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

• Published in: Analytical chemistry (Washington) Vol. 93; no. 48; pp. 16166 - 16174
• Main Authors: Vallejo, Daniel D, Kang, Jukyung, Coghlan, Jill, Ramírez, Carolina Rojas, Polasky, Daniel A, Kurulugama, Ruwan T, Fjeldsted, John C, Schwendeman, Anna A, Ruotolo, Brandon T
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.12.2021
• Subjects: Analytical chemistry; Assessments; Biosimilar Pharmaceuticals; Bridges; Calorimetry; Chemistry; Classification; Conjugation; data collection; Datasets; Degradation; Differential scanning calorimetry; Disulfide bonds; Glycosylation; Heat stress; Heat tolerance; Heat-Shock Response; Infliximab; Ionic mobility; Mass Spectrometry; Mass spectroscopy; Monoclonal antibodies; Protein Unfolding; rapid methods; Reproducibility of Results; spectroscopy; Stability; therapeutics; TNF inhibitors; Tumor necrosis factor-α
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.1c03946

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.