Credit: Steve Gschmeissner / Science Photo Library

After the discovery of insulin in 1921 (Milestone 1), the use of porcine or bovine insulin to control blood glucose levels in individuals with diabetes became widespread and saved many lives. However, insulin derived from animal pancreases had several limitations, including erratic effects on glucose levels and allergic reactions, both resulting from the production of insulin antibodies by the patient’s immune system. This immunogenicity was thought to be the result of contamination of insulin with other pancreatic substances and small differences in amino acid composition between human and animal insulin. Purified animal insulins were developed and reduced the occurrence of allergic reactions, but further improvement was needed.

In the 1970s, advances in DNA synthesis and recombinant DNA technology raised the possibility that bacteria could be genetically altered to produce human insulin. A major step towards this goal was made in 1977 when, for the first time, a functional polypeptide was generated from a chemically synthesized gene. Itakura et al. synthesized the gene for the hormone somatostatin and incorporated it into a plasmid. The plasmid was inserted into Escherichia coli and the resulting strain produced a polypeptide containing the somatostatin amino acid sequence. Cyanogen bromide was used to cleave somatostatin from this larger protein in vitro. Somatostatin is just 14 amino acids long and was chosen in part because of its size — the time-consuming nature of chemical gene synthesis was considered a barrier to the synthesis of longer polypeptides such as the 51-amino-acid insulin.

Nevertheless, just 2 years later, Goeddel et al. reported the first successful generation of fully synthetic human insulin. Native human insulin consists of two amino acid chains — the A chain and the B chain — that are linked by two disulphide bonds. The team chemically synthesized the DNA fragments encoding the two chains and used plasmids to express the A-chain gene in one strain of E. coli and the B-chain gene in another.

Similar to the somatostatin experiment, the team designed the plasmids so that the insulin chains would be produced as part of a large, relatively inactive protein. After purifying the E. coli products, they cleaved the A and B chains from this precursor protein using cyanogen bromide treatment and joined the two chains together using an existing method. The resulting product was tested using several approaches, including the relatively new technique of reversed-phase high-performance liquid chromatography, and behaved in the same way as human insulin.

The next big step was to establish the safety and efficacy of this new insulin product in humans. In 1980, Keen et al. published the results of a study investigating the effect of synthetic insulin in 17 healthy men. No adverse reactions to cutaneous, subcutaneous or intravenous administration of the synthetic insulin were identified. Furthermore, the synthetic insulin depressed blood glucose levels to a similar degree and with a similar trajectory to highly purified porcine insulin. These positive results indicated that synthetic insulin could provide a viable alternative to animal insulin and in 1982 synthetic insulin became the first genetically engineered product to be approved by the FDA. Despite the novelty of the product, review and approval took just 5 months, at a time when the average approval time for new drugs was more than 2 years. From 1986, the original method was replaced with one that used a plasmid containing the gene for human proinsulin.

Evidence soon indicated that the new synthetic human insulin was indeed less immunogenic than animal-derived insulin. In 1983, Fineberg et al. published the results of a study in 221 individuals with diabetes who had undergone 12 months of insulin treatment. Participants treated with synthetic human insulin had lower levels of insulin antibodies in blood than participants treated with porcine insulin.

The generation of fully synthetic human insulin was a major step forwards in the diabetes field and an important breakthrough in medical biotechnology, paving the way for the FDA approval of many more therapeutic recombinant proteins — more than 100 to date.

Further reading

Itakura, K. et al. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198, 1056–1063 (1977).

Crea, R. et al. Chemical synthesis of genes for human insulin. Proc. Natl Acad. Sci. USA 75, 5765–5769 (1978).

Johnson, I. S. Human insulin from recombinant DNA technology. Science 219, 632–637 (1983).

Fineberg, S. E. et al. Immunogenicity of recombinant DNA human insulin. Diabetologia 25, 465–469 (1983).