title: 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.
abstract: 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.
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg, France.
authors.forename: Nassur
authors.initials: N
authors.lastname: Said
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg, France; Division of BioAnalytical Chemistry, AIMMS Research Group BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Rabah
authors.initials: R
authors.lastname: Gahoual
authors.affiliation: Plateforme Protéomique Strasbourg-Esplanade, Institut de Biologie Moléculaire et Cellulaire, FRC 1589, CNRS, Université de Strasbourg, Strasbourg, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Kuhn
authors.affiliation: Centre d'immunologie Pierre Fabre, Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg, France. Electronic address: yfrancois@unistra.fr.
authors.forename: Yannis-Nicolas
authors.initials: YN
authors.lastname: François
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS), UDS-CNRS UMR 7140, Université de Strasbourg, Strasbourg, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Leize-Wagner
mesh_terms: D000079963:Brentuximab Vedotin; D019075:Electrophoresis, Capillary / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018796:Immunoconjugates; D000079963:Brentuximab Vedotin
keywords: Antibody-drug conjugate; Drug to antibody ratio; Drug-loaded peptides; Sheathless capillary electrophoresis-mass spectrometry; vcMMAE-ADC