Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies

Oscar Hernandez-Alba (1) , Elsa Wagner-Rousset (2) , Alain Beck (2) , Sarah Cianférani (1)

1 DSA-IPHC - Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires
2 CIPF - Centre d'Immunologie Pierre Fabre

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 by its specificity to form half mAbs that recombine into bispecific (bsAbs) molecules, through a process termed fab-arm exchange (FAE). We report here the complementarity of native mass spectrometry (MS), ion mobility (IM), 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 confirming/invalidating the occurrence of FAE as a function of these different types of IgG4. 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 demonstrated that S228P mutation stabilizes gas phase conformations of hsIgG4, 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, unfolding signature of the bsAb being 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 to be included in MS-based analytical workflows for conformational/structural characterization of mAb formats in early development phases and for multiple attribute monitoring.

Domaines: Chimie

Oscar Hernandez-Alba, Elsa Wagner-Rousset, Alain Beck, Sarah Cianférani. Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies. Analytical Chemistry, 2018, 90 (15), pp.8865-8872. ⟨10.1021/acs.analchem.8b00912⟩. ⟨hal-02327964⟩