A multiparameter optimization in middle‐down analysis of monoclonal antibodies by LC–MS/MS

Jonathan Dhenin (1, 2, 3) , Mathieu Dupré (1) , Karen Druart (1) , Alain Krick (3) , Christine Mauriac (3) , Julia Chamot-Rooke (1)

1 UTechS MSBio - Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology
2 ED562 - BioSPC - École Doctorale Bio Sorbonne Paris Cité [Paris]
3 Sanofi Aventis R&D [Chilly-Mazarin]

In antibody-based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle-down and topdown approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.

Keywords: MS/MS; middle-down mass spectrometry; monoclonal antibodies; Orbitrap MS

Domains: Analytical chemistry

Jonathan Dhenin, Mathieu Dupré, Karen Druart, Alain Krick, Christine Mauriac, et al.. A multiparameter optimization in middle‐down analysis of monoclonal antibodies by LC–MS/MS. Journal of Mass Spectrometry, 2023, 58 (3), pp.e4909. ⟨10.1002/jms.4909⟩. ⟨pasteur-04102833⟩