Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility – Mass Spectrometry

16 October 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Antibody-based therapeutics continue to expand both in the number of products and their use in patients. These heterogeneous proteins challenge traditional drug characterization strategies, but ion mobility (IM) – mass spectrometry (MS) approaches have eased the challenge of higher-order structural characterization. Energy-dependent IM-MS, e.g., collision-induced unfolding (CIU), has been demonstrated to be sensitive to subtle differences in structure. In the present study, we combine a charge-reduction method, cation-to-anion proton-transfer reactions (CAPTR), with energy-dependent IM-MS and varied solution conditions to probe their combined effects on the gas-phase structures of IgG1κ and IgG4κ from human myeloma. CAPTR paired with MS-only analysis improves the confidence of charge-state assignments and the resolution of interfering protein species. Collision cross-section distributions were determined for each of the charge-reduced products. Similarity scoring was used to quantitively compare distributions determined from matched experiments analyzing samples of the two antibodies. Relative to workflows using energy-dependent IM-MS without charge-state manipulation, combining CAPTR and energy-dependent IM-MS enhanced the differentiation of these antibodies. Combined, these results indicate that CAPTR can benefit many aspects of antibody characterization and differentiation.

Keywords

Mass Spectrometry
Ion Mobility
Ion Chemistry
Antibody

Supplementary materials

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Supporting Information
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Additional description of data analysis procedures and similarity score calculations, as well as figures of additional experimental results.
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