Charge heterogeneity characterization of next-generation
antibodies using capillary electrophoresis and mass
spectrometry
Zuzana Demianova, 1Fang Wang, 2Peter Holper, 2Zoltan Szabo 2Sahana Mollah
SCIEX, Brea, CA, USA, 2SCIEX, Redwood City, CA, USA

11Introduction
This technical note illustrates a rapid profiling method for charge
variant profiling of next-generation antibodies during productdevelopment, manufacturing, stability and release testing using
capillary zone electrophoresis (CZE) on PA 800 Plus
Pharmaceutical Analysis System. Subsequently, the ZipChip
System coupled to the TripleTOF® 6600 LC-MS/MS System
can be used for deeper characterization of the molecule andidentification of the variants.
Next-generation antibodies, such as engineered fusion proteinsand bi-specific antibodies carry great potential in clinicalapplications. Similar to mAbs, they are generally cell-originatedand are subject to post-translational modifications that canintroduce charge heterogeneity to the molecule. Monitoring ofthese variants is required throughout manufacturing to assess
drug purity and heterogeneity and to ensure the safety andefficacy of the drug.

A) :CE-UV0.05

In earlier work, we demonstrated two simple kits-based assaysthat provide highly similar charge variants profiles for routine
monitoring with CZE-UV and peak identification with CZE-MS
for traditional mAb molecules. Here, we used two commerciallyavailable molecules (emicizumab-kxwh and blinatumomab) toshow that the same sets of tools could also be used for charge
variant monitoring and in-depth characterization in nextgeneration biologics development.

Key features• Reliable, high-resolution, rapid charge variant profiling (<15minutes) of next-generation biologics using CZE under nativeconditions• Seamless bridge from peak profiling using CZE-UV for routine
product monitoring followed by deeper peak characterization
with ZipChip MS providing good profile alignment• Easy and ready-to-use kits-based platforms with minimal
sample preparation• Straightforward data analysis and spectrum reconstructionB) :CE-MSMPCEMPAU

0.04
0.03
0.02

B1

0.01

A1A2

B10.00456

7Minutes

89A110Figure 1. A side-by-side comparison between the charge variants separation with CZE-UV and CZE-MS assay for emicizumab-kxwh. A) CE-UVcharge variant profile using CZE Rapid Charge Variant Analysis Kit on a PA 800 Plus Pharmaceutical Analysis System; B) CE-MS charge variant profile
using Charge Variant TOF Kit on a ZipChip coupled to the TripleTOF 6600 system. Detailed MS identification is shown in Figure 3.

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Methods
Sample reconstitution: Emicizumab-kxwh and blinatumomab
antibody were reconstituted to a concentration of 30 μg/μL and0.2 μg/μL with water, respectively. Stock solutions were stored
at -80°C.
Sample preparation: A buffer exchange/sample concentrate
step was performed for blinatumomab in both capillaryelectrophoresis (CE) with optical (UV) and mass spectrometry(MS) detection due to its low concentration. Buffer exchange for
emicizumab-kxwh was only performed for CE-MS analysis.
emicizumab-kxwh was directly diluted to 1 mg/mL with waterfrom 30 mg/mL stock solution before CE-UV analysis.
Buffer exchange: Before analysis, the antibody stock solutions
were desalted and buffer exchanged into sample diluent from theCharge Variant TOF kit (908 Devices) using centrifugal MWCO
filters (emicizumab-kxwh 50 kDa and blinatumomab 30 kDa
MWCO filter). First, the filters were equilibrated with 500 μL ofdiluent and spun at 12,000×g for 5 min. Each antibody sample
was then dispensed into the filter device and spun at 12,000×g
until the sample was concentrated to a volume of 100 μL or less;five consecutive buffer exchanges were performed by adding
diluent up to 500 μL per spin. Emicizumab-kxwh andblinatumomab samples were diluted with diluent to a final
concentration of 1 μg/μL and 0.8 μg/μL, respectively.
Table 1. Mass spectrometry methods.
Parameter

Blinatumomab

Emicizumab-kxwh

MS mass range (m/z)

2000 – 5 000

3000 – 6000

MS accumulation time (s)0.4

GS 1 (psi)4

Curtain gas(psi) :10Polarity:

positiveSource temperature:

100°C

Delustering potential (DP)120 V

Collision energy (CE)30 V

was injected onto the chip. The separation was performed at
500 V/cm with pressure assistance turned on after 0.5 min using
the predefined method “intact proteins.” The total analysis time
was set to 15 or 20 min depending on the sample. The Zipchip
system is controlled with 908 Devices software, while theTripleTOF 6600 System was controlled with Analyst® TF
Software 1.8.1 Detailed MS parameters can be found in Table 1.
Data processing CE-MS: SCIEX OS Software 2.0.0 was used
to visualize data, and BioPharmaView™ Software was applied
to match the post-translational modification on the emicizumabkxwh.
Capillary electrophoresis-UV: The capillary electrophoresis
instrument used was a PA 800 Plus equipped with a UV
detector and a 214 nm bandpass filter (SCIEX P/N 144437).
Separations were performed on a pre-assembled bare fusedsilica cartridge (SCIEX P/N A55625) at 500 V/cm field strength
to improve the resolution. The sample was introduced into thecapillary via pressure injection for 10 seconds at 0.5 psi. The
separation buffer used was from the CZE Rapid Charge Variant
Analysis Kit (SCIEX P/N C44790). Instrument control and dataacquisition was made using 32 Karat Software 10.0.060

Results and discussion
As biopharmaceutical companies face increasing pressure todecrease the overall drug development timeline, analyticalresearchers find the need to finalize methods within shorter
timelines and with more limited samples. Quickly finding arobust, stability-indicating method that can monitor charge
variants is an ongoing analytical challenge. The most popular
capillary isoelectric focusing (cIEF) charge variant methods
generally analyze biotherapeutics in a denatured state and also
face great challenges for peak characterization.1-2 In general,
the identification of peaks in a cIEF profile requires developmentof orthogonal methods. In contrast, CZE combines the50 V

Collisionally activateddissociation (CAD)

7VSum to bin

100

Capillary electrophoresis and mass spectrometry: The CEMS analysis was performed using the ZipChip system (908Devices Inc.) consisting of an optional autosampler coupledwith the TripleTOF 6600 System. A “high-resolution native”(HRN) chip and the Charge Variant TOF kit containing premixed
buffers were used here. For each analysis, 2 nL of the sample

Figure 2. Quick charge heterogeneity profiling of nextgeneration antibody followed by identification of peaks
using the ZipChip system coupled to the TripleTOF 6600System.
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MS profile for blinatumomab. Both analyses are carried out
according to the kit's instruction without extensive optimization.
Similar charge variant profiles were observed for both molecules,
demonstrating the possibility of using CZE-UV workflow routinely
and performing peak characterization when deemed necessary.

benefits of a native state analysis with the speed and resolution
expected of a capillary electrophoresis method. Using a widely
universal buffer requires little to no method development, andhas a simple sample preparation and a high analyticalthroughput.3-4 More importantly, the Charge Variant TOF kit andZipchip system provide a seamless bridge for intact level peakidentification (Figure 2).
Emicizumab-kxwh charge variant characterization: Similar to
traditional mAbs, the formulated sample has a concentration of30 mg/mL, allowing thirty-fold dilutions before being subject toCZE-UV and CZE-MS analysis. The simple buffer exchange step
provides better spectral quality (decreased peak/valley ratio) in
the CZE-MS results, but no significant change was observed for
the CZE-UV analysis.
Blinatumomab charge variant characterization: Due to thelow protein concentration in the sample, formulation buffer
excipients showed great interference in both CE-UV and CE-MSanalysis (Figure 2). After buffer exchange/sample concentration
significantly improved charge variant separation observed in CEUV, the actual protein charge envelop rather than polymer
excipients were observed in CE-MS (Figure 3).
Figure 1 shows the side-by-side comparison between CZE-UVand CZE-MS profile for emicizumab-kxwh. Figure 3 panels band d show the side-by-side comparison between CZE-UV andCZE0.011-A close look at the MS results indicates that neither of the two
next-generation mAbs shows the typical C-terminal lysine
variants observed in traditional mAb as the basic variants.5-6 The
dominant acidic variants are consistent with various deamidation
numbers (Figure 4 and Figure 5). The glycosylation pattern for
emicizumab-kxwh is confirmed at intact MS levels and is labeledin Figure 4. The reconstructed MS for blinatumomab is shown in
Figure 5. The lack of mass shift between main peak(MP) and acidic 1 (A1) species indicates the separation between
them might occur due to potential conformational difference,
which results in differences in surface charges in solution. Thisis consistent with the native-like conditions in both workflows.
Additionally, a charge variant at ~ 3% (A2) also shows good MS
spectrum quality, allowing confident spectrum reconstruction andidentification.
Both CE-UV and CE-MS workflows can be completed within 30
min for a sample with a total volume of 20 µL samples(concentration range 0.8 – 1.0 µg/µL) for multiple injections,providing a great opportunity for performing charge variantanalysis at early stages of product development.

C) CZE-MS TIC before buffer exchangeA) CZE-UV before buffer exchange

AU0.0120.0130.01B) CZE-UV after buffer exchange

0.00AUD) CZE-MS TIC after buffer exchange

MP-0.01

MPA1 A2
B1-0.02

A123456789A210MinutesFigure 3. A side-by-side comparison between the charge variants separation with CE-UV and CE-MS assay for blinatumomab. CE-UV chargevariant profile A) before and B) after buffer exchange; B) CE-MS charge variant profile using charge variant TOF Kit on a ZipChip coupled to theTripleTOF 6600 system.

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+18 Da+5 Da

Obs M:
148491 Da

Figure 4. CE-MS charge variant characterization and identification for emicizumab-kxwh. Top), total ion chromatograph of emicizumab-kxwh
charge variant with peak labels and a zoomed-in charge envelop as an insert. Bottom): reconstructed mass for each charge variant.

MPA1A1MP

A1v

A2A2A2+4 Da

Figure 5. CE-MS charge variant characterization and identification for blinatumomab. Top), total ion chromatograph of blinatumomab charge
variant with peak labels and a zoomed-in charge envelop as an insert. Bottom): reconstructed mass for each charge variant.

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Conclusions• Rapid charge variant analysis times (<10 minutes) of nextgeneration antibody using CE-UV• Ready to use kits and pre-assembled cartridge enables
streamlined analysis• Seamless bridge from peak profiling with CE-UV to peakcharacterization with ZipChip CE-MS with good dataalignment• Platform method for intact protein analysis with flexibility formethod development and optimization and minimal samplepreparation• Excellent MS sensitivity for identification of low abundancecharge variant peaks

References
1.

Righetti, P.G., Determination of the isoelectric point ofproteins by capillary isoelectric focusing. J Chromatogr A,
2004. 1037(1-2): p. 491-9.

2.

Sosic, Z., et al., Application of imaging capillary IEF forcharacterization and quantitative analysis of recombinant
protein charge heterogeneity. Electrophoresis, 2008. 29(21):p. 4368-76.

3.

He, Y. et al., Rapid analysis of charge variants ofmonoclonal antibodies with capillary zone electrophoresis in
dynamically coated fused-silica capillary, J. Sep. Sci. 2011,34, 548–555.

4.

Native state charge variant analysis of commercialized
monoclonal antibodies in minutes. SCIEX technical note,RUO-MKT-02-10446-A.

5.

Characterization of charge heterogeneity of monoclonal
antibodies using capillary electrophoresis (CE) coupled tomass spectrometry (MS). SCIEX technical note, RUOMKT-02-10949-A.

6.

Profiling and characterization of antibody charge
heterogeneity using capillary electrophoresis and massspectrometry. SCIEX technical note, RUO-MKT-02-12774A.

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