Rapid structural discrimination of IgG antibodies by multicharge-state collision-induced unfolding

Abstract

A simplified multicharge-state collision-induced unfolding (CIU) method was proposed for rapid differentiation of IgG isotypes that differ in terms of the numbers and patterns of disulfide bonds.

Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies

“…As IM-based approaches are particularly well adapted for the characterization of proteins containing disulfide bonds, we took advantage of the diversity of mAb IgG-based formats (Figure A) as a fit for purpose application to illustrate the interest of IM-MS/CIU for this particular family of disulfide-rich proteins. ,, …”

“…CIU patterns of the most native charge states (i.e., lower charge states) are generally preferred for CIU analysis, , as Coulomb repulsions are minimized, and so we mainly focused on the 27+ charge state of intact mAbs, which offers the best compromise between “near-native” conformation, number of unfolding states, and signal intensity. For intact elotuzumab (IgG1), six features are detected with CIU-cIM (Figure A).…”

“…As IM-based approaches are particularly well adapted for the characterization of proteins containing disulfide bonds, we took advantage of the diversity of mAb IgG-based formats (Figure 1A) as a fit for purpose application to illustrate the interest of IM-MS/CIU for this particular family of disulfide-rich proteins. 20,31,37 Gaussian Fittings as a Way of Deciphering cIM-MS Profiles. In order to provide a semi-quantitative interpretation of our HR-IM-MS data, Gaussian fits were used as a means to visualize the highly populated conformational landscapes of mAbs, aiming to (i) evaluate if HR-IM-MS can enhance the separation of multiple conformers and (ii) provide a relative comparison between our samples.…”

“…We focused here on two aspects: (i) the potential gain in resolution afforded by cIM-MS to enhance the separation of mAbs’ conformers and (ii) the impact of higher IM resolving powers for CIU experiments. As disulfide-bonded proteins are known to be well adapted for IM-MS and CIU-based studies, ,,− we then used IgG subclassification as a fit for purpose application to benchmark cyclic versus linear TWIMS instruments. We show that increasing the resolution with multiple passes in the cIM cell affords only limited improvements for the direct measurement of ATDs of native mAbs because an intrinsic peak broadening occurs, presumably related to the coexistence of several protein conformations that lead to a nearly continuous range of CCSs.…”