Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations.

PURPOSE: Isolation and identification of unknown impurities of recombinant insulin lispro (produced at IBA) formed during accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formulations of recombinant human insulin (Humulin S®; Lilly), recombinant insulin lispro (Humalog®; Lilly), recombinant insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recombinant insulin detemir (Levemir®; Novo Nordisk) and recombinant insulin glargine (Lantus®; Sanofi-Aventis) were analyzed.

METHODS: The impurities of insulin analogs were isolated by RP-HPLC and identified with peptide mass fingerprinting using MALDI-TOF/TOF mass spectrometry.

RESULTS: The identified derivatives were N-terminally truncated insulin analog impurities of decreased molecular mass of 119, 147 and 377 Da related to the original protein. The modifications resulting in a mass decrease were detected at the N-terminus of B chains of insulin lispro, insulin aspart, human insulin, insulin glargine, insulin detemir in all tested formulations. To our knowledge it is the first time that these impurities are reported.

CONCLUSIONS: The following derivatives formed by truncation of the B chain in insulin analogs were identified in pharmaceutical formulations: desPheB1 -N-formyl-ValB2 derivative, desPheB1 derivative, pyroGluB4 derivative.