Elucidating chemical and disulfide heterogeneities in rituximab using reduced and non‐reduced peptide mapping

Peptide mapping by liquid chromatography-mass spectrometry is the gold standard to characterize post-translational modifications (PTMs) and disulfide bonds. The structural integrity, heterogeneity, and quality of biotherapeutic proteins are evaluated via reduced and non-reduced peptide mapping methods. However, non-enzymatic artifacts are often induced during sample preparation when alkaline pH conditions are used. To minimize these artifacts, methods using various acidic pH conditions have been developed by multiple researchers. However, these may lead to missed and non-specific cleavages during the analysis. In this study, an improved reduced and non-reduced peptide mapping method has been proposed to characterize endogenous chemical modifications and native disulfide bonds of monoclonal antibody-based products. Solubilization has been carried out at acidic pH conditions under high temperature, followed by the addition of tris (2-carboxyethyl) phosphine as a reducing agent and a low alkylating agent. It was observed that the non-enzymatic PTMs and non-native disulfide scrambled peptides significantly reduced under trypsin plus endoproteinase Lys-C digestion conditions at acidic pH as compared to the traditional methods. The results demonstrate that the proposed reduced and non-reduced peptide mapping method using trypsin plus Lys-C could identify and quantify various chemical and disulfide heterogeneities with minimal artifacts.