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

•Enables confident analysis of native disulfide connectivity.•Significantly reduces disulfide scrambling with high reproducibility compared to conventional methods .•Maintains the advantages of tryptic digestion at alkaline pH .•Robust with high digestion efficiency and LC/UV-MS compatibility.•Provides a robust approach for assessing disulfide bonding integrity and stability in different states of mAb development.