Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry |
| |
| Authors: | Rabah Gahoual Alicia Burr Jean-Marc Busnel Lauriane Kuhn Phillipe Hammann Alain Beck Yannis-Nicolas Fran?ois Emmanuelle Leize-Wagner |
| |
| Institution: | 1.Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS); UDS-CNRS UMR 7140; Université de Strasbourg; Strasbourg, France;2.Beckman Coulter Inc.; Brea, CA, USA;3.Plateforme Protéomique; Université de Strasbourg; Institut de Biologie Moléculaire et Cellulaire; Strasbourg, France;4.Centre d’immunologie Pierre Fabre; Saint-Julien-en-Genevois, France |
| |
| Abstract: | Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms. |
| |
| Keywords: | monoclonal antibody sheathless capillary electrophoresis mass spectrometry CESI-MS/MS peptide mapping N-linked glycosylation posttranslational modifications microvariant microheterogeneity |
|
