Fiche publication
Date publication
septembre 2008
Auteurs
Membres identifiés du Cancéropôle Est :
Mme SCHAEFFER-REISS Christine
,
Dr VAN DORSSELAER Alain
Tous les auteurs :
Wagner-Rousset E, Bednarczyk A, Bussat MC, Colas O, Corvaia N, Schaeffer C, Van Dorsselaer A, Beck A
Lien Pubmed
Résumé
Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC-ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip-MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.
Référence
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Sep 1;872(1-2):23-37.
