ABSTRACT
The covalent insulin-protamine product molecules formed by heat stress in Neutral Protamine Hagedorn formulations of insulin and the insulin analogue [LysB28,ProB29] were examined by mass spectrometry. The results demonstrated that the covalent cross-link between insulin and protamine was not caused by linkage through the protamine N-terminal amino group, as had been previously thought. Our results indicate that the linkage was formed between the side chain of a protamine arginine and a histidine in the insulin B chain, resulting in a net mass change of -5 Da, compared to the sum of the protamine and insulin molecular masses. A mechanism for this new type of covalent cross-linking reaction is proposed.
Subject(s)
Cross-Linking Reagents/chemistry , Insulin/analogs & derivatives , Protamines/administration & dosage , Protamines/chemistry , Chemistry, Pharmaceutical , Chromatography, Gel , Delayed-Action Preparations , Insulin/administration & dosage , Insulin/chemistry , Mass Spectrometry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The separation of antibody-antigen complexes by free-solution capillary zone electrophoresis (CZE) has been demonstrated. The antigen, a monoclonal antibody specific for the antigen, and the complex were well resolved. The entire separation was achieved in less than 10 min using on-column UV detection. The pI values for the three species were estimated separately by isoelectric focusing (IEF) experiments on polyacrylamide gels. Reasonably good agreement was found between the relative migration times measured by CZE and the pI values. Both IEF and high-performance size-exclusion chromatography of the antibody-antigen mixtures confirmed the formation of the complexes observed by CZE. This study demonstrates the utility of CZE as a new and complementary technique for the characterization of antibody-antigen complexes.