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. 2025 Jan;42(1):173-184.
doi: 10.1007/s11095-024-03805-z. Epub 2025 Jan 23.

A Novel Peptide Mapping Method Utilizing Cysteine as a Reducing Agent

Jun-Ting Fang  1   2 , Si-Tao Wang  1 , Haibin Wang  3 , Wei-Jie Fang  4   5   6
Affiliations

A Novel Peptide Mapping Method Utilizing Cysteine as a Reducing Agent

Jun-Ting Fang et al. Pharm Res. 2025 Jan.

Abstract

Purposes: In the peptide mapping reduction process for monoclonal antibodies (mAbs) and other proteins, the conventional reducing reagents β-mercaptoethanol (β-ME) and dithiothreitol (DTT) pose challenges due to their strong odor and toxicity at high concentrations. Cysteine (Cys), an essential amino acid for new protein synthesis, is an overlooked, nontoxic, and odorless reducing agent. This study presents a novel peptide mapping method using Cys as the reducing agent.

Methods: We explored the reducing effect of Cys at different concentrations and pH levels for peptide mapping analysis of a specific mAb (mAb-1), using DTT as a positive control. RP-HPLC analysis, including UV chromatogram comparison and overall similarity calculation, was conducted for comparison. LC-MS analysis was subsequently utilized to characterize the primary sequence of mAb-1. We also applied the method to other mAbs or proteins to demonstrate its wide applicability.

Results: The UV chromatogram and overall similarity of Cys as a reducing agent at concentrations ranging from 10 to 40 mM and pH levels between 7.0 and 11.0 were consistent with those of the positive control. Reduced concentrations of Cys or lower pH levels compromised reducing efficacy. This novel reducing method proficiently characterized the primary sequence of mAb-1, achieving an overall sequence coverage of 97%. In the analysis of other mAbs or proteins, the peptide mapping results also showed high consistency.

Conclusions: Cys exhibits a reducing ability comparable to DTT and possesses the advantageous characteristics of being nontoxic and odorless, making it a potential alternative for disulfide bond reduction and peptide mapping analysis of proteins and mAbs.

Keywords: cysteine; dithiothreitol; monoclonal antibody; peptide mapping; reducing agent.

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Conflict of interest statement

Declarations. Conflicts of Interests: The authors have no conflicts of interest to declare.

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