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Updated: Mar 28, 2026
Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
Published on: January 30, 2018
Investigation of Color in a Fusion Protein Using Advanced Analytical Techniques: Delineating Contributions from
Hangtian Song1, Jianlin Xu2, Mi Jin2,3
1Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Bloomsbury, New Jersey, USA. hangtian.song@bms.com.
Purpose:
Discoloration of protein therapeutics has drawn increased attention recently due to concerns of potential impact on quality and safety. Investigation of discoloration in protein therapeutics for comparability is particularly challenging primarily for two reasons. First, the description of color or discoloration is to certain extent a subjective characteristic rather than a quantitative attribute. Secondly, the species contributing to discoloration may arise from multiple sources and are typically present at trace levels. Our purpose is to development a systematic approach that allows effective identification of the color generating species in protein therapeutics.
Methods:
A yellow-brown discoloration event observed in a therapeutic protein was investigated by optical spectroscopy, ultra-performance liquid chromatography, and mass spectrometry (MS).
Results:
Majority of the color generating species were identified as oxidatively modified protein. The location of the oxidized amino acid residues were identified by MS/MS. In addition, the impact of process-related impurities co-purified from media on discoloration was also investigated. Finally a semi-quantitative scale to estimate the contribution of each color source is presented, which revealed oxidized peptides are the major contributors.
Conclusions:
A systematic approach was developed for identification of the color generating species in protein therapeutics and for estimation of the contribution of each color source.
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