Kinetic and structural characterization of therapeutic albumin chemical functionalization using complementary mass spectrometry techniques

Autor: Daniel Scherman, Yannis-Nicolas François, Gilles Clodic, Pascal Houzé, Rabah Gahoual, Nathalie Mignet, Gérard Bolbach, Ikram Ould-Melha
Přispěvatelé: Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS - UM 4 (UMR 8258 / U1022)), Institut de Chimie du CNRS (INC)-Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: Journal of Pharmaceutical and Biomedical Analysis
Journal of Pharmaceutical and Biomedical Analysis, Elsevier, 2020, 185, pp.113242. ⟨10.1016/j.jpba.2020.113242⟩
ISSN: 0731-7085
DOI: 10.1016/j.jpba.2020.113242⟩
Popis: International audience; Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reac-tion implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conju-gates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionaliza-tion kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystal-lography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.
Databáze: OpenAIRE
Externí odkaz: