Stepwise “bridge-to-bridge” reduction of monoclonal antibodies and light chain detection: case studies of tenatumomab and trastuzumab

Monoclonal antibodies are pharmaceutical products that because of their biological origin are characterized by the presence of countless variants, which are generated by post‐translational modifications, during purification or storage. Among these modifications the disulfide bridges variants are of considerable importance, for example their status can affect the correct Y‐shape of antibody. An analytical method, to characterize the oxidized/reduced state of disulfide bridges and to monitor the formation of free light chains followed by chemical reduction, has been developed and here presented. The chromatographic set‐up employs a macroporous polystyrene‐divinylbenzene stationary phase in ion‐pair reversed‐phase high‐performance liquid chromatography and a double UV/high‐resolution mass spectrometry detector. The appearance of increasing amount of reduced light chains can be observed upon incubation of antibodies with the reducing agent tris(2‐carboxyethyl)phosphine. Specifically, three isoforms can be identified at different reaction times: light chain, partially reduced light chain+2H, with one of two disulfide bridges opened in the constant or variable region, and totally reduced light chain+4H. The method was applied in the case of two monoclonal antibodies, namely, tenatumomab and trastuzumab, resulting in a potential tool to detection disulfide bridges variants of other biopharmaceutical products during drug development and release.