Native Mass Spectrometry, Ion Mobility and Collision Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies.
Fiche publication
Date publication
juin 2018
Journal
Analytical chemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CIANFERANI Sarah
Tous les auteurs :
Hernandez-Alba O, Wagner-Rousset E, Beck A, Cianférani S
Lien Pubmed
Résumé
Although the majority of FDA- and EMA-approved therapeutic monoclonal antibodies (mAbs) are IgG1, the number of IgG4-based formats reaching the market is increasing. IgG4 differs from other mAb isotypes due to its specificity to form half mAbs, which recombine into bispecific (bsAbs) molecules, through a process termed fab-arm exchange (FAE). Here, we report on the complementarity of native mass spectrometry (MS), ion mobility (IM-MS) and collision-induced unfolding (CIU) experiments for the structural characterization of members of the IgG4 subfamily (wild-type (wt), hinge-stabilized (hs, S228P mutation) and the resulting bsAb IgG4s). Native MS allows for confirming/invalidating the occurrence of FAE as a function of IgG4 categories. While IM-MS was unable to distinguish iso-cross-section IgG4 species, CIU experiments provide unique specific structural signatures of each individual IgG4, based on their specific unfolding pathways. Common CIU features of IgG4 formats include the observation of three conformational states and two transitions. In addition, CIU experiments demonstrate stabilized gas-phase conformations of hs-IgG4, in agreement with increased stability related to more rigid hinge regions. CIU patterns also appear to be more informative than IM-MS for bsAB structural characterization, while the unfolding signature of the bsAb is intermediate to the ones of the former parent wt-IgG4s, highlighting that bsAb CIU profiles keep the memory of their origins. Altogether, our results demonstrate that CIU patterns can serve as mAb-specific structural signatures and are mature enough to be included in MS-based analytical workflows for conformational/structural characterization of mAb formats in early development phases and for multiple attribute monitoring.
Mots clés
Antibodies, Monoclonal, Humanized, chemistry, Humans, Immunoglobulin G, chemistry, Ion Mobility Spectrometry, methods, Mass Spectrometry, Models, Molecular, Natalizumab, chemistry, Nivolumab, chemistry, Point Mutation, Protein Conformation, Protein Stability, Protein Unfolding
Référence
Anal. Chem.. 2018 Jun 29;: