Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies

Evolène Deslignière (1, 2) , Simon Ollivier (3) , Alain Beck (4) , David Ropartz (5) , Hélène Rogniaux (5) , Sarah Cianférani (6)

1 LSMBO - Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg]
2 IPHC - Institut Pluridisciplinaire Hubert Curien
3 BIBS - BioInformatique et BioStatistiques
4 CIPF - Centre d'Immunologie Pierre Fabre
5 BIA - Unité de recherche sur les Biopolymères, Interactions Assemblages
6 DSA-IPHC - Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires

Monoclonal antibodies (mAbs) currently represent the main class of therapeutic proteins. mAbs approved by regulatory agencies are selected from IgG1, IgG2, and IgG4 subclasses, which possess different interchain disulfide connectivities. Ion mobility coupled to native mass spectrometry (IM-MS) has emerged as a valuable approach to tackle the challenging characterization of mAbs’ higher order structures. However, due to the limited resolution of first-generation IM-MS instruments, subtle conformational differences on large proteins have long been hard to capture. Recent technological developments have aimed at increasing available IM resolving powers and acquisition mode capabilities, namely, through the release of high-resolution IM-MS (HR-IM-MS) instruments, like cyclic IM-MS (cIM-MS). Here, we outline the advantages and drawbacks of cIM-MS for better conformational characterization of intact mAbs (∼150 kDa) in native conditions compared to first-generation instruments. We first assessed the extent to which multipass cIM-MS experiments could improve the separation of mAbs’ conformers. These initial results evidenced some limitations of HR-IM-MS for large native biomolecules which possess rich conformational landscapes that remain challenging to decipher even with higher IM resolving powers. Conversely, for collision-induced unfolding (CIU) approaches, higher resolution proved to be particularly useful (i) to reveal new unfolding states and (ii) to enhance the separation of coexisting activated states, thus allowing one to apprehend gas-phase CIU behaviors of mAbs directly at the intact level. Altogether, this study offers a first panoramic overview of the capabilities of cIM-MS for therapeutic mAbs, paving the way for more widespread HR-IM-MS/CIU characterization of mAb-derived formats.

Mots clés: MOBILITY MASS-SPECTROMETRY; ION MOBILITY; HUMAN IGG; IMMUNOGLOBULIN; GLYCOSYLATION; DYNAMICS; INSIGHTS

Domaines: Chimie analytique; Sciences pharmaceutiques

Evolène Deslignière, Simon Ollivier, Alain Beck, David Ropartz, Hélène Rogniaux, et al.. Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies. Analytical Chemistry, 2023, 95 (8), pp.4162-4171. ⟨10.1021/acs.analchem.2c05265⟩. ⟨hal-04236674⟩