학술논문
Enhancing affinity purification of monoclonal antibodies from human serum for subsequent CZE-MS analysis.
Document Type
Academic Journal
Author
Reinert T; Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 (Unistra-CNRS), Université de Strasbourg, France; Université Paris Cité, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), CNRS, Inserm, Faculté de sciences pharmaceutiques et biologiques, Paris, France. Electronic address: tessa.reinert@etu.unistra.fr.; Houzé P; Université Paris Cité, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), CNRS, Inserm, Faculté de sciences pharmaceutiques et biologiques, Paris, France; Laboratoire de Toxicologie Biologique, Hôpital Lariboisière, Assistance Publique - Hôpitaux de Paris (AP-HP), Paris, France.; Francois YN; Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 (Unistra-CNRS), Université de Strasbourg, France.; Gahoual R; Université Paris Cité, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), CNRS, Inserm, Faculté de sciences pharmaceutiques et biologiques, Paris, France.
Source
Publisher: Elsevier Country of Publication: Netherlands NLM ID: 101139554 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-376X (Electronic) Linking ISSN: 15700232 NLM ISO Abbreviation: J Chromatogr B Analyt Technol Biomed Life Sci Subsets: MEDLINE
Subject
Language
English
Abstract
Due to the separation technique employed, capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis performances are significantly influenced by the chemical composition and the complexity of the sample. In various applications, that impact has prevented the use of CE-MS for the characterization and quantification of proteins in biological samples. Here we present the development and evaluation and a sample preparation procedure, based on affinity purification, for the specific extraction of the monoclonal antibody (mAbs) infliximab from human serum in order to perform subsequent proteolytic digestion and CE-MS/MS analysis. Three distinctive sample preparation strategies were envisaged. In each case, the different steps composing the protocol were thoroughly optimized and evaluated in order to provide a sample preparation addressing the important complexity of serums samples while providing an optimal compatibility with CE-MS/MS analysis. The different sample preparation strategies were assessed concerning the possibility to achieve an appropriate absolute quantification of the mAbs using CE-MS/MS for samples mimicking patient serum samples. Also, the possibility to perform the characterization of several types of post-translational modifications (PTMs) was evaluated. The sample preparation protocols allowed the quantification of the mAbs in serums samples for concentration as low as 0.2 µg·mL -1 (2.03 nM) using CE-MS/MS analysis, also the possibility to characterize and estimate the modification level of PTMs hotspots in a consistent manner. Results allowed to attribute the effect on the electrophoretic separation of the different steps composing sample preparation. Finally, they demonstrated that sample preparation for CE-MS/MS analysis could benefit greatly for the extended applicability of this type of analysis for complex biological matrices.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)