Investigation of Color in a Fusion Protein Using Advanced Analytical Techniques: Delineating Contributions from Oxidation Products and Process Related Impurities
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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.
A yellow-brown discoloration event observed in a therapeutic protein was investigated by optical spectroscopy, ultra-performance liquid chromatography, and mass spectrometry (MS).
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.
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 WORDScolor histidine mass spectrometry oxidation process development tryptophan ultra-performance liquid chromatography
Advance glycation end products
Chinese hamster ovary
- ICP- OES
Inductively coupled plasma-optical emission spectrometry
Reactive oxygen species
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.
- 1.European Medicines Agency, ICH Topic Q6B Specifications: Test procedures and acceptance criteria for biotechnological/biological products, 1999.Google Scholar
- 2.U.S. Food and Drug Administration, Guidance for Industry: Q6B Specifications: Test procedures and acceptance criteria for biotechnological/biological products, 1999.Google Scholar
- 3.Cromwell MEM, Carpenter JF, Scherer T, Randolph TW. Opalescence in antibody formulations is a solution critical phenomenon. The 236th ACS National Meeting; August 17–21, 2008; Philadelphia, PA2008.Google Scholar
- 8.Lucas K, Maloney K: Methods for inhibiting yellow color formation in a composition US20120183531 A1. 2012.Google Scholar
- 24.Lam XM, Oeswein JQ, Ongpipattanakul B, Shahrokh Z, Wang SX, Weissburg RP, et al. Antibody formulation US6171586 B1. 2001.Google Scholar
- 27.Le Brun V, Friess W, Bassarab S, Mühlau S, Garidel P. A critical evaluation of self-interaction chromatography as a predictive tool for the assessment of protein–protein interactions in protein formulation development: a case study of a therapeutic monoclonal antibody. Eur J Pharm Biopharm. 2010;75(1):16–25.CrossRefPubMedGoogle Scholar
- 30.Butko M, Pallat H, Cordoba A, Yu XC. Recombinant antibody color resulting from advanced glycation end product modifications. Anal Chem. 2014.Google Scholar
- 36.Institute of Ophthalmology UCL. CIE (2008) physiologically-relevant 2-deg V(λ) luminous efficiency functions 2008 [cited 2014 06/20/2014]. Available from: http://www.cvrl.org/.
- 60.Itakura K, Uchida K, Kawakishi S. Selective formation of oxindole- and formylkynurenine-type products from tryptophan and its peptides treated with a superoxide-generating system in the presence of iron(III)-EDTA: a possible involvement with iron-oxygen complex. Chem Res Toxicol. 1994;7(2):185–90.CrossRefPubMedGoogle Scholar
- 63.Vijayasankaran N, Varma S, Yang Y, Mun M, Arevalo S, Gawlitzek M, et al. Effect of cell culture medium components on color of formulated monoclonal antibody drug substance. Biotechnol Prog. 2013.Google Scholar