Procedures

Unified Workflow for Monoclonal Antibody Charge
Heterogeneity, Purity, and Molecular Weight Analysis
Separation and Online Detection of Intact mAb Variants and Impurities using CESI-MS
Bryan Fonslow, Marcia Santos, Jose-Luis Gallegos-PerezSCIEX Separations, USA

Overview
Biopharmaceutical development and manufacturing ofmonoclonal antibody (mAb) therapies requires routine analyses
and monitoring of various physiochemical properties. Here, we
describe a unified capillary electrophoresis and electrosprayionization with mass spectrometry (CESI-MS) workflow forcharacterization of mAb samples. The workflow combines theunique separation capabilities of capillary zone electrophoresis(CZE) with the advantages of an MS-based detector to provide
high resolution structural information and accurate molecularweight information. Small amounts (~10 ng) of samples
containing intact (non reduced) or reduced mAbs can beanalyzed to determine charge heterogeneity (includingcharge variants, glycoforms, and clipped species), purity,and molecular weight.

CESI 8000 Plus High Performance Separation Module coupled toa TripleTOF® 6600 System via a Nanospray III source, CESI adapter,and neutral-coated OptiMS capillary cartridge

Who Should Read This: Senior Scientists and Lab Directors
Focus: Analytical testing of mAb samples during druguse of reduced mAb samples instead of intact mAb samples,development or manufacturing. Potential applications of theidentification of clipping events is challenging. Missed clipping

workflow include mAb characterization and purity testing during

events may lead to incorrect assumptions regarding sample

mAb candidate screening, manufacturing process development,

handling, storage conditions, and stability information.

and commercial production quality control.

Results: A single CESI MS workflow was developed and tested

Goal: Determine charge heterogeneity, purity, and molecularfor determination of charge heterogeneity, purity, and molecularweight of therapeutic mAb samples using a single CESI-MS

weight information in intact and reduced mAb samples. Identified

workflow that has improved performance and utility comparedspecies in mAb samples included the intact mAb, variants (eg,with existing industry-accepted methods such as CE-SDS andcharge variants, clipped species, glycoforms), impurities, andcapillary isoelectric focusing (cIEF)

excipient components. Results were similar to those determinedusing a combination of industry-accepted methods (ie, CE-SDS

Problem: mAbs are susceptible to modification and degradation
events that can change their physicochemical properties
and ultimately impact the stability, efficacy, and safety of mAb
therapies. Impurities and excipients introduced duringand cIEF). Notably, the CESI-MS results provided additional
structural information of species which allowed for identificationof potential mAb clipping events that may have been missedby traditional methods.

development and manufacturing can also have similar effects.
Because of this, thorough characterization at all stages of

Key Benefits:development and manufacturing is required. Currently, industry• Simplified workflow with a single CESI-MS analysis for
multiple mAbs characterization insights

relies on multiple methods to obtain charge heterogeneity, purity,and molecular weight information. Together, these methods canconsume relatively large quantities of expensive and masslimited samples. Further, because most methods rely on the• Glycoforms, clipped species, impurities, and excipients
all separated and identified by the same method withaccurate mass detectionp1

Procedures

Introduction

Materials and Methods

Immunoglobulin gammas (IgGs) are the most common molecules

Sample Preparation: For CESI-MS experiments, IgG1, IgG2,

used for generation of therapeutic monoclonal antibodies (mAbs).

and IgG4 molecules (20 mg/mL) were desalted and buffer

The ability to characterize these molecules with high sensitivity

exchanged into 50 mM ammonium acetate, pH 4 using Zeba

and comprehensiveness is essential to their development and forspin columns (Thermo Fisher Scientific). For reduced analysis,regulation of their efficacy, bioavailability, and biosafety. Charge

IgG molecules were incubated for 45 min at 60° C in 10 mM

heterogeneity, purity, and molecular weight analyses are powerful

DTT and 0.1% Rapigest SF Surfactant (Waters). Rapigest was

CE- and MS-based methods for mAb characterization. Intact andcleaved (0.5% formic acid, 37° C, 10 min) and spun down with

reduced analysis of mAbs by CE are used in the mAb screening,

precipitants (14K x g). Concentrated (2 M) ammonium acetate,analytical development, and quality control processes. Combining

pH 4 was added for a 50 mM final concentration. For stand-alone

a similar CE separation with mass spectrometric (MS) detection

CE experiments, IgG molecules were either diluted into a urea-of intact mAbs allows for unification of these three methods

gel-ampholyte solution (cIEF-based analysis) or SDS gel solution

into one. Additionally, the high resolution and mass accuracy(CE-SDS analysis). Reductions in SDS gel solutions wereMS detection can facilitate identification of unknown CE

performed at 60° C for 10 min in 10 mM DTT.

peaks and may also provide more accurate and sensitive
purity and molecular weight measurements than with opticaldetection alone.

CESI 8000 Plus MS Mode Conditions: CESI experiments
were carried out with a SCIEX CESI 8000 Plus system (P/NA98089) equipped with a temperature controlled auto sampler

The integration of capillary electrophoresis (CE) and electrospray

and a power supply with the ability to deliver up to 30 kV. An

ionization (ESI) into one process (CESI) provides theseOptiMS Neutral Surface Cartridge (P/N B07368) with a porous

possibilities while also lowering the sample mass analysis

tip was used. Solutions of 3% and 10% acetic acid wererequirements. We present the analysis of intact and reduced

employed as background electrolytes (BGE) and conductive

IgGs using a single CESI-MS method which provides charge

liquids, respectively. Pressure (5 psi) was applied for 10 sec

heterogeneity, purity, and molecular weight information similar

to generate ~7.5 nL sample injections. Sample stacking after

to the multiple current industry-accepted CE-SDS, capillarypressure injection was performed by transient isotachophoresis

isoelectric focusing (cIEF), and capillary zone electrophoresis(tITP) due to the 50 mM ammonium acetate sample buffer. CESI(CZE) analysis methods combined.

1-3

Notably, the intact andreduced IgG analyses by CESI-MS demonstrate the capabilities
for quickly screening and characterizing candidate or developed
therapeutic IgG molecules which may have either charge and/orsize heterogeneity, particularly due to stress or instability.
The CESI-MS results are compared to existing industryaccepted, CE-based charge heterogeneity, purity, and molecularweight analyses. Charge heterogeneity separations by CESIMS using a capillary zone electrophoresis (CZE)-based

separations were performed at 30 kV with 2 psi for 7 min (preseparation), then 10 psi for 10 min (separation and ESI).
MS Conditions: A SCIEX TripleTOF® 6600 system with aNanoSpray III source and CESI adapter (P/N B07363)
were used. Intact protein detection was performed with MSscans from 400 – 4500 m/z.
Data Analysis: High resolution MS spectra were analyzed usingSCIEX PeakView® and BioPharmaView™ software.

separation mechanism showed similar profiles to a cIEF-based

CESI 8000 Plus Stand-Alone CE Mode Conditions: CE-SDS

method which has been previously demonstrated using opticaland cIEF experiments were performed using SCIEX PA 800 Plus

detection methods.3-6 With the MS-based detection, moleculardetectors (P/N B68372), kits (P/Ns A10663 & A80976),

changes that cause charge heterogeneity, such as glycoforms,and protocols.

were associated with CE migration shifts. Other peaks within
the charge heterogeneity separation could also be primarily
attributed to potential clipped IgG impurities within the samples.
Collectively, the results demonstrate the advantages of using MS
as the detector for a CZE-based charge heterogeneity analysis
since it also provides molecular weight and purity information.

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Basic, higher MW
intact IgG1 specie
and glycoforms

Main intact IgG1
specie andglycoforms

Potential clipped
IgG HC-HC specie

Potential clipped
IgG1 species

8.0

Potential clipped
IgG1 species

8.5

9.0
9.5
Time, min

10.0

10.5Potential clipped
IgG1 species

Figure 1: Charge heterogeneity separation of IgG1 with MS detection. Extraction and deconvolution of non-reduced IgG1 species MS spectrafrom the CESI separation. The extracted ion electropherograms (XIEs) from the most abundant charge states (~3 – 6) from the same charge
state envelopes were summed. Spectra were integrated using 15 – 30 sec from each electrophoretic peak using their full width at half
maximum height as the integration window.

p3species were detected. The main charge variant had an averagemolecular weight of 146,900 Da while the basic IgG1 charge
variant was 151,005 Da. In addition to average molecular weight
differences, their glycoform profiles were different. Further data
analysis using the high mass accuracy deconvoluted molecularweights would facilitate identification of candidate sequence andglycoform matching. While two main IgG1 variants were detected
as electrophoretic peaks and intact MS spectra, their glycoform
profiles and partially split electrophoretic peaks also imply that
additional variants are also present in the sample. Additional
deconvolution analysis indicates a mixture of spectra from due
deamidations, disulfide breaks, and glycosylation variants (datanot shown). Four other IgG1 species were also detected by MS
which appeared to be impurities from potential in-solution clipping
events. The IgG1 impurities included abundant molecular weightsof 28,608, 36,687, and 46,585 Da. The highest molecular weight
impurity was 100,545 Da which had a molecular weight andglycosylation profile consistent with a heavy chain dimer (HCHC) impurity. Notably, the CESI-MS-based charge heterogeneity
analysis provided results consistent with a cIEF (Figure 2A) andCE-SDS (Figure 2B) analysis of the same IgG1 sample. For
example, a similar separation profile and number of species weredetected between the cIEF and CESI-MS charge heterogeneity
separations. Additionally, the number of molecular weight
impurities from CE-SDS analysis is also consistent with thenumber of species detected with unique molecular weights by theCESI-MS based analysis. Thus, the CESI-MS analysis combines
both aspects of the cIEF and CE-SDS analyses through a CZEbased charge heterogeneity separation and molecular weight
determination by MS. Ultimately, the CESI-MS analysis allows
for direct detection and candidate identification of potential
IgG1 clipping events that might be otherwise be misinterpreted
as intact IgG1 charge isoforms or difficult to identify by UV
detection alone. Representative raw MS data used for spectral
deconvolution is shown for each electrophoretic peak in Figure 3.
Figure 2: (A) cIEF analysis of non-reduced IgG1 molecules with UV
detection. (B) CE-SDS analysis of non-reduced, alkylated (blacktrace) and reduced (blue trace) IgG1 molecules with PDA detection.

The CZE-based method can also be used for reduced mAbanalysis. Figure 4 shows the separation and detection of reduced
IgG1 molecules. One IgG1 heavy chain was detected while twoIgG1 light chain species were detect. This was also consistent

Results and Discussion
CESI-MS based analyses were performed at the non-reduced(intact) and reduced levels using the neutral-coated CESIcapillary. The CZE-based separation of non-reduced IgG1 (Figurewith the three main reduced IgG1 species detected by the CESDS analysis. The molecular weight measurement of the twoIgG1 light chain species contributes to the understanding of themolecular weight differences of the intact IgG1 forms.

1) generated a charge heterogeneity separation and facilitatedThat is, the higher molecular weight IgG1 light chain is likely

accurate molecular weight determination of IgG1 species andpresent in the acidic, higher molecular weight intact IgG1

impurities through spectral deconvolution. Two intact IgG1charge variant. In combination with the high mass accuracyp4

measurement by the MS, further data analyses could be usedof each in both cases. Similarly, potential clipped IgG2 andto confirm the different intact IgG1 forms using the reduced

IgG4 species both migrate before their intact forms, allowing for

IgG1 analysis.

sensitive identification of the impurities. In the case of the IgG2analysis, a potential formulation excipient (e.g. polysorbate 80)

The same CESI-MS intact charge heterogeneity analyses werealso performed on representative IgG2 and IgG4 molecules

was well resolved from IgG2 that might otherwise detrimentallyaffect MS spectral quality by nanoESI- or LC-MS-based(Figures 5 and 6, respectively). Just as with the IgG1 analyses,
multiple intact and clipped IgG2 and IgG4 species were detectedin each analysis. Even though the intact IgG2 and IgG4 co-analyses. Similar results with other non-ionic detergents would
be expected since they will have the same electrophoretic
mobilities and migration times.

migrate with their respective forms, the spectral deconvolution
process allows for identification of two different glycoforms

8.0

8.5

9.0
9.5Time, min

10.0

10.5

Figure 3: Extraction of representative raw MS spectra from non-reduced IgG1 species from the CESI separation used for generationof deconvoluted spectra in Figure 1. Spectra were integrated 15 – 30 sec from each electrophoretic peak using their full width at half
maximum height as the integration window.
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IgG1 heavy chain
IgG1 light chain
Higher MW IgG1 light chain

Figure 4: Reduced IgG1 analysis with MS detection. Extraction and deconvolution of reduced IgG1 species MS spectra from the CESIseparation. The extracted ion electropherograms (XIEs) from the most abundant charge states (~3 – 6) from the same charge stateenvelopes were summed.

IgG2 glycoforms

Potential excipient polymer

Potential clipped
IgG2 specie

Potential clipped
IgG2 species

Figure 5: Charge heterogeneity separation of IgG2 with MS detection. Extraction and deconvolution of non-reduced IgG2 species MS spectrafrom the CESI separation. The extracted ion electropherograms (XIEs) from the most abundant charge states (~3 – 6) from the same charge
state envelopes were summed.

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AB

Potential clipped
IgG4 species

CIgG4 glycoforms

Figure 6: (A) Charge heterogeneity separation of IgG4 with MS detection. The extracted ion electropherograms (XIEs) from the mostabundant charge states (~3 – 6) from the same charge state envelopes were summed. (B) Extraction and deconvolution of potential
clipped IgG4 species MS spectra from the CESI separation. Deconvoluted MS spectra colors correspond to the XIE peaks of the samecolor. (C) Extraction and deconvolution of non- reduced IgG4 species MS spectra from the CESI separation. The left and right deconvoluted
spectra correspond to spectral extraction of the left and right side of the orange, split XIE peak.

Conclusion
The combined CESI-MS and stand-alone CE

References
1.

Nunnally, B. et al., A series of collaborations between
various pharmaceutical companies and regulatory
authorities concern- ing the analysis of biomolecules
using CE, Chromatographia,
a. 2006, 64, 359-368.

2.

Salas-Solano, O., et al., Intercompany study to evaluate
the robustness of C-IEF technology for the analysis ofmonoclonal antibodies, Chromatographia, 2011, 73,1137-1144.

3.

Moritz, B. et al., Evaluation of capillary zone
electrophoresis for charge heterogeneity testing ofmonoclonal antibodies, J. Chrom. B, 2015, 983–984,101–110.

functionality of the CESI 8000 Plus system provides
powerful methods to characterize intact and reduced
IgG forms. Both analyses deliver charge heterogeneity,purity, and molecular weight information. When coupledwith the TripleTOF® 6600 system, the CESI- based
analysis using a neutral-coated capillary generates highresolution separations with high mass accuracy
molecular weight information for characterization ofmAb charge variants, glycoforms, and impurities.
Additionally, the high sensitivity CESI- MS analyses areachieved from small sample amounts (~10 ng),
particularly useful in the mass-limited development
phase.

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4.

He, Y. et al., Analysis of Identity, Charge Variants, andDisulfide Isomers of Monoclonal Antibodies withCapillary Zone Electrophoresis in an Uncoated Capillary
Column, Anal. Chem. 2010, 82, 3222–3230.

5.

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.

6.

Santos, M. Analysis of Monoclonal Antibody Charge
Variants by Capillary Zone Electrophoresis, Beckman
Coulter Technical Information Bulletin, IB-17031A.

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Document number: RUO-MKT-02-2757-A

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