Historical review of the analytical control of insulin

Abstract

Following the discovery of insulin in 1922, the lack of knowledge of its chemical structure caused biological methods of assay based on the measurement of the hypoglycaemic response in rabbits and the convulsive response in mice to be developed in order to assess the potency of the hormone. These classical methods, the evolution of which contributed largely to the establishment of biometrics, are still the only acceptable pharmacopoeial methods for the determination of insulin potency. By using these methods four International Standards for insulin were established during the period 1926 to 1959. Other in vivo methods were developed so as to increase the sensitivity of the assay.

Attempts to replace the in vivo assays by in vitro assays have been made, based mainly on the observation in 1940 that insulin increased glucose uptake and glycogen deposition in the isolated rat diaphargm and on the later observation in 1958 that insulin enhanced glucose uptake and oxidation in isolated rat epididymal fat. While methods utilising these parameters are sensitive they lack precision and also lack specificity when used as in-process assays for monitoring the isolation of insulin from the pancreas.

Following the establishment of radioimmunoassays for the measurement of the amount of insulin in blood, attempts during the last decade have been made to replace the pharmacopoeial assays by radioimmunoassay. This method of assay is, however, not always correlated with biological potency.

Assays based on the physico-chemical properties of insulin have been developed in the last 20 years, involving the use notably of chromatographic and electrophoretic techniques. In general they are less sensitive and give less reproducible quantitative results from one laboratory to another than biological assays but have been invaluable in assessing the purity of insulin by revealing the presence of any associated polypeptides that could give rise to unwanted immunological responses in man.

Insulin preparations with prolonged action have been developed from 1936 onwards, in which insulin is rendered insoluble at neutral pH by complexing it with a protein such as protamine or globin (PZI or GZI) or by crystallising it in the presence of excess of zinc (Lente Insulins). Pharmacopoeial tests to ensure uniformity of the preparation may include a test to demonstrate prolongation of action in rabbits or guinea pigs.

In further defining the purity of insulin, other analytical controls are applied including the determination of nitrogen, zinc, ash, moisture and glucagon. No comprehensive pharmacopoeial monograph yet exists for crystalline insulin but all pharmacopoeias contain monographs to control the quality and concentration of insulin in formulated preparations.

Insulin in clinical use is largely bovine or porcine in origin but other species insulins have been used. The biological activity of various species insulin is discussed.

Insulin has also been modified chemically, usually by acylation, in an attempt to increase potency, prolong activity or reduce immunogenicity. The chemical and biological control of these insulins is also discussed.

Since the discovery of the linear and tertiary structures of insulin, attempts have been made to determine active regions of the molecule with a view to their synthesis and preservation of the tertiary structure. The synthesis of insulin itself has also been achieved, although not yet commercially. The analytical controls that will have to be exercised on synthetic insulin and insulin-like molecules are discussed.