Insulin aggregation assessment by size-exclusion chromatography and capillary gel electrophoresis

Abstract

Size-exclusion chromatography (SEC) is the method of choice for the analysis of protein aggregates in biopharmaceuticals. Aggregates could be formed during production and storage of formulations and may lead to complications in insulin therapy. For that reason, they are important parameters to investigate for insulin quality control. The United States and European Pharmacopoeias currently use a SEC method with acidic mobile phase for the quantification of aggregates in insulin formulations. However, changes in aggregates assessment have been reported when the mobile phase composition differs from the sample dissolution medium. [1] To investigate the impact of mobile phase on human insulin aggregates analysis, the aggregated human insulin samples were analyzed by SEC using neutral (nSEC) or acidic mobile phases (aSEC). The aggregated samples were obtained by dissolving human insulin in acidic media, followed by agitation for 8, 16, 24, 32, 40 and 48 hours respectively. During SEC method development, the impact of arginine and acetonitrile addition to the mobile phase was pointed out. Additionally, an orthogonal capillary-gel electrophoresis method (CGE) for the assessment of insulin aggregates was developed. The optimal pH 8.1 CGE buffer was formulated without SDS in order to preserve the non-covalent aggregates. After the optimization of the methods, human insulin and aggregated samples were analyzed using nSEC, aSEC and CGE. A similar increase of dimers percentage with incubation time was noticed by both nSEC and CGE, while no significant increase of dimers content was observed by aSEC. However, an insulin polymeric complex was detectable for some samples with aSEC. The three methods were used to analyze an insulin formulation and a similar tendency was observed. The results obtained emphasize the importance of mobile phase choice in SEC. The good correlation between dimers percentage obtained by nSEC and CGE suggests that these technics most probably enable the detection of the species initially present in the sample and do not change the composition of the sample during analysis. The developed CGE method is a fast and reliable tool for the study of the complex process of insulin aggregation. Furthermore, the CGE method could be easily applied to other proteins since it proved to be highly reproducible and has the advantages of low sample consumption, and no expensive column or organic solvents are required