title: Metal-mediated protein oxidation: applications of a modified ELISA-based carbonyl detection assay for complex proteins.
abstract: OBJECTIVE
  Therapeutic proteins are prone to oxidative modification during manufacturing, processing, and storage that may lead to degradation, aggregation, and immunogenicity. Protein carbonylation is an irreversible oxidative modification and has been identified as a hallmark of severe oxidative stress but not extensively studied for its impact on the stability and activity of therapeutic proteins.
  
  
  METHODS
  We describe the application of a modified ELISA-based method to quantify global levels of carbonyl modification of complex proteins. We investigated protein oxidation of large protein molecules (transferrin, rabbit IgG, or β-glucosidase) and complex protein samples (human plasma) that were either stored in different buffer formulations, with varying amounts of divalent iron, or under different storage temperatures to determine the impact of different physicochemical stresses on carbonyl modifications.
  
  
  RESULTS
  The modified ELISA allows for sensitive and specific carbonyl quantification with measurements that closely match those determined with the conventional spectrophotometric method. The method was useful for complex protein mixtures such as cell lysates without the need for additional procedures to remove DNA and RNA. Our findings demonstrate significant oxidative modification of each of the proteins stored in commonly used buffers and excipients at 37°C, 23°C, and 4°C. The carbonyl levels were further exacerbated with addition of trace amounts of Fe(2+). We also measured the extent of protein aggregation under oxidizing conditions.
  
  
  CONCLUSIONS
  Collectively, our results indicate the importance of better characterizing carbonyl modification of proteins during their storage and use.
authors.affiliation: Laboratory of Chemistry, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 29 Lincoln Drive Bldg 29A, Room 2A-11, Bethesda, Maryland, 20892, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uehara
authors.forename: V Ashutosh
authors.initials: VA
authors.lastname: Rao
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D066329:Protein Aggregates / Q000187:drug effects / Q000502:physiology*; D050050:Protein Carbonylation / Q000187:drug effects / Q000502:physiology*; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D008670:Metals; D066329:Protein Aggregates; D012710:Serum Albumin, Bovine