Collision-Induced Unfolding Reveals Stability Differences in Infliximab Therapeutics under Native and Heat Stress Conditions

Abstract

Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis. We subject each infliximab sample to forced degradation through heat stress and observe broadly similar yet subtly different stability patterns for these three biotherapeutics. We find that CIU is capable of tracking differences in mAb higher-order structure (HOS) imparted during forced heat stress degradation and that DSC is less sensitive to these alterations in comparison. Furthermore, we collected our comprehensive IM-MS and CIU data across two instrument platforms (Waters G2 and Agilent 6560), with both producing similar abilities to differentiate mAbs while also revealing minor differences between the results obtained on the two instruments. Finally, we demonstrate that CIU-based heatmaps and classification allow for rapid assessment of the most differentiating charge states for the analysis of infliximab, and using multiplexed classification, we conservatively estimate a 30-fold improvement in the time required to perform mAb stability and HOS measurements over standard DSC tools.

Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer

“…CIU has long been proposed as an ideal approach for inclusion in biotherapeutic pipelines, with a substantial body of work supporting its ability to characterize relevant mAb structures. 12,14,15,46,52,53 A high level of technical reproducibility is required to conduct comparative analyses across mAb subtypes. Although the BSA results discussed above can be used to positively project the reproducibility of CIU experiments for protein-based pharmaceuticals, mAbs are over two times larger (∼150 kDa), and thus pose unique challenges for high-precision IM-MS and CIU.…”

“…To date, our work has demonstrated the ability of this IM-MS platform to produce CIU data similar to those reported previously. 9,15,45,46 In this study, we evaluated the reproducibility of CIU fingerprints on three geographically distinct DTIM-MS instruments equipped with prototype high-energy source hardware of the same design. Furthermore, we improved upon previously described methods, achieving higher-energy CIU of large proteins and protein complexes using a standard ESI source without the requisite addition of heavier dopant gases (i.e.…”

Ion Mobility Mass Spectrometry for the Separation and Characterization of Small Molecules

“…Regarding conformational isomers, IM-MS and collision-induced unfolding (CIU) analyses have been shown to be sensitive to disulfide bond structure, glycosylation patterns, and small-molecule binding levels. , Wei et al can quickly determine the type of isomer by electronic excitation dissociation and TIMS to avoid mistaking conformers for isomers. Recently, Panczyk et al analyzed the structure of protein complexes by TIMS and SID.…”

Performance Evaluation of In-source Ion Activation Hardware for Collision-Induced Unfolding of Proteins and Protein Complexes on a Drift Tube Ion Mobility-Mass Spectrometer

“…CIU has long been proposed as an ideal approach for inclusion in biotherapeutic pipelines, with a substantial body of work supporting its ability to characterize relevant mAb structures. 12,14,15,46,52,53 A high level of technical reproducibility is required to conduct comparative analyses across mAb subtypes. Although the BSA results discussed above can be used to positively project the reproducibility of such CIU experiments for protein-based pharmaceuticals, mAbs are over two times larger (~150 kDa), and thus pose unique challenges for high-precision IM-MS and CIU.…”

“…To date, our work has demonstrated the ability of this IM-MS platform to produce CIU data similar to those reported previously. 9,15,45,46 In this study, we present the reproducibility of CIU fingerprints on three geographically distinct DTIM-MS instruments each equipped with prototype highenergy source hardware of the same design. Furthermore, we improved upon previously described methods, achieving higher-energy CIU of large proteins and protein complexes using a standard ESI source without the requisite addition of heavier dopant gases (i.e.…”