Development of a simple non-reduced peptide mapping method that prevents disulfide scrambling of mAbs without affecting tryptic enzyme activity

Non-reduced peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis is a commonly used method for disulfide linkage characterization to assess structural integrity and quality of therapeutic monoclonal antibodies (mAbs). However, disulfide scrambling artifacts induced during sample preparation are often observed when basic pH and high temperatures are used during denaturation and digestion. To minimize disulfide scrambling artifacts, methods using various acidic pH conditions have been developed by multiple groups. However, lower pH conditions increase missed and non-specific cleavages, which complicates disulfide bond analysis because the majority of enzymes used in protein characterization are most efficient at alkaline pH. Here, we developed a non-reduced peptide mapping method for mAb characterization that minimizes disulfide scrambling at basic pH by adding an oxidizing agent, cystamine, and a low concentration of iodoacetamide (IAA) alkylating agent. Two human IgG1 mAbs, one with kappa light chain and another one with lambda light chain, were used as model proteins to develop and optimize the method. Using this novel method, disulfide scrambled peptides related to light chain-heavy chain (LC-HC) inter-disulfide disruption were significantly reduced with high reproducibility compared to conventional methods. Results demonstrated that the cystamine-added method is robust and minimizes disulfide scrambling artifacts produced during sample preparation.