The reduction of the number of animals used in the biological assay of insulin.

The biological control of insulin is vital as it is administered daily to a large proportion of the population during the major part of their lives. The development of the biological assay of insulin will be described with the progressive evolution of methods eventually to dispense with the need for animals. From the quantal Mouse Convulsion Method using around 600 mice per sample to the quantitative Mouse Blood Glucose method using an average of 120 mice per sample in which the fall in the blood glucose levels is used to estimate the potency of insulin by its hypoglycaemic effect. This was followed by the development of the in vitro Radioreceptor Method which is a procedure based on competition between the binding of insulin and [125I]-insulin to cells of the cultured human IM-9 cell line. The production of highly purified insulin by the Pharmaceutical Industry has made possible the recent introduction of new High Performance Liquid Chromatography technology.

HPLC as a replacement for the animal response assays for insulin.

The work describes the assay of the potency of insulin (drug substance) and its formulations by gradient elution reversed-phase high-performance liquid chromatography (HPLC). The methods are shown to differentiate between insulins of bovine, porcine and human sequence, and to be both reproducible and stability-indicating. The HPLC assay results show good agreement with those obtained by the mouse blood glucose assay. The advantages of the HPLC assays over the animal response assays are discussed. It is suggested that the animal response assays should now be replaced by the HPLC assays.

Stability of bovine insulin.

A stability experiment on purified crystalline bovine insulin showed that it deteriorates at normal refrigeration temperature (5 degrees C) and that storage at -20 degrees C is necessary to ensure stability. Purified insulin degrades by two mechanisms: deamidation and polymerization. Immunochemical potency determinations alone are insufficient to determine the stability of insulin as they may be little affected by the chemical changes which occur on storage. The activation energy for the polymerization of insulin was found to be 66 kJ mol-1 and for deamidation 45 kJ mol-1. It is recommended that crystalline bovine insulin be stored at -20 degrees C.