Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility–Mass Spectrometry

[Image: see text] Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins but can also alter the conformation or even biological functions. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. Innovative analytical strategies should be developed to map these PTMs as well as explore possible induced conformational changes. Cation-exchange chromatography (CEX) coupled with native mass spectrometry has already emerged as a valuable asset for the characterization of mAb charge variants. Nevertheless, questions regarding protein conformation cannot be explored using this approach. Thus, we have combined CEX separation with collision-induced unfolding (CIU) experiments to monitor the unfolding pattern of separated mAbs and thereby pick up subtle conformational differences without impairing the CEX resolution. Using this novel strategy, only four CEX–CIU runs had to be recorded for a complete CIU fingerprint either at the intact mAb level or after enzymatic digestion at the mAb subunit level. As a proof of concept, CEX–CIU was first used for an isobaric mAb mixture to highlight the possibility to acquire individual CIU fingerprints of CEX-separated species without compromising CEX separation performances. CEX–CIU was next successfully applied to conformational characterization of mAb glyco-variants, in order to derive glycoform-specific information on the gas-phase unfolding, and CIU patterns of Fc fragments, revealing increased resistance of sialylated glycoforms against gas-phase unfolding. Altogether, we demonstrated the possibilities and benefits of combining CEX with CIU for in-depth characterization of mAb glycoforms, paving the way for linking conformational changes and resistance to gas-phase unfolding charge variants.