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

Abstract

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 interchains 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 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.

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

“…Altogether, these results suggest that the chemical conjugation process does not drastically affect the overall global conformation of the mAb. However, drawing clear-cut conclusions solely from nIM-MS measurement for mAbs with very close conformations remains challenging at both intact and middle-up levels due to the low resolution of linear TWIMS [ 50 ]. The preliminary CCS measurements are the rationale for performing further CIU experiments, as an alternative to tackle small conformational variations that would result in differences in CIU patterns.…”

“…These results demonstrate that CIU approaches offer clear benefits over standard nIM-MS experiments to detect subtle conformational differences that translate into different CIU patterns. In addition, CIU data have been reported to correlate with unfolding patterns observed using differential scanning calorimetry (DSC), suggesting a solution-phase memory effect of mAbs products in the gas phase [ 33 , 50 ]. CIU offers significant benefits over DSC, with improved sensitivity and selectivity, and thus, appears as an appealing approach to acquire conjugation-dependent gas-phase stability shift information for biotherapeutics.…”

Toward Automation of Collision-Induced Unfolding Experiments through Online Size Exclusion Chromatography Coupled to Native Mass Spectrometry

“…Altogether, SEC-CIU fingerprints provide the same level of information as nanoESI-or ESI-CIU. Previous works have demonstrated that mAbs subclasses, which namely differ in their number and bridging of disulfide bonds, could be classified based on their unfolding patterns 7,28 . To benchmark our SEC-CIU strategy, we further applied it to the analysis of three mAbs belonging to different subclasses, adalimumab (IgG1), panitumumab (IgG2) and reslizumab (IgG4).…”

“…In particular, Tian et al 7 demonstrated that subtle differences between intact mAbs subclasses, with different numbers and patterns of disulfide bridges, could be distinguished based on their gas-phase unfolding. Recently, Botzanowski et al expanded this strategy to the middle-level classification of mAbs subclasses, which provides a more clear-cut categorization than the intact level 28 .…”

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb

“…This technique appeared to be highly valuable for therapeutic mAb products. 18 , 19 , 27 − 30 In an attempt to automate CIU workflows, Deslignière et al have proposed a SEC–CIU setup that allows recording CIU fingerprints in a rapid manner. 31 …”