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
Autor: | Alain Beck, Rabah Gahoual, Yannis-Nicolas François, Nassur Said, Lauriane Kuhn, Emmanuelle Leize-Wagner |
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Přispěvatelé: | Université de Strasbourg (UNISTRA), Tectonique Moléculaire du Solide (TMS), Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie de Coordination Organique, Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Strasbourg, Centre National de la Recherche Scientifique (CNRS)-Université Louis Pasteur - Strasbourg I-Institut de Chimie du CNRS (INC), Laboratoire de Chimie Quantique, Centre National de la Recherche Scientifique (CNRS), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre |
Jazyk: | angličtina |
Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
Spectrometry Mass Electrospray Ionization Antibody-drug conjugate Immunoconjugates Mass spectrometry Tandem mass spectrometry 01 natural sciences Biochemistry Analytical Chemistry 03 medical and health sciences Capillary electrophoresis Tandem Mass Spectrometry [CHIM.ANAL]Chemical Sciences/Analytical chemistry Environmental Chemistry Spectroscopy Brentuximab Vedotin chemistry.chemical_classification Chromatography Biomolecule 010401 analytical chemistry Electrophoresis Capillary 0104 chemical sciences Characterization (materials science) 030104 developmental biology chemistry Mass spectrum Conjugate |
Zdroj: | Analytica Chimica Acta Analytica Chimica Acta, Elsevier Masson, 2016, 918, pp.50-59. ⟨10.1016/j.aca.2016.03.006⟩ |
ISSN: | 0003-2670 |
Popis: | 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 com-plexity 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 tech-niques for complete structure assessment. We report the development of complementary approaches imple-menting sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the differ-ent 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 anti-body 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 pay-loads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hy-drophobic 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 multi-level characterization of these complex biomolecules |
Databáze: | OpenAIRE |
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