Comprehensive analysis of low abundant mannose
glycopeptides in peptide mapping of adalimumab
Featuring CESI 8000 Plus and TripleTOF® 6600+ LC-MS/MS System
Zuzana Demianova1, Miloslav Sanda2, Zoe Zhang3, Sean McCarthy4, and Sahana Mollah3
1 SCIEX, Brea, CA; 2 Georgetown University, Washington, DC; 3 SCIEX, Redwood City, CA; 4 SCIEX,Framingham, MA

Glycosylation is a common modification that occurs during theproduction of antibodies or other protein therapeutics. The
glycan structures attached to the proteins can directly affectprotein stability, bioactivity, and immunogenicity. Therefore,glycan variants of a glycoprotein product must be adequately
analyzed and controlled throughout development and production
to ensure product quality. Reversed phase-liquid
chromatography-mass spectrometry (RP-LC-MS) has become a
primary technique for antibody and protein therapeutics
characterization. However, it is challenging tochromatographically resolve some types of low abundant
glycoforms, such as high mannose glycopeptides (Figure 1).
CESI-MS provides a separation mechanism orthogonal to LCMS based approaches for separation, identification, andquantification. It provides the exceptionally high resolution powerof capillary zone electrophoresis (CZE) in conjunction with thesample identification capability of mass spectrometry. CZE isuniquely able to separate peptides based on charge difference,unlike a reversed phase HPLC, and provides sensitive andreproducible results. The ultra-low flow rate of CESI (~20 nL/min)is highly beneficial in maximizing ionization efficiency andminimizing ion suppression. In addition, the open tubular formatof CESI without any solid stationary phase, allows elution of all

CESI 8000 Plus and TripleTOF 6600+ System with processing
software SCIEX OS Software and BYOS

peptides from the capillary (both hydrophobic and hydrophilic),
thus providing better sequence coverage of the antibody.
In this technical note, bottom-up peptide mapping of adalimumab(Humira) and its post-translational modifications (PTMs) wereperformed by CESI-MS analysis. We were able toelectrophoretically separate various classes of glycopeptides andconfirm using diagnostic ions in the MS/MS spectra.

Key features of CESI 8000 Plus coupled tothe TripleTOF 6600+ SystemFigure 1. Separation of highly mannose glycopeptides by CESI-MSand LC-MS.

•Capability of electrophoretically separate various
glycopeptides including a high mannose species•Minimum sample required (nanoliters) to obtain highsequence coverage•High sensitivity of CESI allows for identification of low-levelPTMs•

Data Processing using BYOS software (Protein metrics) for
identification with MS/MS confirmation of short andhydrophilic peptides which are often missed in LC-MS
p1

Methods
Sample preparation and mAb digestion: Adalimumab (1mg/ml) was digested with trypsin (gold trypsin, Promega) in a
1:20 (v/v) enzyme-to-antibody sample ratio for 2h at 37°C. Prior
to trypsin digestion, antibody was reduced with 20 mM
dithiothreitol (DTT) at 56°C for 30 min and alkylated with 20 mM
iodoacetamide (IAM) in the dark for 30 min. The alkylation
reaction was quenched with 10 mM DTT. A detailed protocol isdescribed in a CESI peptide digest preparation technical note.1
Capillary electrophoresis and mass spectrometry: CESI
method parameters: Trypsin digested sample was diluted in a
1:2 (v:v) ratio with 100 mM ammonium acetate at pH 4. For theanalysis, a Bare-Fused Silica Surface OptiMS Cartridge wasused (P/N B07367). Sample was injected hydrodynamically at 5psi for 60 s, resulting in approximately 44 nL injected volume,
which is approximately 39 ng of adalimumab on the cartridge.
Separation conditions are listed in Table 1.
Table 1. CESI separation conditions used for the peptide
mapping of adalimumab

MS method parameters: The TripleTOF 6600+ System wascoupled with the NanoSpray® III Ion Source with CESI adapter(P/N B07366) and controlled by Analyst® Software 1.8.1. The
peptide mapping of adalimumab was performed using data
dependent acquisition (DDA) with 10 MS/MS cycles (150 and 50
ms accumulation times for MS and MS/MS, respectively). Theacquisition mass range was from 100 to 2250 m/z with cycle timeof 0.8 s. The precursors with charges from +1 to +5 and intensity
above 100 cps were triggered for MS/MS fragmentation.
Selected precursors were fragmented using a rolling collisionenergy (CE) with spread of ± 5 V. The CE curve slope (Table 2)
was modified for better glycopeptide fragmentation.2 Source
parameters were set to ion spray voltage 1850 V, source
temperature 30°C and curtain gas 5 psi.
Table 2. The CE curve slope used to calculate CE for rolling
collision energy
ChargeSlopeIntercept

Unknown

0.037-110.038520.037-1

Time Pressure Voltage Direction(min) (psi)(kV)

Solution/Note30.036-2

1 Rinse2.5100Forward

0.1 M NaOH

40.038-2

2 Rinse2.5100Forward

0.1 M HCI

50.038-2

3 Rinse4100

ForwardWater

4 Rinse375Reverse

10% Acetic acid

5 Rinse4100

Forward

10% Acetic acid

6 Injection

60 s5Forward

10% Acetic acid

7 Injection

25 s

0.5

Forward

10% Acetic acid

8 Separation

50015

Forward

10% Acetic acid

9 Separation

501

Forward

5 min ramp down

EventLiquid chromatography and mass spectrometry: The Waters
CSH C18 column (2.1×150 mm, particles 1.7 µm) was used toseparate 2 µg of trypsin digested adalimumab using ExionLCTM
AD System coupled to a X500B QTOF system. The 0.1% formic
acid in water (mobile phase A) and 0.1% formic acid inacetonitrile (mobile phase B) were used. The LC gradient wasset accordingly, from 1 to 5 min, 1% of mobile phase B, from 5 to6 min, 1 to 10% of mobile phase B, from 6 to 32 min, 10% to25% of mobile phase B, 32 to 65 min, 25% to 40% of mobile

Figure 2. Separation of high mannose glycopeptides of adalimumab using CESI-MS and LC-MS techniques. A) CESI-MS separation, B) RP-LCMS separation and C) fragmentation spectra of the Man5 glycoform with adjusted rolling CE curve (Table 2) for maximum recovery of the y-ions. *interfering ionp2Figure 3. Separation of fucosylated glycopeptides of adalimumab using CESI-MS and LC-MS techniques. A) CESI-MS separation, B) RP-LC-MS
separation and C) fragmentation spectra of the G0F glycoform using adjusted rolling CE (Table 2)phase B, from 65 to 70 min, 40 to 60% of mobile phase B andfrom 70 to 70.5 min, 60 to 90% of mobile phase B. The analysis
length was 75 min using DDA with 8 MS/MS cycles (250 and 40ms accumulation times for MS and MS/MS, respectively). The
MS parameters were ion spray voltage 5500 V, gas 1 60 psi, gas
2 60 psi, curtain gas 55 psi, source temperature 350°C, androlling collision energy.
Data processing: Data were visualized using SCIEX OS
Software and processed with a BYOS (Protein Metrics). Searchparameters were set for two miss-cleavages, trypsin digestionand +/- 5 ppm mass error.

Results and discussion
The biologics segment is growing significantly within thetherapeutic industry.3 As biotherapeutic products are derived
from recombinant DNA technology and produced in hostorganisms, monitoring of CQAs (Critical Quality Attributes) is
crucial to ensure the consistency of product quality, safety, andefficacy. Glycosylation is considered as one of the crucial CQAof mAbs and biosimilars. A unique feature of CESI compare toLCMS analysis is the ability to separate various glycopeptides
and characterize the mAb in a single run. In this study, we focus
on full characterization of adalimumab.
Separation and identification of glycopeptide of adalimumab
A high-resolution CESI-MS technique is well suited for theanalysis of glycopeptides as it separates peptides based on theircharge-to-mass ratio. Of these glycopeptides, high mannose
glycopeptides are considered to be some of the most challenging
to analyze due to their low abundance in the samples. Figure 1and 2A show a comparison of CESI-MS and LC-MS separationof high mannose glycopeptides. Man3, Man4, Man5, Man6,
Man7 and Man8 were electrophoretically separated from eachother, which allowed for the identification of low abundantspecies compared to the LC analysis (Figure 2B). Furthermore,
Figure 2C illustrates fragmentation spectra of Man5
glycopeptide. The y-ion 793 m/z represents the loss of fivemannose residues, leaving the two core GlcNAc residues
attached to the peptide backbone. The y-ions 1041 m/z and 879
m/z are losses of 2 an 4 mannose residues, respectively.

Figure 4. Minor glycopeptides of adalimumab separated using CESI-MS and LC-MS techniques. A) CESI-MS separation, B) RP-LC-MS
separation and C) fragmentation spectra of the G0F-GlNAc
p3

Table 3. Glycoforms observed with high confidenceshows three fucosylate glycopeptides (G0F, G1F and G2F) well
separated in comparison to the LC-MS analysis (Figure 3B),
which missed the low abundant G2F glycopeptide. Figure 3C
represents the MS/MS spectrum of G0F glycopeptide.
In addition to higher abundant glycopeptides, we were able toconfirm a few minor species, such as G0, G0F-GlNAc and G1FGlNAc (Figure 4). Altogether, we have confidently identified 12
different glycopeptides of adalimumab with their relative
abundances (Table 3).
Our findings are in good alignment with results from release Nglycan analysis using the SCIEX C100HT Biologics Analyzer.5
CESI-MS peptide mapping and post-translational
modifications

Sequential loss of oligosaccharides together with precursor
provided confirmatory evidence for the presence of the Man5glycoform. To obtain a good fragmentation pattern forglycopeptides as well as peptides with the DDA, the rolling
collision energy curve slope was lowered compared to
instrument pre-set values (Table 2).2 In addition to y-ions,common glycan fragments (m/z 204, 366 and 528) are found in
the spectra (Figure 2C, 3C and 4C). These fragments have ahigh sensitivity, but low specificity as they are common for all Nglycopeptides.4
Fucosylate glycopeptides is another class that is important tomonitor as a CQA. This group is normally identified by LC-MS,
but are challenging to separate using a RP column, which iscommonly used for peptide mapping. In Figure 3A, CESI-MS

CESI-MS is an effective and complementary technology tocharacterize mAbs on the peptide level with a high sequence
coverage; in the case of adalimumab, we confidently map 100%of its sequence and have confirmed 12 various glycopeptides.
For the next step, we focused other PTMs, such as deamidationand spontaneous cyclization of N-terminal glutamic acid. Figure
5 illustrates examples of the separation between the unmodifiedand deamidated form of VGNALQSGNSQESVTEQDSK peptide.
The deamidated peptide was determined to be 10.8% of the total
amount of peptide in the sample. Deamidation is one of the mostcommon PTMs occurring in therapeutic proteins produced using
recombinant DNA technology.
Another example of common PTM is pyro-glutamate. Itspresences is frequently detected and mainly attributed tospontaneous cyclization during fermentation, purification andstorage. An example is shown in Figure 6, the unmodified

Figure 5. Unmodified vs. peptide with deamidated asparagine are separated by CESI-MS.

p4

Figure 5. Unmodified N-terminal glutamic acid vs. peptide with N-terminal pyroglutamate are separated by CESI-MS.

EVQLVEGGGLVQPGR peptide contains 2.1% of the N-terminal
pyro-glutamate.
In addition, CESI-MS is capable of analyzing challenging short
peptides or hydrophilic peptides, that could be easily missed byLC-MS. 6 Figure 7 shows identification with MS/MS confirmationof short and hydrophilic peptide (VSNK [M+H]+1).

Conclusions
Peptide mapping remains a key analytical methodology tocharacterize biotherapeutics from drug discovery throughproduction and it provides the ability to assign specific locations
to product attributes. It allows monitoring of amino acidsubstitution as well as post-translation modifications that are
critical for a biotherapeutic’s safety and efficacy.
CESI-MS peptide mapping is valuable orthogonal technologythat provides additional sequence coverage compared to LC-MS.
In this study, we characterized several CQAs, such as
glycosylation, deamidation and oxidation of adalimumab
peptides, within the same run. A high sequence coverage(>95%) was obtained from less than 40 ng of adalimumab
loaded into the CESI cartridge which seamless data processing
using BYOS software (Protein Metrics)
CESI-MS additionally separated 12 diverse glycopeptides
including 6 low abundant high mannose glycopeptides ofadalimumab. These are in good agreement with literature
examples.5 Adjusted CE slope allowed MS/MS confirmation ofglycopeptides based on their specific y-ions as well as N-glycan
diagnostic fragments without sacrificing results of adalimumab
peptide mapping.

ReferencesFigure 7. Identification of hydrophilic peptide VSNK [M+H]+1.

1.

RUO-MKT-02-11835-A, Optimized digestion procedure andcharacterization for monoclonal antibodies and proteins byCESI-MS

2.

https://sciex.com/community/applicationdiscussions/proteomics/swath/datap5

acquisition/crowdsourcing-optimized-rolling-collision-energycurves-for-id-and-swath-acquisition
3.

Jognson D. E., Biotherapeutics: Challenges andOpportunities for Predictive Toxicology of Monoclonal
Antibodies, Int J Mol Sci., 2018, 19 (11), pp. 3685

4.

Sanda M., Goldman R., Data Independent Analysis of IgG
Glycoforms in Samples of Unfractionated Human Plasma,
Anal Chem, 2016, 88, pp.1018

5.

RUO-MKT-02-9755-A, Analysis of Fluorophore Labeled Nglycans by the Multicapillary C100HT Biologics Analyzer
and HILIC-UPLC: Emphasis on Neutral Structure,https://sciex.com/Documents/tech%20notes/applications/ph
arma/c100ht-vs-uplc-neutralpdf.pdf

6.

Lew C., Gallegos-Perez JL., et al., Rapid Level-3
Characterization of Therapeutic Antibodies by Capillary
Electrophoresis Electrospray Ionization Mass Spectrometry,
J Chrom Science, 2015, 53(3), pp.443

The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales
representative or refer to https://sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures. Trademarks and/or registeredtrademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain other countries.
© 2020 DH Tech. Dev. Pte. Ltd. DOC RUO-MKT-02-11834-B
AB SCIEX™ is being used under license.

p6