In the biotechnology industry, traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
has been widely used to
monitor the integrity and purity of
therapeutic proteins during formulation
and process development, and for
lot release and stability testing. In this
technique, the proteins are bound to
SDS at a ratio of 1.4 g of SDS to 1 g of
protein.1 This constant binding leads
to similar free solution mobilities for all
protein molecules2 and, therefore, their
separation based on hydrodynamic size.
Recently, there has been a growing
interest in using capillary electrophoresis
(CE)-SDS to replace traditional
SDS-PAGE in the therapeutic protein
area. The applications of CE-SDS in
recombinant GB virus-C protein,3
recombinant carboxypeptidase B,4 collagen,
5 a protective antigen protein
(the major component of human
anthrax vaccine),6 and monoclonal
antibodies7–10 have been demonstrated.
The potential advantages of CE-SDS
over SDS-PAGE include faster analysis,
the ability for quantification, full
automation, low sample consumption,
and potentially better resolution. In
this paper, the authors will present the
development of a CE-SDS method and
will evaluate its feasibility for the stability
testing of a heavily glycosylated
protein: Protein X. Over 40% of the
total mass in Protein X is composed of
carbohydrates. The high heterogeneity
of the molecule poses extra challenges
for the development of the CE-SDS
method, since the main peak of the
protein is expected to be broad, and
therefore may compromise the sensitivity
of the assay.
The method development discussed
in this paper is focused primarily on
optimizing electrophoresis conditions
and improving sensitivity by a variety
of means, including the use of
extended light path capillaries. A
homemade sample buffer was successfully
implemented to replace the commercial
sample buffer in order to minimize
sample dilution, which further
improved sensitivity. In addition,
accelerated stability studies were performed
by protease cleavage of Protein
X, or by incubation of the protein at
elevated temperatures in buffers at
various pH levels. The results demonstrated
that the described CE-SDS
method is capable of monitoring the
degraded species quantitatively.
Experimental
Reagents and materials
Protein X was produced in transfected
Chinese hamster ovary cells (Amgen
Inc.,Thousand Oaks, CA). The CE-SDS
protein kit including CE-SDS sample
buffer, CE-SDS protein run buffer, 0.1 N
NaOH, 0.1 N HCl, and internal standard
(IS) benzoic acid (1 mg/mL) was acquired
from Bio-Rad Laboratories (Hercules,
CA). Unless otherwise stated, all other
chemicals were purchased from Sigma
(St. Louis, MO). Both the extended light
path capillary (50 μm i.d. × 33 cm, bubble
factor 3) and standard capillary (50 μm
i.d. × 33 cm) were obtained from Agilent
Technologies (Palo Alto, CA).
Sample preparation using Bio-Rad sample
buffer: Protein X was mixed with sample
buffer at a ratio of 1:8 (v/v) followed
by the addition of benzoic acid at 5%
(v/v). β-Mercaptoethanol (BME) was
further added at 2.5% (v/v) to the mixture
to reduce the protein. The mixture
was incubated at 95 °C for 10 min.
Sample preparation using homemade
sample buffer: Protein X was incubated with 20% or 10% SDS (w/v, in H2O)
at a mass ratio of 1:1.7 (protein:SDS)
with benzoic acid added as an IS at 5%
(v/v). BME was further added to the
mixture at 2.5% (v/v) to reduce the
protein. The sample mixture was
heated at 95 °C for 10 min.
CE-SDS conditions: CE-SDS was performed
on a G1600 HPCE instrument
(Agilent Technologies) equipped with
a UV detector. Unless stated separately,
for electrophoretic injection,
the sample was introduced at 20 kV
(negative polarity) for 20 sec. For pressure
injection, the sample was introduced
at 3 bar for 24 sec. A negative
voltage of up to 25 kV was applied
across the capillary and the sample was
electrophoresed for 30 min. Signal collection
was conducted at 220 nm.
Results and discussion
Selection of capillary
Figure 1 - Comparison of the bubble-cell capillary and standard capillary. CE-SDS conditions:
electrophoretic injection at –10 kV for 20 sec; run voltage at –15 kV.
Method development was started by
using the CE-SDS protein kit. The
lower trace shown in Figure 1 is a typical
electropherogram for Protein X at 0.5
mg/mL, using a standard bare silica capillary
with an internal diameter of 50 μm.
The signal intensity obtained was low,
and such low sensitivity would be a problem
for stability monitoring since any
degraded species are expected to grow at
a much lower concentration relative to
the main peak. In order to improve the
sensitivity, extended light path capillaries
(also called bubble-cell capillaries)
were used instead of standard capillaries.
The unique feature of the bubble-cell
capillary is that its detection window is
blown to the shape of a bubble to
increase the optical light path (Figure 1,
inset). It can be seen from the figure that
the signal intensity of Protein X
obtained by using the bubble-cell capillary
is almost 3× higher than that
obtained by using the standard capillary.
The increased intensity is a direct result
of the increased light path. Another
advantage of the bubble-cell capillary is
that the running current stays comparable
with the standard capillary because
the inner diameter of the capillary is
only increased at its detection window
while the rest of the capillary remains
unchanged. Therefore, the Joule heating
effect is minimal. However, the bubble-cell
capillary resulted in a slight decrease
in the resolution of the isoforms under
the main peak, which could be microheterogeneity
contributed from the carbohydrate
structures.
Optimizing CE-SDS
running conditions
Next, the effect of run voltage at –15,
–20, and –25 kV on the separation
was investigated. It was found that
higher voltages offered faster separation
and sharper peak shape. However,
as the run voltage increased, the
baseline became less stable. Under the
consideration of both separation time
and baseline, –20 kV was chosen as
the optimum run voltage.
For electrophoretic injection, injection
time and voltage both have an impact
on the signal intensity. As far as the
injection time is concerned, the longer
the injection time, the higher the signal
intensity. It was found that at a
fixed injection voltage of –20 kV, the
peak became wider as the injection
time was increased from 5 to 20 sec due
to the larger sample plug introduced. In
the end, an optimum injection time of
20 sec was chosen. An injection time
longer than 20 sec was not considered
due to the wider peak width.
A similar effect was found for injection
voltage. The signal intensity was
increased as the injection voltage was
increased from –10 to –20 kV. Based
on the same consideration for the
peak width, –20 kV was chosen as the
optimum injection voltage.
Optimizing sample
preparation
Sample preparation is an important
aspect to consider for the CE-SDS
development. One crucial component
for the sample preparation is the sample
buffer, which contains SDS for protein
binding. The original sample preparation
was performed by mixing Protein X
with the Bio-Rad sample buffer at a
ratio of 1:8, which resulted in at least a
ninefold dilution of the protein. If the
protein concentration is low, such dilution
could be a concern for monitoring
species in the stability samples. The
authors have developed a homemade
sample buffer consisting of 20% SDS
(w/v, in H2O), and its binding capability
with Protein X was investigated.
The concentration of SDS was made
high so that a very minimal volume of
SDS solution could be used in order to
minimize the dilution from the sample
buffer. Another reason to use homemade
buffer is that the buffer composition
is known so that it is possible to
manipulate the binding ratio to
improve the method sensitivity.
Figure 2 - Comparison of the Bio-Rad sample buffer with the homemade sample buffer. The data
have been offset for better visual comparison. The numbers represent the mass binding ratio of SDS to
protein. CE-SDS conditions: electrophoretic injection at –20 kV for 20 sec; run voltage at –20 kV.
Shown in Figure 2 are the electropherograms
comparing the Bio-Rad
sample buffer with the homemade
SDS buffer. The binding ratio of SDS
to Protein X based on mass, as indicated by the numbers in Figure 2,
ranges from 0.9 to 20.7. These binding
ratios correspond to the dilution factor
from 1.05 to 1.6, compared to 9 for
the Bio-Rad sample buffer. One
notable point is that the final concentration
for the protein sample was
made the same (0.2 mg/mL) by adding
an appropriate amount of H2O to the
mixture of the protein and sample
buffer in order to compare the signal
intensity. It can be seen that the signal
intensity is the lowest for the Bio-Rad
sample buffer. For the homemade
buffer, the intensity increases as the
binding ratio decreases from 20.7 to
1.7 and changes very little thereafter.
One possible reason for the increased
signal intensity could be that the
excess SDS in the sample may have an
adverse effect on the electrophoretic
injection of the protein (i.e., it lowers
the loading efficiency, which results in
the lower signal). For later method
development, a binding ratio of 1.7
was used. This ratio is similar to that
used in SDS-PAGE, in which a binding
ratio of 1.4 is typically used.
It was also found that a 10% SDS
solution provided sufficient signal
intensity while maintaining minimum
dilution of the sample. Therefore, a
10% SDS solution was used as the
sample buffer for the subsequent
method development.
Effect of salt concentration
Figure 3 - Effect of buffer salt concentration on electrophoretic injection and pressure injection.
a) Buffer salt effect on electrophoretic injection; b) comparison of pressure and electrophoretic injection
under the same salt matrix.
During the experiments, it was noticed
that the salt concentration in the sample
had an effect on the signal intensity
when the sample was injected electrophoretically.
Figure 3a shows an
example in which the injection was performed
at –20 kV for 20 sec. The blue
trace represents the signal for Protein X
at 5.8 mg/mL in the buffer of 20 mM
NaPi and 140 mM NaCl. A higher signal
was obtained when the sample was
diluted fivefold, as shown by the red
trace under the sample injection conditions.
This comparison clearly demonstrates
that high salt content in the sample
reduces the loading efficiency of the
protein. In the red trace, although the
protein was more dilute compared to
that in the blue trace, the salt concentration
was 5× lower than that in the
blue trace. Therefore, more protein was
loaded in the capillary, which resulted
in the higher signal in the red trace.
In contrast to electrophoretic injection,
pressure injection has no bias toward the
sample in terms of loading efficiency.
Shown as the red trace in Figure 3b is the
electropherogram for Protein X at 1.0
mg/mL in the buffer of 20 mM NaCl and
140 mM NaPi, where the sample was
injected using pressure at 3 bar for 24 sec. It can be seen that, although the salt
concentration was as high as that in the
blue trace (Figure 3b), much higher signal
intensity was obtained.
The recommendation for the sample
preparation is to desalt if the salt concentration
is higher than 50 mM for the
electrophoretic injection. However,
pressure is a preferred injection mode in
order to minimize sample manipulation.
In the authors’ subsequent experiments,
the injection was performed
using pressure at 3 bar for 24 sec.
CE-SDS for stability testing
Figure 4 - Electropherograms of Protein X control and Protein X incubated with a protease at 37
°C for 24 hr. CE-SDS conditions: pressure injection at 3 bar for 24 sec; run voltage at –20 kV.
To generate degraded protein species for
accelerated stability testing, Protein X was
incubated with a protease that the protein
is known to be sensitive to. As shown in
Figure 4, clipped species were identified by
comparing the electrophoretic profile to
that of the control sample. The level of
clips was determined to be 4.8% relative
to the total protein peak area. Additional protein cleavage was
induced by incubating Protein X at
elevated temperatures (37 and 55 °C)
in 20 mM buffers at pH 5, 6, and 8
over time. Table 1 summarizes the
effect of temperature and pH on the
generation of the clipped species, with
the protein sampled at intervals of 5,
7, and 9 weeks. It is obvious that the
protein is most stable in the pH 6
buffer, and least stable in the pH 8
buffer. Higher temperature appears to
accelerate the protein degradation.
Conclusion
A CE-SDS method has been developed
using the Agilent HPCE instrument for
Protein X. In order to improve the
method sensitivity for this highly heterogeneous
glycoprotein, a bubble-cell
capillary was used. Compared to the
standard capillary, the signal intensity
was increased threefold. For the sample
preparation, a homemade sample buffer
was employed to minimize the sample
dilution, which allowed further
improvement to sensitivity. It was also
found that pressure injection was able to
withstand higher salt content in the
sample than electrophoretic injection,
and also required very minimal sample
treatment. Finally, in order to qualify
the method for stability testing, clipped
species were induced by incubation of
the protein with a protease, or at elevated
temperatures in various pH buffers
for up to 9 weeks. The results demonstrated
that the CE-SDS method was
stability-indicating and that the method
may be used in formulation development.
In the future, the feasibility of
using a fluorescent tag7 will be explored
to further improve the method sensitivity.
It is believed that the method will
have a wider range of application in
addition to stability testing, such as
impurity detection for process development,
with the increased sensitivity.
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The authors are with the Department of Process
and Analytical Sciences Operations, Amgen
Inc.,One Amgen Center Dr., Thousand Oaks,
CA 91320, U.S.A.; tel.: 805-447-1000; fax:
805-499-0126; e-mail: [email protected].