Eliminating liquid chromatography-induced methionine oxidation of monoclonal antibodies using high-concentration TCEP injections: The "anTCEPtic" column cleaning method

  • J Pharm Sci. 2026 Jun;115(6):104298. doi: 10.1016/j.xphs.2026.104298.
Andrew Kleinberg  1 ,  Joy Yoon  2 ,  Yuan Mao  3 ,  Ning Li  2
Affiliations
  • 1. Analytical and Biological Mass Spectrometry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, United States. Electronic address: [email protected].
  • 2. Analytical and Biological Mass Spectrometry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, United States.
  • 3. Analytical and Biological Mass Spectrometry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, United States. Electronic address: [email protected].
PMID: 42034288 DOI: 10.1016/j.xphs.2026.104298
Abstract

Artificial oxidation of methionine residues during LC/MS analysis of peptide mapping samples is a common issue arising from multiple possible contamination sources. Accurate monitoring of post-translational modifications (PTMs) is critical for ensuring the safety, stability and efficacy of biologics. Herein, we demonstrate that high-concentration LC injections of tris(2-carboxyethyl)phosphine (TCEP) can effectively clean unwanted Reactive Oxygen Species (ROS) from LC systems, thereby minimizing artificial oxidation of methionines, and enabling quantitation and reporting of correct PTM levels. This simple process alleviates troubleshooting time and saves significant costs associated with purchasing new LC columns and preparing fresh buffers.

Keywords
Liquid chromatography-mass spectrometry; Methionine oxidation; Monoclonal antibodies; Peptide mapping; Post-translation modifications; Reactive oxygen species; Tris(2-carboxyethyl)phosphine.
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