title: Investigation of Color in a Fusion Protein Using Advanced Analytical Techniques: Delineating Contributions from Oxidation Products and Process Related Impurities.
abstract: OBJECTIVE
  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.
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Bloomsbury, New Jersey, USA. hangtian.song@bms.com.
authors.forename: Hangtian
authors.identifier: http://orcid.org/0000-0001-5924-2360
authors.initials: H
authors.lastname: Song
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, East Syracuse, New York, USA.
authors.forename: Jianlin
authors.initials: J
authors.lastname: Xu
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, East Syracuse, New York, USA.|, Teva Pharmaceuticals, 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
authors.forename: Mi
authors.initials: M
authors.lastname: Jin
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, East Syracuse, New York, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Huang
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Hopewell, New Jersey, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Bongers
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Hopewell, New Jersey, USA.
authors.forename: He
authors.initials: H
authors.lastname: Bai
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Bloomsbury, New Jersey, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Hopewell, New Jersey, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Ludwig
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, Devens, Massachusetts, USA.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Li
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Hopewell, New Jersey, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.affiliation: Biologics Molecular and Analytical Development, Bristol-Myers Squibb, Hopewell, New Jersey, USA.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Das
mesh_terms: D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003116:Color; D003412:Cricetulus; D004340:Drug Contamination; D006639:Histidine / Q000032:analysis; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D004364:Pharmaceutical Preparations / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods; D014364:Tryptophan / Q000032:analysis
chemical_list: D010455:Peptides; D004364:Pharmaceutical Preparations; D011506:Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D014364:Tryptophan
keywords: color; histidine; mass spectrometry; oxidation; process development; tryptophan; ultra-performance liquid chromatography