title: Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product.
abstract: This report summarizes the identification of six cyclic imide [Asu] and two deamidation products from a sample of pramlintide final drug product that had been stressed at 40 degrees C for 45 days. The pramlintide degradation products were isolated by cation exchange high-performance liquid chromatography (HPLC) followed by reversed-phase HPLC. The isolated components were characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessary, by enzymatic (thermolysin) digestion followed by liquid chromatography/mass spectrometry (LC/MS) and sequence analysis. The isolated products were identified as [Asu14]-pramlintide, [Asu21]-pramlintide, [Asu22]-pramlintide, [Asu35]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-succinimide-pramlintide. Also identified were [Asp35]-pramlintide, the deamidation product of pramlintide at Asn35, and [Tyr37-OH]-pramlintide, the deamidation product of the pramlintide amidated C-terminal Tyr. Together these data support those presented earlier (C. Hekman et al., Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product. Pharm Res 1998;15:650-9) indicating that the primary mechanism of degradation for pramlintide in this pH 4.0 formulation is deamidation, with six of the eight possible deamidation sites observed to undergo deamidation. Gln-10 and Asn-31 are the only two residues subject to deamidation for which none is observed. The data indicate that the cyclic imide products account for approximately 20% of the total thermal degradation while the deamidation products account for 64%. The remaining degradation is due to peptide backbone hydrolysis.
authors.affiliation: Amylin Pharmaceuticals Incorporated, San Diego, CA 92121, USA. chekman@cabrillolabs.com
authors.forename: C M
authors.initials: CM
authors.lastname: Hekman
authors.forename: W S
authors.initials: WS
authors.lastname: DeMond
authors.forename: P J
authors.initials: PJ
authors.lastname: Kelley
authors.forename: S F
authors.initials: SF
authors.lastname: Mauch
authors.forename: J D
authors.initials: JD
authors.lastname: Williams
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000032:analysis* / Q000138:chemical synthesis; D002851:Chromatography, High Pressure Liquid; D004304:Dosage Forms / Q000592:standards; D006358:Hot Temperature; D007004:Hypoglycemic Agents / Q000032:analysis* / Q000138:chemical synthesis / Q000592:standards; D007094:Imides / Q000032:analysis*; D058228:Islet Amyloid Polypeptide; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D013820:Thermolysin
chemical_list: D000682:Amyloid; D004304:Dosage Forms; D007004:Hypoglycemic Agents; D007094:Imides; D058228:Islet Amyloid Polypeptide; C105254:pramlintide; D013820:Thermolysin