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Related Experiment Video

Updated: May 5, 2026

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Published on: June 21, 2021

Artificial Oxidation: A Major Challenge in Implementing Multi-Attribute Methods for Therapeutic Protein Analysis.

Yaokai Duan¹, Michael Lanzillotti², Dylan L Riggs²

¹Process Development, Amgen Inc., Thousand Oaks, CA 91320, USA.

Pharmaceuticals (Basel, Switzerland)

|May 4, 2026

Summary

Artificial oxidation during therapeutic protein analysis using multi-attribute methods (MAM) is a significant challenge. This study highlights unpredictable oxidation issues, impacting quality control and necessitating enhanced control strategies for reliable results.

Keywords:

LC-MS artificial oxidation metal-induced oxidation methionine oxidation multi-attribute method peptide mapping therapeutic proteins

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Last Updated: May 5, 2026

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Area of Science:

Biopharmaceutical analysis
Analytical chemistry
Mass spectrometry

Background:

Multi-attribute methods (MAM) using mass spectrometry offer comprehensive therapeutic protein analysis.
Widespread adoption of MAM is limited by challenges, particularly artificial oxidation during sample preparation and analysis.

Purpose of the Study:

To report long-term operational observations and case studies on artificial oxidation in therapeutic protein analysis using MAM.
To identify sources and impacts of artificial oxidation in a regulated quality control environment.

Main Methods:

A high-resolution liquid chromatography-mass spectrometry (LC-MS) MAM workflow was applied to Fc-fusion proteins and antibody-based therapeutics.
Investigation involved analyzing a frozen reference standard for system suitability and trending, comparing various consumables, and LC-MS configurations.

Main Results:

A seven-year trending record revealed a 5-fold increase in methionine oxidation in a reference standard, traced to microcentrifuge tubes.
Methionine oxidation in an antibody-drug conjugate was significantly affected by sample preparation.
On-column oxidation in other antibodies was reduced by EDTA flushing, suggesting metal ion involvement.

Conclusions:

Artificial oxidation remains an unpredictable obstacle for robust implementation of MAM in regulated quality control.
Enhanced control strategies, including consumable qualification, metal management, and data analysis, are crucial for reliable therapeutic protein analysis.