title: Identification of site-specific heterogeneity in peptide drugs using intact mass spectrometry with electron transfer dissociation.
abstract: BACKGROUND
  Protamine sulfate is a peptide drug product consisting of multiple basic peptides. As traditional high-performance liquid chromatography (HPLC) separation methods may not resolve these peptides, as well as any possible peptide-related impurities, a method utilizing top-down mass spectrometry was developed for the characterization of complex peptide drug products, including any low-level impurities, which is described in this study.
  
  
  METHODS
  Herring protamine sulfate was used as a model system to demonstrate the applicability of the method. Direct infusion mass spectrometry and tandem mass spectrometry (MS/MS) on a high-resolution, mass accurate instrument with electron transfer dissociation (ETD) were used to identify all the species present in the herring protamine sulfate sample. Identifications were made based on mass accuracy analysis as well as MS/MS fragmentation patterns.
  
  
  RESULTS
  Complete sequence coverage of the three abundant herring protamine peptides was obtained using the top-down ETD-MS/MS method, which also identified a discrepancy with the published herring protamine peptide sequences. Additionally, three low-abundance related peptide species were also identified and fully characterized. These three peptides had not previously been reported as herring protamine peptides, but could be related to the published sequences through amino acid additions and/or substitutions.
  
  
  CONCLUSIONS
  A method for the characterization of protamine, a complex peptide drug product, was developed that can be extended to other complex peptide or protein drug products. The selectivity and sensitivity of this method improves a regulator's ability to identify peptide impurities not previously observed using the established methods and presents an opportunity to better understand the composition of complex peptide drug products.
authors: U.S. Food and Drug Administration, CDER/OPS/OTR Division of Pharmaceutical Analysis, 10903 New Hampshire Ave, Silver Spring, MD, 20993, USA.
authors: Ashley C
authors: AC
authors: Gucinski
authors: Michael T
authors: MT
authors: Boyne
mesh_terms: D000595:Amino Acid Sequence; D004340:Drug Contamination / Q000517:prevention & control*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004579:Electron Transport; D008969:Molecular Sequence Data; D011479:Protamines / Q000032:analysis* / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011479:Protamines