Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination.

Human antibodies of the IgG2 subclass exhibit complex inter-chain disulfide bonding patterns that result in three structures, namely A, A/B, and B. In therapeutic applications, the distribution of disulfide isoforms is a critical product quality attribute because each configuration affects higher order structure, stability, isoelectric point, and antigen binding. The current standard for quantification of IgG2 disulfide isoform distribution is based on chromatographic or electrophoretic techniques that require additional characterization using mass spectrometry (MS)-based methods to confirm disulfide linkages. Detailed characterization of the IgG2 disulfide linkages often involve MS/MS approaches that include electrospray ionization or electron-transfer dissociation, and method optimization is often cumbersome due to the large size and heterogeneity of the disulfide-bonded peptides. As reported here, we developed a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. We established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.