biostudies-literatureLin YNational Institute of Allergy and Infectious DiseasesNIAID NIH HHSPfizerNational Institute of General Medical SciencesNIGMS NIH HHS16840-16849https://www.ebi.ac.uk/biostudies/studies/S-EPMC10909587biostudies-literatureUnknown95(46)Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.Analytical chemistryCharacterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.PMC1090958775N93019C00062R24GM136766R24 GM136766P41 GM103422Liu HRouse JCLiu LLZhang YGross MLBeaumont VAKim HYMoyle ABLin YPolleck SShi HfalseCharacterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.2023-01-01T00:00:00Z2023 Nov2025-04-18T17:08:59.772Z2025-04-07T04:45:59.215ZS-EPMC109095873793395410.1021/acs.analchem.3c02422