CESI-MS Comparison of Tryptic Digests from Different
Monoclonal Antibodies in an Assessment of Biosimilarity
Stephen Lock,1 Rabah Gahoual,2 Michael Biacchi, Alain Beck,3Yannis-Nicolas François2 and Emmanulle Leize-Wagner2
1 SCIEX, Warrington, UK
2 Laboratoire de Spectrométrie de Masses des Interactions et des Systémes (LSMIS),
Université de Strasbourg, Strasbourg, France
3 Centre d’immunologie Pierre Fabre, Saint-Julien-en-Genevois, FranceIntroduction
Several of the 10 top selling drugs are antibody-based
therapeutics that have lost or will lose patent protection soon.1As highly complex glycoproteins, monoclonal antibodies (mAbs)
have a wide range of micro-variants. Therefore, mAbs require
extensive characterization through multiple methods for structureassessment, manufacturing control, and biosimilarity studies.
Biosimilars contain a version of an active substance that isalready an authorized original biological drug. Biosimilars require
additional analytical and statistical characterizations for FDA
approval.2 As drugs lose patent protection these biosimilars
have started to enter the market.
Capillary electrospray Ionization (CESI) is the integration ofcapillary electrophoresis (CE) and electrospray ionization (ESI)
into a single process in a single device (Figure 1).3 CESI-MS
operates at low nL/min flow rates and offers several advantages,including increased ionization efficiency and a reduction inion suppression. CESI-MS separates analytes by their charge
and size, and it is therefore a separation mechanism that iscomplementary to more traditional techniques, such asreversed-phase LC. CESI-MS has been previously used toFigure 1. The OptiMS cartridge provides an ultra-low-flow ESI interface.

Materials and Methods
Chemicals: All chemicals were reagent grade and purchasedfrom Sigma Aldrich or alternative suppliers. Trastuzumab andcetuximab samples are European Medicines Agency (EMA)
approved products purchased from Roche (Penzberg, Germany)and Merck KGaA (Darmstadt, Germany), respectively. The
biosimilar versions were produced at Pierre Fabre (Saint-Julienen-Genevois, France) for analytical method development.
RapiGest SF surfactant was purchased from Waters (Milford, MA).
Sample Preparation: Samples were first diluted with Milli-Q

characterize mAbs.4

water, 0.1% RapiGest, then reduced and alkylated using

This document summarizes the work recently published by thedigested using trypsin (overnight at 37 °C). After digestion, 1%

research group at LSMIS at the Université de Strasbourg.5 In
this application note we will show how CESI-MS can be used tocharacterize and compare a marketed reference mAb with otherbiosimilar candidates. In a single injection the CESI-MS will be
used to simultaneously detect post translational modifications(PTMs) as well as provide sequence coverage. This data willthen be used to compare the reference mAb with a biosimilar.

dithiothreitol followed by iodoacetamide. The proteins were then(v/v) formic acid (FA) was added to cleave the surfactant andsamples were diluted to a final concentration of 2.2 µM of proteinusing 50 mM ammonium acetate (pH 4.0).5
CESI-MS method: For the analysis, a bare fused silica OptiMS
cartridge (30 μm ID x 91 cm) was used, and was thermostatted
using recirculating liquid coolant (20 °C). Samples were injectedhydrodynamically (10 psi, 60 s) and the peptides separated usinga background electrolyte of 10% acetic acid (Table 1).

p1

For MS analysis, a SCIEX TripleTOF® 5600+ LC-MS/MS System

The same experiment was performed for cetuximab andwas fitted with the NanoSpray® III Ion Source. Gas 1 and 2 were

cetuximab-B, and once again 100% sequence coverage was

not used, the curtain gas was set to 5 psi and the temperature ofobtained for the reference mAb and the biosimilar (cetuximab-B),

the interface was set at 150 °C. Ionization at these very low-flow

but in this case the sequences of these 2 mAbs were identical.

rates occurred by simply applying the ionspray voltage (1,750 V).
MS data was acquired using a TOF survey scan (accumulation
time 250 ms, m/z 50–2,000 amu), which triggered MS/MS
acquisition (accumulation time 100 ms per scan, 100–2,000 amu)
and led to a total duty cycle time of 1.75 sec.

To confirm these results, experiments were repeated in triplicate
and the same results were obtained each time. In addition,
more than 70% of the y/b fragment ions in MS/MS spectra of thepeptides were observed, and in the case of trastuzumab, it wasmore than 90%. This high MS/MS fragment ion coverage wasImportant:• A separation current above 5 μA might cause permanent
damage to the separation capillary• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

ActionTime(min)PressureDirection(psi)

Voltage(kV)

Solution

Rinse

3.5

100

Forward00.1 M NaOH

Rinse

1.5

Rinse

3100

Forward0

Water100

Forward00.1 M HClRinse

5100

Forward010% acetic acid

Rinse

275

Reverse010% acetic acid

Injection

60 s

10Forward0Sample vial

Injection

10 s

5Forward

010% acetic acid

Separation

350Forward

20

10% acetic acid

Voltage

210Forward

110% acetic acid

Table 1. CESI separation conditions used for the analysis of tryptic digests.

Results

Figure 2. Sequence coverage obtained by CESI-MS/MS for trastuzumab(left-hand side) and trastuzumab-B (right hand side).

Amino acid sequence is a key criterion for demonstrating

only possible due to the high ionization efficiency of CESI-MS/MS

biosimilarity, so in this study, 2 reference mAbs (trastuzumab(running at a flow of < 40 nL/min) in combination with the shortand cetuximab in their commercial formulation) were compared

duty cycle time of the MS system. This combination is well suited

to 2 potential biosimilars (trastuzumab-B and cetuximab-B). For

for the detection of post translation modifications (PTMs), which

trastuzumab and trastuzumab-B, a single analysis of a tryptic

often help to distinguish reference mAbs from biosimilars.

digest of each protein provided 100% sequence coverage on
both the heavy chain (HC) and light chain (LC), confirming theresults of a previous study that demonstrated CESI-MS/MS iscapable of 100% coverage in a single analysis (Figure 2).4

Glycosylation is important to the structural conformation andstability of mAbs. In this study, glycan release was not performed
during sample preparation, so glycans could be characterized
directly by detecting the glycopeptides, enabling identification ofWhen these sequences were compared, only 1 difference

the site of glycan modification. In the case of the trastuzumab/

between the primary sequence of the biosimilar and thetrastuzumab-B analysis, the injection of the reference mAbreference mAb could be identified on the HC at position

identified 16 different glycoforms. When the reference mAbs and217. Analysis of MS/MS spectra was able to confirm thatbiosimilars were semi-quantitatively compared (Figure 4), bothtrastuzumab-B, in position 217, underwent a lysine residue

biosimilars exhibited different glycoforms distribution compared

substituted by an arginine (Figure 3).

to the reference mAb.

p2

In addition to glycosylation, other critical quality attributes(CQAs) are considered important in biosimilarity assessment.
These CQAs, referred to as PTM hot spots, may influence
the immunogenicity and the PK/PD of the protein2 and include
N-terminal glutamine/glutamic acid cyclization, leading to theformation of N-terminal pyroglutamic acid (pE), asparagine
deamidation (deaN), methionine oxidation (oxiM) and aspartic
acid isomerization (isoD) on various positions, depending on
the mAb. The CESI-MS/MS data was also used to characterizethese PTM hot spots on the different mAbs. Table 2 highlightsthat hot spots could be monitored successfully in the cetuximab/
cetuximab-B comparison.

Modification

Figure 3. MS/MS spectra for trastuzumab (1) and trastuzumab-B (2),showing the absence of the fragmentation ions m/z 517.3095 (1+) fortrastuzumab-B and the presence of m/z 314.6937 (2+) characterizing theamino acid substitution of lysine to arginine.

Q1 / pQ1
N161 /
deaN161
D282 / isoD282
N386 /
deaN386
D403 / isoD403
N41 / deaN41
N158 /
deaN158

Cetuximab distribution

Biosimilar distribution% mod.

% unmod.

% mod.

% unmod.

1000100

0

25.6

74.4

59.5

40.5

95.2

4.8

96.3

3.745.654.4

95.7

4.3

100

0100087.8

12.2

94.7

5.3

96.6

3.4

100

0Table 2. Summary of the CESI-MS/MS study of PTM hot spots occurrence
levels for cetuximab/cetuximab-B. Percent distribution and modifications
were determined through signal intensity for each targeted PTM.

Figure 4. Glycoform profiling results obtained from the CESI-MS/MS
data for the Fc peptide of trastuzumab/trastuzumab-B and cetuximab/cetuximab-B.

Figure 5. Extracted ion electropherogram (EIE) corresponding tom/z (839.408, 2+) for digested peptide HT22 (position 278-291), which
experiences aspartic acid isomerization.

p3

In CQA analysis, the change in structure by a PTM often
significantly changes the electrophoretic mobility of the peptide(e.g., from 0.5 min in the case of deaN to several minutes for pE),
aiding separation and identification by CESI-MS/MS. The useof CE as a separating technique is clearly of great interest. Thisis emphasized in Figure 5, where it is possible to separate thesame peptide differing solely by the conformation of its asparticacid, which is not possible by MS alone, as they exhibit the same
mass. Again, all identifications were aided by the high-quality
MS/MS spectra obtained in this study.
Throughout this study, it was important that sequence coverage,
PTM hot spot detection and glycosylation site mapping
information was obtained by just a single injection of eachdigested mAb.

Conclusions
A CESI-MS/MS protocol for biosimilarity studies of mAbs has
been developed. Using this protocol, and with a single injectionof a tryptic digest of an mAb, several important structuralproperties could be obtained, including:• 100% amino acid sequence coverage.

References
1. Udpa, N., Million, R. Monoclonal antibody biosimilars. Nat Rev
Drug Discov (2016), 15: 13–14. doi:10.1038/nrd.2015.12.
2. Epstein, M.S., Ehrenpreis, E.D., Kulkarni, P. M. Biosimilars:
the need, the challenge, the future: the FDA perspective.
Am J Gastroenterol. (2014, Dec), 109(12): 1856–9.
3. Busnel, J-M., Schoenmaker, B., Ramautar, R., CarrascoPancorbo, A., Ratnayake, C., Feitelson, J.S., Chapman, J.D.,
Deelder A.M., and Mayboroda, O.A. High Capacity Capillary
Electrophoresis-Electrospray Ionization Mass Spectrometry:
Coupling a Porous Sheath-less Interface with TransientIsotachophoresis. Anal. Chem. (2010), 82: 9476–9483.
4. Gahoual, R., Busnel, J.M., Beck, A., Francois, Y.-N.,
Leize-Wagner, E. Full antibody primary structure and microvariant characterization in a single injection using transientisotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry. Anal. Chem. (2014), 86: 9074–9081.
1. Gahoual, R., Biacchi, M., Chicher, J., Kuhn, L., Hammann, P.,
Beck, A., Francois, Y.-N., Leize-Wagner, E. Monoclonal
antibodies biosimilarity assessment using transientisotachophoresis capillary zone electrophoresis-tandem
mass spectrometry. MAbs (2014), 6(6):1464–1473.
doi:10.4161/mabs.36305.

• Glycosylation site mapping and identifications.
• PTM hot spot mapping and identification.
For further information on this topic, we would like to referreaders to the full scientific publication on which this applicationnote is based.5

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 registered trademarks
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. RUO-MKT-02-4401-B
AB SCIEX™ is being used under license. Beckman Coulter® is being used under license.

Headquarters
500 Old Connecticut Path | Framingham, MA 01701, USA
Phone 508-383-7700
sciex.com

International Sales
For our office locations please call the division
headquarters or refer to our website atsciex.com/offices