Monoclonal antibodies primary structure and biosimilarity assessment in a single analysis usingtransient isotachophoresis sheathless capillary electrophoresis-tandem mass spectrometry
Rabah Gahoual1, Jean-Marc Busnel2, Johana Chicher3, Lauriane Kuhn3, Phillipe Hammann3, Alain Beck4, Yannis Nicolas François1, Emmanuelle Leize-Wagner1
1Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), CNRS – UMR7140, University of Strasbourg, Strasbourg, France; 2Beckman Coulter Inc., Marseille, France; 3Institut deBiologie Moléculaire et Cellulaire (IBMC), University of Strasbourg; 4Centre d’immunologie Pierre Fabre, Saint-Julien-en-Genevois, FranceOverview
Monoclonal antibodies (mAbs) represent currently the therapeutic agent category experiencing the most important
progression due to their therapeutic potency and specificity. Some patents regarding mAbs are going to end in thenear future, giving the opportunity to alternative actors to produce and market the same protein which is defined inResults
Amino acids sequence characterization / comparison

Cetuximab glycoprofiling / profiles comparison
Glycosylation site
dependant characterization

biopharmaceuticals as biosimilars. Here, capillary zone electrophoresis coupled to tandem mass spectrometry by
the intermediate of an ultra-low flow interface (CESI-MS/MS) was used to characterize marketed mAbs and theirrespective candidate biosimilar simultaneously over different facets of their primary structure using a sole sample
injection of 200 fmol of digested peptides. CESI-MS/MS data enabled to obtain simultaneously 100% sequence
coverage, structures of 15 glycoforms and the characterization of all PTMs hot-spots present on the studied mAbs
samples. CESI-MS/MS allowed to conclude regarding the biosimilarity study between approved mAbs andbiosimilar candidate. Characterization results allowed to specifically point out the facets of the candidate which were
not complying to be considered as a biosimilar.

Minor differences exposed
between original mAb andbiosimilar

Introduction
Monoclonal antibodies (mAbs) have taken a major market share in the pharmaceutical industry and theirdevelopment is constantly increasing. Several patents pending upon early approved mAbs are going to end in thenext few months, giving the opportunity to different companies to produce “copies” of the concerned antibody, thosecopies are referred as biosimilars or follow-on biologics. Since a few years, regulatory agencies have beenworking toward establishing guidelines designed to determine critical criteria that must be common between aninnovator mAb and its biosimilar. mAbs are highly complex proteins that display a wide range of micro
heterogeneities that requires multiple analytical methods for full structure assessment and quality control [1]. As aconsequence, the characterization of mAbs on different level is particularly product and time-consuming.
In this work, we have developed a transientisotachophoresis CE-ESI-MS/MS methodology, bymean of the CESI-MS system, in order to obtain in
the first place the characterization of severalmAbs. This characterization, over different level ofthe protein, was performed in a single analysis ofeach sample. In a second time, each mAb wascompared to a candidate biosimilar in order toestablish if the methodology developed could beused to assess the biosimilarity between twosamples.

100 % sequence coverage(no miscleavages / PTMs)

1 AA difference characterized
for trastuzumab biosimilar

Trastuzumab glycoprofiling / profiles comparison
Different glycoforms characterized between cetuximab andbiosimilar (glycans containing murine sialic acids)
Conclusions

Methods
Studied samples. Trastuzumab, cetuximab in their final formulation and respective candidate biosimilar
trastuzumab-B and cetuximab-B were characterized by CESI-MS/MS and the obtained data confronted forbiosimilarity assessment
Samples treatment. Tryptic digestion in-solution of mAbs. Digestion buffer: 50mM bicarbonate ammonium,reducing reagent: dithiothreitol 100mM, alkylation reagent: iodoacetamide 100mM. Sample peptide final
concentration : 0.3 µg/µL

Structures deduced from MS/MS spectra

15 glycosylations
structures characterized

Glycoforms relative abundances established

Glycoforms distribution
comparison for biosimilarity

PTMs hot-spots characterization / abundance comparison
CE conditions. Instrument: Sciex separation CESI-MS prototype (Brea, CA, US) equipped with a bare-fused silica
capillary cartridge (90 cm, 30 µm i.d.), injection volume: 100 nL (220 fmol of digest), BGE: 10% acetic acid,separation voltage: +20 kV, duration: 50 min. CESI-MS interface for MS coupling

CESI allowed separation ofpeptides with PTMs

Mass spectrometry. MS/MS was performed using an ESI-Qq-TOF 5600 tripleTOF ® (AB Sciex, San Francisco, CA)
MS/MS method: IDA (top15 precursor ions), dynamic exclusion (0.15 min after 2 spectra), mass range 100-2000
m/z, duty cycle 1.75 sec, capillary voltage -1.75 kV, curtain gain 5, Source temperature 160°C.

Trastuzumab

Trastuzumab-B

Cetuximab

Cetuximab-B

sequence coverage

100%

100%

100%

100%identified glycosylations

15101016

glutamic acid cyclization

1/1

1/1

1/1

1/1

methionine oxidation

2/2

2/2

0/0

0/0

asparagine deamidation

4/4

2/2

4/4

4/4

aspartic acid isomerization

6/6

3/3

2/2

2/2

other PTMs hotspots

Data obtained from solely one injection of each sample tryptic digest allowed to characterize each mAbs
simultaneously over several levels defining their primary structure. Biosimilarity study results indicated that inboth cases, candidate biosimilars exhibited minor differences compared to the originator mAb. In the caseof trastuzumab-B, one AA difference compared to the original mAb leaded to this conclusion while in the case ofcetuximab-B uncontrolled expression of murine glycoforms toxic to human metabolism ended up in the rejection
candidate biosimilar.

Acknowledgements
Authors would like to thank AB Sciex for the loan of a prototype CESI system and a 5600 TripleTOF ® mass spec. The[1] Beck A, Sanglier-Cianférani S, Van Dorsselaer A. , " Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry.” AnalChem 2012, 84,4637-4646[2] Gahoual R., Biacchi M., Chicher J., Kuhn L., Hammann P., Beck A., François Y-N., Leize-Wagner E., " Monoclonal antibodies biosimilarity
assessment using transient isotachophoresis preconcentration-capillary zone electrophoresis-tandem mass spectrometry “ 2014, submitted.

Relative quantitation of each PTMs
hot-spots obtained

authors would like to express their gratitude to Dr. M. Anselme (Sciex, Les Ullis, France) for his support and Dr.
Joseph Vlasak (Merck, West Point, PA) for critical discussions around antibody structural characterization.