Structural and Functional Characterization of Disulfide Bond Isomers of Therapeutic Immunoglobulin G2 Antibody

Human immunoglobulin G2 (IgG2) is a crucial therapeutic monoclonal antibody (mAb). IgG2 possesses a unique short hinge region characterized by four pairs of interheavy chain disulfide bonds, generating multiple disulfide-bonded isomers, including IgG2-A, IgG2-B, and the intermediate IgG2-A/B. Despite their biological relevance, the characterization of isomers has primarily focused on IgG2-A and IgG2-B, with IgG2-A/B overlooked. In this work, using ion-exchange chromatography and mass spectrometry, we purified isomers of recombinant IgG2κ mAb, identified the conversion mechanism of disulfide bonds, and assessed their biological potencies. The potencies of the isomers were found to be IgG2-A > IgG2-A/B > IgG2-B. These differences may correlate with the increased hydrodynamic radius of IgG2-A and IgG2-A/B compared with IgG2-B. Disulfide bond isomers should be categorized as a critical quality attribute for IgG2 mAb due to the significant potency differences. Our work provides a strategy for purifying a particular disulfide isomer of therapeutic IgG2.