Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab')₂ and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab')₂ domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab')₂ regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab')₂ and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab')₂ domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.