title: Site-specific tryptophan oxidation induced by autocatalytic reaction of polysorbate 20 in protein formulation.
abstract: OBJECTIVE
  Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction.
  
  
  METHODS
  Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively.
  
  
  RESULTS
  PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation.
  
  
  CONCLUSIONS
  A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants.
authors.affiliation: Department of Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA. lam@gene.com
authors.forename: Xanthe M
authors.initials: XM
authors.lastname: Lam
authors.forename: William G
authors.initials: WG
authors.lastname: Lai
authors.forename: Edwin K
authors.initials: EK
authors.lastname: Chan
authors.forename: Victor
authors.initials: V
authors.lastname: Ling
authors.forename: Chung C
authors.initials: CC
authors.lastname: Hsu
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D005609:Free Radicals / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010545:Peroxides / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D005609:Free Radicals; D007140:Immunoglobulin Fab Fragments; D010545:Peroxides; D011136:Polysorbates; D006639:Histidine; D014364:Tryptophan