Study of insulin aggregation by SEC and CGE

Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient but only the monomeric form is active once dissociated in the bloodstream. Insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolded proteins results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. Moreover, during its production, insulin is often subjected to extreme conditions making lack of aggregates an important parameter to be controlled during its quality control.
United States and European Pharmacopoeias use both size exclusion chromatography (SEC) to assess the level of covalent high molecular weight species. This technique is reproducible, and easy to use but shows many drawbacks including possible changes in the aggregates composition by dilution into the HPLC system or adsorption of sample onto the stationary phase. For those reasons other techniques have been considered in the literature for studying aggregation of insulin. Optical microscopy, electron microscopy, dynamic light scattering, turbidimetry, Fourier Transform infrared spectroscopy, Raman spectroscopy, thioflavin T fluorescence and circular dichroism spectroscopy are some of them. In any cases, the use of orthogonal techniques is essential to assess the relevance of the results.
Results: In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by SEC and capillary electrophoresis (CE). CE shows many advantages in terms of sample and solvent consumption and enables analysis of samples under their native form. We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method.