title: Qualitative and quantitative comparison of brand name and generic protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.
abstract: The capability of iTRAQ (isotope tags for relative and absolute quantification) reagents coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) as a qualitative and quantitative technique for the analysis of complicated protein pharmaceutical mixtures was evaluated. Mixtures of Somavert and Miacalcin with a small amount of bovine serum albumin (BSA) as an impurity were analyzed. Both Somavert and Miacalcin were qualitatively identified, and BSA was detected at levels as low as 0.8mol%. Genotropin and Somavert were compared in a single experiment, and all of the distinct amino acid residues from the two proteins were readily identified. Four somatropin drug products (Genotropin, Norditropin, Jintropin, and Omnitrope) were compared using the iTRAQ/MALDI-MS method to determine the similarity between their primary structures and quantify the amount of protein in each product. All four product samples were well labeled and successfully compared when a filtration cleanup step preceded iTRAQ labeling. The quantitative accuracy of the iTRAQ method was evaluated. In all cases, the accuracy of experimentally determined protein ratios was higher than 90%, and the relative standard deviation (RSD) was less than 10%. The iTRAQ and global internal standard technology (GIST) methods were compared, and the iTRAQ method provided both higher sequence coverage and enhanced signal intensity.
authors.affiliation: U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Division of Pharmaceutical Analysis (DPA), St. Louis, MO 63101, USA. hongping.ye@fda.hhs.gov
authors.forename: Hongping
authors.initials: H
authors.lastname: Ye
authors.forename: John
authors.initials: J
authors.lastname: Hill
authors.forename: John
authors.initials: J
authors.lastname: Kauffman
authors.forename: Xianlin
authors.initials: X
authors.lastname: Han
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000737:chemistry*; D002417:Cattle; D004340:Drug Contamination; D016568:Drugs, Generic / Q000032:analysis / Q000737:chemistry*; D019382:Human Growth Hormone / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry*; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D012710:Serum Albumin, Bovine / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D016568:Drugs, Generic; D019382:Human Growth Hormone; D012710:Serum Albumin, Bovine; C028815:salmon calcitonin; D002116:Calcitonin; C406545:pegvisomant