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

Cite this: Anal. Chem. 2023, 95, 8, 4162–4171
Publication Date (Web):February 13, 2023
https://doi.org/10.1021/acs.analchem.2c05265
Copyright © 2023 American Chemical Society

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    Abstract

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    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.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c05265.

    • Voltages used to set the direction of the ions in the multifunction array region of the cIM-MS spectrometer for each type of cIM event (Table S1); determination of correction factors to derive drift times from raw arrival times (Table S2); intensities obtained on the cIM-MS instrument for extracted ATDs (Table S3); reproducibility of Gaussian fittings (Table S4); %RSDs between technical replicates of mAbs at 1 and 5 passes on the cIM-MS instrument (Table S5); parameters used to evaluate the reproducibility of cIM profiles (Figure S1); intra- and interclass variations (Figure S2); principle of CIU experiments performed in the trap cell of the cIM-MS instrument (Figure S3); comparison of CIU plots obtained for intact deglycosylated nivolumab using a linear versus a cyclic TWIMS cell (Figure S4); cIM-MS experiments for charge state 28+ after 4 passes (Figure S5); analysis of a mixture containing two different mAbs on the cIM-MS instrument, including targeted scheduled CIU subclassification (Figure S6); CIU-cIM fingerprints and IgG classification scores for charge states 27 and 28+ (Figure S7) (PDF)

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    Cited By

    This article is cited by 2 publications.

    1. Theresa A. Gozzo, Matthew F. Bush. Quantitatively Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility-Mass Spectrometry. Analytical Chemistry 2024, 96 (1) , 505-513. https://doi.org/10.1021/acs.analchem.3c04638
    2. Lars Konermann, Zeyuan Liu, Yousef Haidar, Mathew J. Willans, Nicholas A. Bainbridge. On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry. Analytical Chemistry 2023, 95 (37) , 13957-13966. https://doi.org/10.1021/acs.analchem.3c02546