Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis – Tandem mass spectrometry as nanoESI infusion platform and separation method

Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs re...

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Bibliographic Details
Published in:	Analytica chimica acta 2016-04, Vol.918, p.50-59
Main Authors:	Said, Nassur, Gahoual, Rabah, Kuhn, Lauriane, Beck, Alain, François, Yannis-Nicolas, Leize-Wagner, Emmanuelle
Format:	Article
Language:	eng
Subjects:	Analytical chemistry Antibodies Antibody-drug conjugate Capillarity Chemical Sciences Conjugates Drug to antibody ratio Drug-loaded peptides Drugs Electrophoresis, Capillary - methods Immunoconjugates - chemistry Infusion Methodology Nanostructure Platforms Sheathless capillary electrophoresis-mass spectrometry Spectrometry, Mass, Electrospray Ionization - methods Tandem Mass Spectrometry - methods vcMMAE-ADC
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Summary:	Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical techniques for complete structure assessment. We report the development of complementary approaches implementing sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the different aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to antibody ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab')2 subunits incorporating 1, 0 to 4 and 0 to 8 payloads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hydrophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multilevel characterization of these complex biomolecules. [Display omitted] •We confirm the power of sheathless CE/MS coupling for the characterization of ADC.•We demonstrate structural information of Lc, Fab and F(ab')2 subunits incorporating various number of drugs.•We determine the average Drug to antibody ratio (DAR).•We characterize unambiguously the drug-loaded-peptides with seven new diagnostic ions.
ISSN:	0003-2670 1873-4324