
neutralization prior to the extraction of CHO DNA from
samples obtained after the acidic purification step (pH 3.5).
Quantification of CHO DNA by PCR with LightCycler®
System
Quantification of nucleic acids was performed using the
capillary-based LightCycler® 2.0 Instrument or the
microtiter plate-based LightCycler® 480 Instrument using
an external standard curve (4 pg/ml – 400,000 pg/ml of
CHO DNA) for absolute quantification (Roche Diagnostics,
Mannheim, Germany). Each PCR run included a positive
control from the DNA extraction step, and two negative
controls. One negative control was for the extraction, and
one negative control was for the PCR reaction. A calibration
standard for the alignment of the external standard curve
(400 pg/ml CHO DNA) was also used. Each amplification
reaction of 45 cycles, set up with 15 µl PCR master mix
and 5 µl eluate, was run in duplicate.
3
4 Results and Discussion
Determination of Quantitation Limit (QL) and
Detection Limit (DL)
DNA extraction using the MagNA Pure LC System resulted
in highly sensitive PCR quantification of CHO DNA
in solutions of therapeutic antibodies. For standard (up to
25 mg/ml) and high protein concentrations (150 mg/ml),
a quantitation limit (QL) of 4 pg/ml was obtained using
LightCycler® Instruments (see Table 1). The detection
limit (DL) was 0.4 pg/ml.
Our target for the variation coefficient is < 20%, and
for mean recovery is 80-120%. Both were met. Individual
experiments were done on different days, by different
coworkers, and different instruments, to maximize potential
causes for intra-assay variations.
For protein concentrations of up to 25 mg/ml, both the
QIAcube and MagNA Pure LC System showed comparable
results, when the additional manual steps were done
prior to DNA extraction with the QIAcube. However with
high protein concentrations (150 mg/ml), undiluted or with
dilutions < 1:10, even with additional manual Proteinase K
and carrier RNA steps, the QIAcube showed a QL and DL
of 40 pg/ml and 4 pg/ml, respectively. This was a tenfold
higher QL and DL than obtained with the MagNA Pure LC
Instrument.
Sample
[pg/ml]
Sample +
DNA Spike
DL [pg/ml]
Sample +
DNA Spike
QL [pg/ml]
Accuracy
QL [%]
~25 mg/ml Protein
Mean
0.06
0.46
3.97
106
STD
0.11
0.07
0.7
-
~150 mg/ml Protein
Mean
0.01
0.39
3.97
109
STD
0.03
0.14
0.5
-
Table 1: Determination of Quantitation Limit (QL) and Detection Limit
(DL) for the MagNA Pure LC Instrument. Results met the target
acceptance criteria for standard and high protein concentrations.
Real-time PCR was performed using a LightCycler® Instrument.
Determination of linearity
In this exemplary experiment, the linearity for the relevant
targeted dynamic measurement in the range of 4 to 40,000 pg/ml
of spiked DNA in solutions of therapeutic antibodies was
investigated (see Figure 3).
32 30 Crossing Point
Linearity was determined for the different protein
concentrations, using an undiluted protein concentration
of about 25 mg/ml, as well as dilutions of samples with
other types of therapeutic antibodies, thus representing a
wide range of protein concentrations (data not shown).
34 28 26 24 22 20 18 4
40
400
4,000
40,000
DNA Conc [pg/ml]
Figure 3: Samples of a therapeutic antibody solution (25 mg/ml) were spiked
to final concentrations of 4, 40, 400, 4,000 and 40,000 pg/ml CHO DNA
respectively. The samples were purified using a MagNA Pure LC 2.0 Instrument
to examine the linearity of the resulting purified DNA yields.
4
4 Results and Discussion
Determination of accuracy
110 100 Recovery [%]
Nucleic acid extraction using the MagNA Pure LC System
demonstrated excellent recovery rates (see Figure 4a).
For protein concentrations of 25 mg/ml, spiked with 40,
400 and 4,000 pg/ml DNA, mean accuracy was within the target
range of 80-120%. This protein concentration is the
representative final concentration in common protein drug
formulations.
90 80 70 60 40 pg
400 pg
4000 pg
Concentration of DNA Spikes [pg/ml]
Figure 4a: Therapeutic antibody samples (25 mg/ml) were spiked to final
concentrations of 40, 400 and 4,000 pg/ml CHO DNA respectively.
DNA isolation was performed using the MagNA Pure LC 2.0 Instrument.
Results in turquois show acceptance criteria (80-120%) where achieved.
Using the reference method with the QIAcube, recovery rates
were only acceptable when samples were diluted, indicating
negative effects from the sample matrix (see Figure 4b).
100 Recovery [%]
80 60 40 20 0 Undil.
1:2
1:4
1:8
1:10
1:15
1:20
Sample Dilutions
Figure 4b: Therapeutic antibody samples (25 mg/ml) were spiked with
423 pg/ml CHO DNA each. DNA isolation was performed using the QIAcube
Instrument with the additional manual steps, such as prior Proteinase K
digestion. Results in dark gray show acceptance criteria (80-120%) where
met, and results in light gray where they were not.
 Qiagen
 Roche
18000 16000 Concentration [pg/ml]
These findings indicate that quantitative real-time PCR can
be negatively affected by protein in the eluate. Manual
dilution and Proteinase K addition prior to DNA extraction
appears to be required when using the QIAcube Instrument
to prevent protein leakage into the eluate, which then
negatively affects accuracy and sensitivity of qPCR analyses
(see Figure 4c). Note again, that the MagNA Pure LC Total
Nucleic Acid Kit – High Performance procedure incorporates
an automated step for Proteinase K digestion.
14000 12000 10000 8000 6000 4000 2000 0 Undiluted Sample
1:10 Diluted Sample
Figure 4c: Therapeutic antibody samples with protein content (14-17 mg/ml)
were digested with Proteinase K. Undiluted and 1:10 diluted samples were
purified using the MagNA Pure LC Instrument and the QIAcube Instrument.
5
4 Results and Discussion
High Protein Loads
 Roche [%]  Qiagen [%]
120 100 Recovery [%]
For protein loads of 150 mg/ml, DNA extraction using the
MagNA Pure LC Instrument showed a mean recovery
of 109%. DNA extraction using the QIAcube Instrument
required prior dilution and Proteinase K digestion for
these samples. However, the results still did not comply with
our acceptance criteria of 80-120% (see Figure 5).
80 60 40 20 DNA extraction using the MagNA Pure LC Instrument did
not require further dilution of proteinaceous samples.
It produced higher sensitivity and reproducibility in the
downstream PCR analyses, especially at the low DNA and
high protein concentrations in samples from late protein
polishing steps.
0 4
400
Conc. DNA Spike [pg/ml]
Samples Undiluted
400
1:10 Diluted
Figure 5: In-process control samples of another therapeutic antibody with very
high protein content (150 mg/ml) were used undiluted or diluted, and spiked
with CHO DNA. These samples were then purified using either the Roche or
Qiagen automated sample preparation instruments. Relying on existing data,
the MagNA Pure LC Instrument was not tested for 400 pg/ml spike, as it had
worked fine earlier. For Qiagen, the low DNA amounts spiked were also not
tested, since these did not previously meet the target criteria.
Effect of acidic samples
 Without Neutralization
 With Neutralization
6000 Concentration [pg/ml]
Some of our in-process intermediate products are acidic.
Tests examining the need for neutralization to pH 7 prior to
DNA isolation were performed using the MagNA Pure LC
Instrument. Both samples which were neutralized prior to
processing, and samples which were not neutralized showed
similar results (see Figure 6a).
5000 4000 3000 2000 1000 0 1:2
1:4
1:8
Dilution Factor
Figure 6a: Acidic in-process control samples of a column purified
therapeutic antibody are tested for matrix effects using the MagNA Pure LC
Instrument. No manual neutralization step is necessary.
 QiaCube Instrument
20000 15000 10000 5000 0 6
 MagNA Pure LC Instrument
25000 Concentration [pg/ml]
When comparing the Roche and Qiagen instruments, samples
which were not neutralized prior to processing showed
better performance using the MagNA Pure LC Instrument
than with the QIAcube Instrument. When performing
the neutralization step, the QIAcube Instrument was in
a comparable range (see Figure 6b). Based on these
and other data (not shown), we found that this additional
neutralization step can be omitted when using the
MagNA Pure LC Instrument.
Without Neutralization
With Neutralization
Figure 6b: An acidic in-process-control sample of a column purified
therapeutic antibody is tested for matrix effects using the MagNA Pure
LC Instrument and compared to the Qiagen Instrument. For the QIAcube a
neutralization step is necessary.
4 Results and Discussion
Eliminating unnecessary steps: Effect of adding carrier
RNA
2500 Concentration [pg/ml]
Using the MagNA Pure LC Instrument, samples with low
DNA concentration (1:50 diluted) for which carrier RNA had
been added prior to processing (as recommended by Qiagen
for the QIAcube), and samples without carrier RNA showed
similar results (see Figure 7). Qiagen recommends adding
carrier RNA for a good purification performance of DNA at
low concentrations in their instructions for use. This
recommendation was confirmed by our experience (data not
shown). We were able to omit the step of manually adding
carrier RNA when using the MagNA Pure LC Instrument.
 Without Carrier-RNA  With Carrier-RNA
2000 1500 1000 500 0 Sample
Sample + low spike Sample + high spike
Figure 7: Sample with a low CHO DNA concentration were tested using the
MagNA Pure LC Instrument for the need to add carrier RNA. The results
showed that the addition of carrier RNA is not needed for purification using
the MagNA Pure LC Instrument.
5 Conclusion
Nucleic acid extraction using the MagNA Pure LC 2.0 System
and MagNA Pure LC Total Nucleic Acid Kit – High
Performance provides a robust automated workflow for
sensitive and reproducible quantification of host-cell
DNA in both in-process controls and final drug substance
samples of protein-based drugs. In contrast to the current
QIAcube, the MagNA Pure LC 2.0 System required no manual
pre-processing, additional Proteinase K, neutralisation or
dilution steps, nor did it need the addition of carrier RNA.
It additionally offers less hands-on time at the same
or higher performance level as the QIAcube instrument.
The system provides a fully automated workflow from DNA
extraction to DNA quantification on the LightCycler® 2.0
and LightCycler® 480 Instruments. Hence it can help to reduce
time-to-analysis, manual work and expedites testing for
development, in process control testing and quality control
testing. The LightCycler® Instruments provide the attractive
option of loading standard curve data, and normalizing
results using just an additional single standard control in
each PCR run. This option means that it is not necessary to
run the many replicate samples required for a standard
curve for each new purification and real-time PCR run.
6 References
1 WHO Expert Committee on Biological Standardization: Highlights of
the 46th WHO Meeting, October 1996, in WHO Weekly Epidemiological
Record, 1997, 72: 141-145
7
For general laboratory use.
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