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

Abstract

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. 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. The isolated products were identified as [iso-Asp21]-pramlintide, [iso-Asp3]-pramlintide, and [iso-Asp22]-pramlintide, the deamidation products of pramlintide with rearrangement at Asn21, Asn3, and Asn22, respectively. Also found were [Asp/iso-Asp14]-pramlintide, and [Asp/iso-Asp35]-pramlintide, the deamidation products at Asn14, and Asn35, and [Asp21]-pramlintide together with [Asp22]-pramlintide. For the deamidations at the 14th and 35th residues, it could not be determined whether the substance corresponded to the Asp or the iso-Asp product. The [Asp21] and [Asp22] 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 His18/Ser19 and Ser19/Ser20, respectively. One other product was isolated and tentatively identified as a cyclic imide intermediate preceeding deamidation. The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.