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Fast in-line bottom-up analysis of monoclonal antibodies: Toward an electrophoretic fingerprinting approach
Authors:Meriem Dadouch  Yoann Ladner  Claudia Bich  Jérôme Montels  Jacques Morel  Catherine Perrin
Institution:1. UMR 5247-CNRS-UM-ENSCM, Institut des Biomolécules Max Mousseron (IBMM), Université de Montpellier, Montpellier, France;2. Département de Rhumatologie, Université de Montpellier, Hôpital Lapeyronie, Montpellier Cedex 5, 34295 France
Abstract:For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom-up level to generate specific fingerprints that can be used to tackle post-translational modifications or ensure production consistency between lots. To circumvent time-consuming and labor-intensive off-line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in-line methodology for the routine quality control of mAbs at the bottom-up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in-line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In-line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS-compatible BGE was used to obtain high resolution separation of released peptides and in-line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE-fingerprints were successfully established for different mAbs and an antibody-drug conjugate.
Keywords:Bottom-up analysis  Capillary electrophoresis  Fingerprints  Monoclonal antibodies  Transverse diffusion of laminar flow profile
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