Disulfide bond assignment of an IgG1 monoclonal antibody by LC–MS with post-column partial reduction

Confirmation of the correct disulfide linkage and demonstration of the lack of a significant level of scrambled disulfide bonds are critical to ensure the appropriate folding and structure of recombinant monoclonal antibodies. Currently these are typically achieved by carrying out multiple experiments, most commonly via the comparison of the samples before and after reduction by LC–MS and MS/MS. The data are then analyzed by searching across all the possible disulfide linkages manually or with the aid of computer algorithms. To eliminate the need of multiple experiments and complicated data analysis, a simple LC–MS-based method coupled with post-column partial reduction was developed. Using a recombinant monoclonal IgG1 antibody as an example, this method demonstrates the ability to confirm the correct disulfide linkage and the ability to detect scrambled disulfide bonds from a single experiment with a simple data analysis strategy.