Chromatographic Analysis of Conformationally Changed Insulin and Its Cytotoxic Effect on PC12 Cells

With the aim of developing a quality control system for insulin formulations in the pharmaceutical industry, we tested the feasibility of chromatographic methods, including reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE), as tools for monitoring the conformational changes in insulin. Insulin samples were dissolved in 0.01 M HCl and incubated at 60 degrees C for 24, 48, and 96 hr to induce aggregation and fibrillation, which were confirmed by studying their circular dichroism spectra and by assaying a fluorescent indicator of beta-sheet structure in aggregate proteins [thioflavine T (ThT)]; the samples were also analyzed by RP-HPLC and CE. In RP-HPLC and CE, the distinct changes in the elution patterns, as reflected by the insulin peaks, revealed the process of conformational change in insulin. With respect to the cytotoxicity of these insulin samples against rat pheochromocytoma (PC12) cells, a significant increase in lactate dehydrogenase activity was observed in the culture medium containing insulin aggregates (50 mu M) as compared to the medium containing native insulin. These results suggest that the cytotoxicity of insulin in terms of the conformational changes in its structure can be rapidly assessed by using RP-HPLC and CE.