Capillary Electrophoresis in Quality Control:
PART I: Application for Therapeutic Proteins
Chantal Felten*1 and Oscar Salas Solano2
*Consulting author
1Alpine Analytical Acadamy, Whistler, British Columbia 2Seattle Genetics, Bothell, WA
T-14326A
Abstract
A decade ago, Capillary Electrophoresis (CE) was
considered a novelty. Today it is commonly used in quality
control worldwide, providing automated, high-resolution
methods, with online detection. In the first part of this
series of articles, we will illustrate an overview of CE
in a modern quality control laboratory. The complete
series of articles will address in depth the following:
Applications in QC; CE-SDS (sodium dodecyl sulfate)
method development and robustness; Application of
ICH Q2 (R1) to CE and finally successful CE method
lifecycle management, i.e. training, transfer and method
replacement.
Electrophoretic techniques such as SDS-PAGE and
IEF gels have traditionally been part of release testing
in the biopharmaceutical industry. However, these slab
gel methods are inconvenient, use toxic reagents, exhibit
high intra- and inter- gel effective mobility variability and
are generally not reproducible due to inconsistency in
staining/destaining steps used for analyte detection.
As a result, many biotech companies like Genentech,
Amgen, Pfizer, and Eli Lilly, to name a few, have sought
out CE methods such as capillary electrophoresis
sodium dodecyl sulfate (CE-SDS), capillary isoelectric
focusing (CIEF) and capillary zone electrophoresis (CZE)
as practical replacements of the slab gel format.1 With
the addition of user-friendly instrumentation, software-
providing tools to enable compliance with CFR 21 Part
11, and assay kits designed specifically for protein and
monoclonal antibody analysis, CE has become a viable
replacement in quality control laboratories for setting
and justification of release specifications for therapeutic
proteins. These specifications are an integral part of the
release of a commercial drug substance/drug product
and generally contain methods to measure safety,
identity, purity and composition of a drug
substance and drug product.
This article describes the advantages of three
CE applications for the analysis of protein-based
pharmaceuticals. The first application illustrates
the application and advantages of a generic and
quantitative CE-SDS assay with UV and or laser-
induced fluorescence (LIF) detection to assess the size
heterogeneity of protein products. The second section
focuses on the use of capillary isoelectric focusing (CIEF)
to determine charge heterogeneity of protein products.
Finally, given the growing importance of glycosylation
characterization, we will review the application of capillary
zone electrophoresis (CZE), coupled with LIF as well as
MS detection for complex glycoprotein analysis.
CE-SDS for Analysis of Therapeutic Proteins
Historically, SDS-PAGE has been applied routinely
to quality control systems for monitoring of
manufacturing consistency and apparent protein
molecular weight. CE-SDS has emerged as a robust
replacement for SDS-PAGE. This assay is used to
determine the apparent molecular weight of proteins
and to evaluate the size heterogeneity, purity and
manufacturing consistency of biologics.2-6 In contrast
to SDS-PAGE, CE-SDS offers direct on-column UV
or fluorescence detection, automation, enhanced
resolution and reproducibility, accurate quantification
of proteins, and determination of molecular weight.7,8
Currently, linear or slightly branched polymers
such as linear polyacrylamide, polyethylene oxide,9
polyethylene glycol, dextran, and pullulan are used
as sieving matrices for CE-SDS.7,10,11 In comparison
to cross-linked polyacrylamide gel matrices, these
polymers add a great deal of flexibility to CE-SDS
Blood Banking
Capillary Electrophoresis
Centrifugation
Flow Cytometry
Genomics
Lab Automation
Lab Tools
Particle Characterization
since they are water-soluble and replaceable between
CE analyses, resulting in enhanced overall precision
and robustness.12
Results regarding the performance of a commercial
rMAb CE-SDS based method were recently reported
by eleven independent biopharmaceutical companies
and a regulatory authority. In this study, the reduced
rMAb standard contained a mixture of light chain (LC),
non-glycosylated heavy chain (NGHC) and heavy
chain (HC) components. The highly resolving nature
of the CE-SDS separation was demonstrated by
showing baseline resolution between HC and NGHC
species present at low levels (less than 5%). The
relative standard deviation (RSD%) for the relative
migration time of those components was ~ 2%.
Moreover, the percentage of peak area quantitation
of the rMAb sample components was comparable
across all organizations in the study with RSD%
values of less than 9%.1
It is important to indicate that an advantage of CE-SDS
over other size-based separation methods such as high-
performance size-exclusion chromatography (HPSEC)
and SDS-PAGE is improved resolution of closely
related size-variants. CE-SDS can be run in a variety
of modes, including reduced and non-reduced sample
preparation, each offering their own advantages to a
QC system. With respect to the non-reduced sample
preparation, CE-SDS is highly capable of resolving
protein fragments in the size range of 10-100kD while
allowing for concurrent detection of the intact antibody
and any aggregates that may be present. This broad MW
separation efficiency makes CE-SDS the ideal tool for
monitoring the intact protein-to-fragment ratio for release
and stability of monoclonal antibodies and proteins alike.
Figure 1 (bottom) illustrates an example electropherogram
of a native rhuMab CE-SDS separation visualizing intact
antibody and common antibody fragments. Additionally,
as seen in Figure 1(top), CE-SDS of reduced monoclonal
antibodies offers a simple solution for reliable and
accurate quantitation of the non-glycosylated heavy chain,
which is generally well separated from the glycosylated
heavy chain variant.2,3
Traditional sample preparation conditions including
heat treatment at elevated temperatures (e.g. 90ºC)
are employed to form SDS-protein complexes prior to
electrophoretic analysis. In the case of non-reduced
rMAbs, this could lead to sample preparation artifacts in
the form of thermally induced fragmentation attributed
to disulfide reduction and exchange reactions.2,6 It
was also reported that high pH conditions during heat
treatment also enhanced the fragmentation of SDS-
rMAb complexes.5 These artifacts significantly alter the
true representation of the size heterogeneity of a protein
and may increase the variability of quantitative CE-SDS
methodologies of non-reduced samples. It is therefore
crucial to not only optimize capillary electrophoresis
conditions, but to invest a significant amount of effort into
defining the correct sample preparation conditions.
Figure 1. Analysis of both reduced (top) and non-reduced (bottom) IgG suitability standard.
Peak identification: 1: Internal standard (10 kDa); 2: Light chain (L); 3: Non-glycosylated (NG) Heavy chain (H);
4: Heavy chain (H); 6: Heavy chain (2H); 7: 2 heavy 1 light chain (2H1L); 8: NG HC; 9: IgG monomer. (Reprinted from
Reference 23, with permission)
2
Figure 2. CE-SDS separations
of (Figure 2A,top) non-reduced
and (Figure 2B, bottom) reduced
preparations of a 5-TAMRA
SE-labeled rMAb sample.
Separation conditions were as
follows: ProteomeLabTM PA 800
instrument equipped with LIF
detection; 50-μm ID, 375-μm OD
uncoated fused-silica capillary
effective length 21.2 cm, total length
31.2 cm; both anode and cathode
buffers were Beckman Coulter
CE-SDS gel solution. Samples
were injected at a constant electric
field of 160 V/cm for 20 s and
electrophoresed at 480 V/cm
(32.5 μA) and 40°C. (Reprinted
from Reference 3, with permission).
Many applications of CE-SDS (reduced and non-reduced)
apply UV detection, as shown in Figure 1, which is
equivalent to SDS-Comassie staining sensitivity.
However, certain companies including Genentech, have
chosen a laser induced fluorescence (LIF) detection
strategy for their CE-SDS based assays.6 Although it
adds additional sample preparation and the potential to
introduce sample artifacts, fluorophore labeling and
subsequent LIF detection does offer two significant
advantages. First, LIF detection offers sensitivity on par
with silver-stained SDS-PAGE.2,6 Figures 2A and 2B
show example data for a CE-SDS-LIF analysis in both
reduced and non-reduced mode. The analysis of
trace-level rMAb variants and process impurities at
levels as low as 50 ppm were reported using CE-SDS
with LIF detection. The second advantage is that while
the capillary is filled with a viscous solution,
gel-interference during the on-column detection will to
some degree affect the baseline noise, and thus, the
integration of minor protein variants. Labeling of proteins
with 5-carboxytetramethylrhodamine succinimidyl ester
(5-TAMRA.SE) can significantly decrease gel interference
during online detection while adding up to a 100-fold
increase in sensitivity.
3
Capillary Isoelectric Focusing for Monitoring
Charged Variants
Regulatory agencies routinely require assessment
of charge heterogeneity for biotechnology products.
Charged variants of recombinant protein products
often include post-translational modifications such as
sialylation, phosphorylation, deamidation and addition
of C-terminal lysine residues. Such variants are often
monitored by widely used ion-exchange chromatography
(IEC) techniques. However, the development of an IEC
method is typically extensive and product-specific. IEC
assays require the optimization of complex separation
parameters including column, mobile phase composition,
pH, salt, temperature, and gradient. CE techniques
such as CZE and CIEF offer the advantages of faster
analysis time and development of generic methods for
multiple products, which is desirable in today’s fast paced
therapeutic protein arena.
In CZE, analytes are separated from each other based
on the differences in their electrophoretic mobilities.
Therefore, this technique also introduced a size-based
element to the separation. Alternatively, the ability for
CIEF to separate solely based on charge may have
an added advantage in isolating charge variants of
recombinant monoclonal antibodies (MAbs). CIEF
separation of a MAb sample illustrates the powerful
separation efficiency of modern CIEF technology
(Figure 3).
CIEF (both by on-column detection and image detection)
as a tool for protein purity analysis is quickly becoming
the method of choice for many biopharmaceutical
companies due mainly to its fast method development
and highly reproducible nature.13 CIEF is usually carried
out in commercial CE instruments employing on-column
UV absorbance detection. Following the focusing of an
analyte in a capillary, protein zones are detected using
chemical, electrophoretic or hydrodynamic mobilization
strategies. Convenient protein and rhuMab chemistry kits
are commercially available facilitating not only method
development but also integration into quality control.
Figure 4 gives an example of the reproducibility obtained
using conventional CIEF technology for charge variant
analysis. As summarized in Table 1, RSDs using this
technology are generally less than 1% for the main peak
and 2-5% for acidic and basic variants, demonstrating
equivalent, if not superior, reproducibility to ion exchange
chromatography methods.14 Additionally, CIEF can
be used as an identity assay on the basis of highly
reproducible measurements of the apparent pI of the
main isoform peak (See Table 1).
4
Figure 3. The three isoform groups and seven signature isoform peaks used for the intermediate precision analysis of a MAb CIEF
separation. Peaks lettered A through G were used to characterize variation in the estimated pI values and isoform group percent
compositions. Peaks denoted with an asterisk were only used to estimate variation of the isoform group percent compositions
(as shown in Table 1) (Reprinted from Reference 14, with permission).
Table 1: Summary of CIEF separation inter-day reproducibility data analysis for 3 MAb molecules (MAb 1, MAb 2 and
MAb 3) (Reprinted from Reference 14, with permission).
Peaks Average Standard deviation %CV
mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 mAb 1 (%) mAb 2 (%) mAb 3 (%)
Estimated p/
A 9.65 8.31 7.79 0.00507 0.00500 0.00493 0.05 0.06 0.06
B 9.58 8.18 7.59 0.00577 0.00577 0.00351 0.06 0.07 0.05
C 9.48 8.13 7.46 0.00640 0.00583 0.00458 0.07 0.07 0.06
D 9.44 8.07 7.43 0.00812 0.00569 0.00289 0.09 0.07 0.04
E 9.33 8.01 7.38 0.00700 0.00583 0.00351 0.08 0.07 0.05
F 9.27 7.90 7.28 0.00645 0.00583 0.00507 0.07 0.07 0.07
G 9.24 7.78 7.12 0.00781 0.00400 0.00458 0.08 0.05 0.06
Isoform group percent composition
Basic 13.94% 30.97% 18.03% 0.42% 0.67% 0.28% 3.04 2.17 1.53
Main 71.97% 45.01% 68.42% 0.46% 0.45% 0.42% 0.64 0.99 0.62
Acidic 14.09% 24.02% 13.55% 0.34% 0.60% 0.34% 2.38 2.50 2.49
5
Figure 4. CIEF intermediate precision data for separation of a MAb. The detector traces are aligned to the peaks of the
pI 9.99 and 7.00 synthetic peptide pI markers. Separation parameters are as follows: Focusing was performed for 15 min
at 25 kV; mobilization using 350mM acetic acid for 20 min at 30 kV. The sample solution contained 2.5M urea, 1.8% w/v
Pharmalyte pI 3–10 Commercial Ampholyte, 1.7mM iminodiacetic acid (IDA), 42mM arginine and 10 mM each of the pI
7.00 and 9.99 synthetic peptide markers, all diluted into Beckman Coulter’s CIEF polymer solution. Quantitative data is
shown in Table 1. (Reprinted from Reference 14, with permission).
Figure 5. Separation of oligosaccharides associated with a recombinant therapeutic MAb. Oligosaccharides were
cleaved from a therapeutic MAb, APTS labeled, and separated by CE using the Beckman Coulter Glycan separation
buffer. A number of oligosaccharide species were resolved from one another (A). In order to identify and help
illustrate resolution between co-migrating glycan species, we spiked the MAb sample with standards. Relative to the
oligosaccharide standards, we were able to quantifiably identify G0, G0F, G1F, G1’F, and G2F. G2 standard was also
spiked into the mixture to indicate the location in the separation at which this oligosaccharide species would reside.
Further experimentation is being performed to identify the additional species present in the electropherogram. Separation
conditions were the same except that injection for the MAb alone was 0.5 psi and that for the MAb + glycan standards
was 1.5 psi. (Reprinted from Reference 24, with permission).
CZE-LIF for N-Linked Glycans Analysis
Glycosylation, one of the many post-translational
modifications present on therapeutic proteins,
has received considerable attention in the field of
proteomics due to its reported role in protein function.
In the biotechnology industry, it is well known that
carbohydrates factor significantly into a glycoprotein’s
activity and efficacy and therefore must be carefully
monitored.15,16 Even minor changes in glycan
distribution can have a notable effect on the activity of a
biopharmaceutical product.17,18 Therefore, advances are
continually being made in the biopharmaceutical industry
to minimize glycan heterogeneity and improve analytical
assays for identification and quantification of minor
changes across products and/or batches.19 However, the
task of glycoprotein characterization remains an analytical
challenge due to the vast heterogeneity of these species.
Capillary Zone electrophoresis (CZE) is an excellent
tool for profiling glycosylation due to the advantages
of automation, short analysis times and the ability to
separate and quantify isomeric species. The coupling
of CE with on-column LIF detection has the advantage
of highly sensitive analysis. The primary assay used
for profiling rMAb glycosylation involves: 1) Releasing
N-linked oligosaccharides with the endoglycosidase
PNGase-F; 2) Labeling with a charged fluorophore; and
3) Sample analysis using CZE-LIF.
Common labeling reagents include 8-aminopyrene-1,3,6-
trisulfonate (APTS), 8-aminonapthalene- 1,3,6-trisulfonate
(ANTS) or analogous negatively charged labels or
alternatively, 2-aminopyridine (2-AP) and
2-aminobenzamide (2-AB) which are net positively
charged labels used for LIF labeling of glycans. This
strategy offers the advantage of low-picomole sensitivity
attainable for LIF-labeled carbohydrates.20,12,22,25
Beckman Coulter provides a Carbohydrate Labeling
and Analysis Kit that includes the necessary reagents
for characterizing glycan populations associated with
therapeutic proteins. This solution utilizes an APTS
derivatization strategy and separation gel buffer with a
separation efficiency allowing for routine separation of
the G1 isomers. Figure 5 shows an example trace for
N-Glycans released from an APTS labeled rMAb. In this
profile, the major glycan species G0 G1, G1’ and G2,
with and without fucose, in addition to several unidentified
minor peaks can be easily resolved at ~24 minutes.
One disadvantage of CE-based methods, which is
especially apparent in glycan analysis, is the lack of
routine detection methods that can provide direct
structural information on migrating species. Global
process transfer for commercial products is a reality for
many biopharmaceuticals and as a result, the ability to
6
2.0
1.8
1.6
1.4
1.2
1.0
RFU
0.8
0.6
0.4
0.2
0.0
-2.0
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-2.0
242322212019
Minutes
18171615
assess minor glycosylation differences using a validated
method is critical. In the glycan assay, characterization can
be achieved using one of two methods: 1) co-migration
with commercially available standards, and 2) exoglycosi-
dase treatment followed by co-elution with standards,
such as shown in Figure 5. These methods can be
particularly labor-intensive and do not provide any direct
information on glycan structure.
Recently, efforts have been undertaken to couple CE to
Mass Spectrometry (MS), and Glycan analysis25, with its
MS-compatible ammonium acetate or aqueous epsilon-
amino-caproic acid (EACA) separation buffers, is an
ideal application for this somewhat novel technology.
By coupling CE with MS or even MS/MS in an online
fashion, complex carbohydrate structures can be resolved
in as simple as three steps: 1) Release of carbohydrate
from protein backbone 2) Labeling of the glycan entities
for concurrent LIF analysis 3) Structural analysis via online
MS/MS analysis.22
The ability to perform complex CE-Glycan characterization
in an online fashion opens new doors for the use of CE
not only as part of QC release, but also as part of process
characterization and validation. The clock for CE-MS in
a quality control environment has already started with a
validated CE-MS assay for glycoprotein analysis, as
presented by Paula Domann from LGC, UK at CE in
Pharmaceutical Analysis, 2008.
Conclusions
Capillary electrophoresis (CE) methods for the analysis of
therapeutic proteins are routinely used in quality control
lot-release testing. This article was meant to introduce the
reader to the advantages of CE-SDS, CIEF and
CZE-Glycan for the analysis of protein therapeutics.
As has been shown, properly optimized CE methods are
robust and reproducible, averaging migration time RSDs
of less than 2% and peak area reproducibility for main
peak of < 5%. In the second part of this series of articles,
we will focus on optimization and robustness testing for
the most common QC CE technology: CE-SDS in
reduced and non-redu
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