Quality control of antibodies using the
2100 bioanalyzer and the Protein 200
Plus assay
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Antibodies are commonly used in diagnostics, as research tools
(for example protein arrays) or as biopharmaceutical therapeutics. In
all cases the successful production of antibodies requires precise testing
and quality control. The key quality criteria include: protein identification,
quantitation and the monitoring of purity and stability. Agilent's
2100 bioanalyzer, the first commercial lab-on-a-chip analysis tool,
developed in collaboration with Caliper Technologies Corporation, was
used to verify the suitability of this technology for antibody quality
control. The system allows sizing and analysis proteins of 5 to 200 kDa,
depending on the application. In addition, it determines the relative
quantitation based on internal standards for each sample, or absolute
quantitation based on user-defined calibration standards. The microfabricated
chips with distinct microfluidic channels allows antibody
analysis under both reducing and non-reducing conditions in a single
run. In this Application Note we demonstrate the use of the system for
the evaluation of a stress stability test of two different antibodies.
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Experiments and Results
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Antibody samples were kindly
provided from a customer in the
pharmaceutical industry. For the
first sample a stress stability test
was performed for one month at
40 °C, for the second sample the
stress test was done for 12 weeks
at 40 °C. The standard samples
and the stress test samples were analyzed under reducing conditions
on the Agilent 2100 bioanalyzer
using a Protein 200 Plus
LabChip® kit and the dedicated
Protein 200 Plus software assay.
All chips were prepared according
to the protocol provided with the
Protein 200 Plus LabChip kit. The
kit includes 25 chips, spin filters
and all the reagents needed for the
experiments including the Protein 200 Plus ladder as well as the
upper and lower marker premixed
in the sample buffer.
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To validate the performance of the
instrument three individual users
measured the samples on two separate
instruments using two lots
of reagents on different days. Thus
a total number of twelve chips
were run. The samples were diluted 1:3 in PBS buffer (Life
Technologies GmbH, Karlsruhe,
Germany) in order to adjust the
concentration of the samples to
approximately 5 mg/ml total
protein concentration. Sample
preparation was performed individually
for each chip. All samples
were loaded twice on each chip.
Therefore, 24 analyses per sample
were evaluated using the following
Protein 200 Plus assay settings
for peak integration: minimum
peak height of 0.1, minimum peak
width of 0.1 and slope threshold of
4.0.
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An example of the performed
analysis is depicted in figure 1.
Figure 1A shows the gel-like
image of the four different antibody
samples (2 standards, 2 test
samples) aligned to the ladder
sample. The ladder includes proteins
of known sizes, was analyzed
on each chip and was used for
size determination. Figure 1B displays
the overlay of the corresponding
electropherograms. Both
standard samples show almost
only two peaks corresponding to
the light (LC) and heavy chain
(HC) of the antibody respectively.
After incubation of the antibodies
at elevated temperatures additional
peaks appear in the electropherograms
(marked with an
arrow) due to degradation of the
antibody or the formation of
aggregates, larger products.
Table 1 shows the results of the
validation study. On average
98.76 % of standard antibody
sample 1 corresponds to the light
and heavy chain of the antibody.
After incubation for one month at
40°C the two chains represent
only 93.78% of the whole protein amount detected. Antibody 2 was
incubated for 12 weeks at 40°C
and the proportion of light and
heavy chain decreases from an
average of 98.38 % to 85.34 %. An
excellent reproducibility below
2 % CV was achieved
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Conclusion
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In this study we show that the Agilent
2100 bioanalyzer can be used
as an ideal tool to study the stability
of antibodies in stress tests
with excellent reproducibility.
This application is commonly used
as a quality control step in QA/QC
departments in order to trigger
typical degradation and aggregation
patterns for a specific antibody.
Those patterns will be used
in the down stream processes of
antibody production and formulations.
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Traditional denaturing sodium
dodecylsulfate-polyacrylamide gel
electrophoreses (SDS-PAGE) still
serves as one of the standard
methods for the QC of antibodies.
SDS-PAGE is labor-intensive, timeconsuming
and difficult to standardize (1-3). In contrast the Agilent
2100 bioanalyzer provides a
fast, standardized method with
automated and detailed data analysis.
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