Insulin Aggregation Assessment using SDS-free Capillary Gel Electrophoresis: Comparison with Size-Exclusion Chromatography

Jacques Crommen, Laboratory for the Analysis of Medicines, Department of Pharmacy, CIRM, University of Liege, Liege, Belgium (jcrommen@uliege.be)
Alice Demelenne, Laboratory for The Analysis of Medicines, Department Of Pharmacy, CIRM, University of Liege, Liege, Belgium
Fabrice Bouillenne, Laboratory of Enzymology And Protein Folding, CIP, University of Liege, Liege, Belgium
Anne-Catherine Servais, Laboratory for The Analysis of Medicines, Department of Pharmacy, CIRM, University of Liege, Liege, Belgium
Marianne Fillet, Laboratory for The Analysis of Medicines, Department of Pharmacy, CIRM, University of Liege, Liege, Belgium

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates. The United States and European Pharmacopoeias currently use a SEC method with an acidic mobile phase to assess the content of aggregates in insulin formulations.

In this study, aggregated human insulin samples were analyzed and under neutral conditions, both SEC (nSEC) and capillary gel electrophoresis (CGE) were found to lead to similar results for aggregate content, unlike SEC under acidic conditions (aSEC) [1]. Polymeric complexes were detected using aSEC while they were not observed with nSEC and CGE. During method development, the effects of arginine and acetonitrile addition on SEC profiles were studied. In CGE, the impact of SDS on disruption of non-covalent insulin aggregates was confirmed and the benefit of sodium deoxycholate addition into the sieving gel was examined. The three methods were applied to the analysis of an insulin formulation and similar results to those obtained for human insulin in pure form were observed. Finally, the CGE method was used to study the stability of human insulin under different storage conditions.

In view of the results obtained, which emphasize the importance of the mobile phase composition and pH in SEC, one may question the relevance of the current pharmacopoeial method for insulin aggregation assessment. The new CGE method is an easy approach for studying non-covalent aggregates of insulin, which could be applied to other proteins.

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Abstract Reference & Short Personal Biography of Presenting Author

Abstract Reference:

A. Demelenne, A. Napp, F. Bouillenne, J. Crommen, A.C. Servais, M. Fillet, Talanta, 199 (2019) 457-463.

Short Personal Biography:

Jacques Crommen is currently Professor Emeritus at the University of Liege, Belgium and Guest Professor at Jinan University, Guangzhou, China. He was Full Professor and Head of the Laboratory of Analytical Pharmaceutical Chemistry at the University of Liege from 1991 to 2010. He was also Guest Professor at the Catholic University of Louvain (UCL), Belgium, from 1997 to 2003. He was Editor of the Journal of Pharmaceutical and Biomedical Analysis from 1999 to 2003. He is Vice President of the Belgian Royal Academy of Medicine, and Honorary Member of the Hungarian Pharmaceutical Society and the Belgian Society of Pharmaceutical Sciences. He was awarded the degree of Doctor honoris causa from Iuliu Hatieganu University, Cluj-Napoca, Romania. His current research interests include chiral separations, analysis of counterfeit drugs by HPLC and CE, and quantification of biomolecules by miniaturized separation techniques coupled to mass spectrometry.