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°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-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 14^th and 35^th 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 l-18]-pramlintide, l-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. Conclusions. The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.