Comprehensive Characterization of IgG2 Disulfide Isoforms Using Native Cation Exchange Chromatography-Mass Spectrometry and Peptide Mapping

The IgG2 antibody subclass is unique in displaying complex disulfide bond connectivity in its hinge region, resulting in three distinct isoforms, namely, A, A/B, and B. Structural elucidation of these isoforms by liquid chromatography-mass spectrometry (LC-MS) is challenging and usually involves denaturing methodologies, such as reversed-phase chromatography or capillary electrophoresis. In this study, we developed a native cation exchange chromatography-mass spectrometry (CEX-MS) method using volatile salts to separate IgG2 disulfide isoforms. This approach was combined with a middle-up strategy focusing on F(ab')₂ fragments of the antibody, where IgG2 disulfide bond rearrangements are localized. By targeting F(ab')₂, this methodology achieves optimal chromatographic separation and high-quality MS spectra while excluding Fc-related heterogeneity. The elution order of IgG2 disulfide isoforms was determined by CEX-MS through redox treatment and site-directed mutagenesis. For the first time, native CEX-MS enabled the characterization of an IgG2 mutant designed to favor agonistic activity through a pseudoisoform B structure stabilized by noncovalent interactions between subunits. To further decipher hinge region disulfide linkage, we also developed an optimized nonreduced peptide mapping that limits missed cleavages. Peptide identification was enhanced by an isotope envelope confidence score, which compares theoretical and experimental isotope distributions. This scoring allowed for confident identification of complex hinge-related peptides, overcoming limitations of conventional peptide mapping methods for the elucidation of IgG2 interchain patterns.