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Pharmaceutical Research

, Volume 33, Issue 4, pp 932–941 | Cite as

Investigation of Color in a Fusion Protein Using Advanced Analytical Techniques: Delineating Contributions from Oxidation Products and Process Related Impurities

  • Hangtian SongEmail author
  • Jianlin Xu
  • Mi Jin
  • Chao Huang
  • Jacob Bongers
  • He Bai
  • Wei Wu
  • Richard Ludwig
  • Zhengjian Li
  • Li Tao
  • Tapan K. Das
Research Paper

ABSTRACT

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.

KEY WORDS

color histidine mass spectrometry oxidation process development tryptophan ultra-performance liquid chromatography 

Abbreviations

AGE

Advance glycation end products

CHO

Chinese hamster ovary

DP

Drug product

DS

Drug substance

ICP- OES

Inductively coupled plasma-optical emission spectrometry

Kyn

Kynurenine

MCO

Metal-catalyzed oxidation

NaBH4

Sodium borohydride

NFK

N-formylkynurenine

OH-Trp

Hydroxytryptophan

PDA

Photodiode array

ROS

Reactive oxygen species

Notes

ACKNOWLEDGMENTS AND DISCLOSURES

We thank Jinmei Fu, Yunping Huang, Peiran Liu, Thomas Slaney, Neil Hershey, Hui Wei, Yemin Xu, Gurusamy Balakrishnan, Kirby Steger, Douglas Weaver for helpful discussion, Bruce Eagan, Sivaprakash Agastin, Lei Zhou, Andrew Chen, Susan E. Egan, Qin He, Erin K. Abbott, Jongchan Lee and Michael Borys for upstream processing, Kelley Ledford, Vasavi Arunachalam and Cherie Strain for downstream processing, Chris A. House and Ves Lesins for the operation of bioreactors and pilot scale purification. The authors declare no competing financial interest.

Supplementary material

11095_2015_1839_MOESM1_ESM.pdf (723 kb)
ESM 1 (PDF 722 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Biologics Molecular and Analytical DevelopmentBristol-Myers SquibbBloomsburyUSA
  2. 2.Biologics Process DevelopmentBristol-Myers SquibbEast SyracuseUSA
  3. 3.Biologics Molecular and Analytical DevelopmentBristol-Myers SquibbHopewellUSA
  4. 4.Biologics Process DevelopmentBristol-Myers SquibbDevensUSA
  5. 5.West ChesterUSA

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