Ultrasensitive disulfide scrambling analysis of mAbs by LC-MS with post-column reduction and glycine signal enhancement

Explicitly confirming the complete disulfide bond linkage pattern of a monoclonal antibody (mAb) presents a challenge in the biopharmaceutical industry. Although proper native disulfide connections are in high abundance for analytical purposes within a peptide mapping digest under non-reducing conditions, disulfide scrambling can also exist but be difficult to detect, let alone characterize, particularly at low levels. Here, we developed an ultrasensitive high-confidence method for identifying explicit disulfide connectivity in mAbs. By applying a post-column addition of tris (2-carboxyethyl)phosphine hydrochloride (TCEP) to the liquid chromatography (LC) eluent of a non-reduced mAb digest, partial reduction of disulfide peptides is achieved after the initial peptide separation, allowing both the parent disulfide and its reduced daughter peptides to co-elute for simultaneous mass spectrometry (MS) detection. Combining this concept with the recently discovered ability of glycine to enhance MS signal when added to the LC eluent, we demonstrate a method for detecting, characterizing and quantifying low-abundance disulfide scrambling in mAbs.