Purpose: Identification of human insulin analogs' impurity with a mass shift +14 Da in comparison to a parent protein.
Methods: The protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing.
Results: The misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs.
Conclusions: Although there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.
Keywords: NMR; insulin analogs; insulin impurities; mass spectrometry; misincorporation.