Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product

Purpose. This report summarizes the identification of nine deamidation and four hydrolysis products from a sample of pramlintide injection final drug product that was subjected to stress at 40 degrees C for 45 days. Methods. The pramlintide degradation products were isolated by strong cation exchange HPLC followed by reversed-phase HPLC. Subsequent to isolation, the molecular weight of each component was determined by liquid chromatography-mass spectrometry (LC/MS). Further characterization was accomplished by amino acid sequence analysis and/or enzymatic (thermolysin) digestion followed by LC/MS and sequence analysis. Results. The isolated products were identified as [iso-Asp(21)]-pramlintide, [iso-Asp(3)]-pramlintide, and [iso-Asp(22)]-pramlintide, the deamidation products of pramlintide with rearrangement at Asn(21), Asn(3), and Asn(22), respectively. Also found were [Asp/iso-Asp(14)]-pramlintide, and [Asp/iso-Asp(35)]-pramlintide, the deamidation products at Asn(14), and Asn(35), and [Asp(21)]-pramlintide together with [Asp(22)]-pramlintide. For the deamidations at the 14(th) and 35(th) residues, it could not be determined whether the substance corresponded to the Asp or the iso-Asp product. The [Asp(21)] and [Asp(22)] products could not be separated from each other chromatographically but were both identified in a single fraction. Two minor degradation products were also identified as deamidated species. However, the sites of deamidation remain unknown. Also identified were [1-18]-pramlintide, [1-19]-pramlintide, [19-37]-pramlintide, and [20-37]-pramlintide, the products of hydrolytic peptide backbone cleavage at amino acids His(18)/Ser(19) and Ser(19)/Ser(20), respectively. One other product was isolated and tentatively identified as a cyclic imide intermediate preceeding deamidation. Conclusions. The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.