Characterization of disulfide linkages at the hinge region of IgG antibodies by HPLC mass spectrometry.

There are two types of disulfide linkages in IgG antibodies at the hinge region: intra- and inter-disulfide linkages. Characterization of intra-disulfide linked isomer will provide important information on the stability of the antibodies and better understanding of the mechanism of Fab-arm exchange. In this report, HPLC coupled with high-resolution mass spectrometry was applied for characterization of disulfide linkages in IgG antibodies at the hinge region. We were able to accurately identify both inter- and intra-disulfide linked peptides and correctly quantify intra-disulfide isomers. It is the first study to quantify intra-disulfide isomers in IgG antibodies with a mass spectrometry approach. It will help to achieve efficient generation of bispecific antibodies with Fab-arm exchange.