Effect of contaminating proteolytic activity upon insulin and insulin-protamine complex.

Abstract

Abstract The proteolytic contamination in batches of various insulin preparations was studied using chromogenic synthetic peptide substrates, synthetic ester substrates, and a test similar to the European Pharmacopoeia test for ‘Limit of Proteolytic Activity’. In order to study the latter test, insulin-protamine complexes were prepared using salmine sulphate and samples of crystalline insulin, some of which contained peptidase activity. All insulin-protamine complexes prepared from insulin crystals in which peptidase activity had been detected using a synthetic chromogenic substrate were degraded on storage at 37 °C, whereas those prepared from insulin crystals in which no peptidase activity could be detected showed no significant loss in weight. Chromatography on Sephadex G50 revealed degradation of insulin in solutions containing traces (∼0·5 p kat mg−1 preparation) of peptidase activity and stored for 2 weeks at 37 °C at pH 7·4, but not of such insulin stored at pH 2·5 at 37 °C for up to 6 weeks. Since insulin preparations formulated at neutral pH are so susceptible to molecular degradation by traces of peptidase contamination, it is suggested that the test using a chromogenic peptide substrate could be applied to bulk crystals before vialing of insulin preparations and could also replace the current test specified in the monograph on Isophane Insulin of the European Pharmacopoeia.