Insulin

With a speed no longer seen in drug discovery and development, insulin was isolated for the first time in 1921 from animal sources and commercialized within 12 months. Decades later, it took just four years for developers to move from expressing recombinant insulin in bacteria to launching the world's first biotechnology drug product.

Scientists Frederick G. Banting and Charles H. Best, working in a lab provided by John J. R. MacLeod at the University of Toronto, isolated the polypeptide hormone and began testing it in dogs. By 1922, with the help of James B. Collip and pharmaceutical company partners, the researchers could purify and produce animal-based insulin in larger quantities.

Insulin is produced by beta cells in the pancreas and is the most important hormone in the body to regulate blood glucose levels. A partial or complete lack of insulin causes diabetes, which, untreated, is often fatal by the teenage years. The World Health Organization reports that an estimated 177 million people worldwide have diabetes. Although not a cure, insulin injections have been the standard treatment since 1924.

Before insulin was discovered, diabetes was managed through diet, which allowed patients to survive, but generally for just a few years after diagnosis. Remarkable medical results were achieved with the first insulin injections. Doctors finally had a means to offer patients a nearly normal quality of life, and it quickly became necessary to increase insulin production.

The Toronto scientists had trouble, however, with consistently isolating and purifying the drug. Connaught Laboratories in Canada, now part of Sanofi-Aventis, assisted, and Eli Lilly & Co. proposed developing large-scale production methods. The university initially rebuffed offers from Lilly, but an agreement was reached in May 1922. By that summer, Lilly was supplying the quantities needed for clinical trials.

To expand the supply, the university gave many royalty-free licenses. Among the licensees was Danish Nobel Laureate August Krogh, whose wife was diabetic. In December 1922, production started in Copenhagen, and the first patient was treated there in March 1923. Krogh later founded the Nordic Insulin Laboratory, now part of the drug firm Novo Nordisk, with Danish physician Hans C. Hagedorn.

Insulin was first commercialized in Great Britain in May 1922, according to the University of Toronto, and by October 1923, it was being sold in the U.S. and Canada. By 1924, large U.S. and British companies were marketing insulin worldwide. It became a major product for Lilly and remains the foundation of Novo Nordisk's business.

According to accounts from the time, the scientists' fame rose rapidly. MacLeod and Banting were awarded the 1923 Nobel Prize for Medicine or Physiology, which they shared independently and respectively with Collip and Best. The Nobel Prize was a first for Canadian scientists, but the relationship between Banting and MacLeod was strained over differences about their contributions to the discovery.

Since then, the science, production, and delivery of insulin have been widely studied. It was the first protein for which the chemical structure and molecular weight were determined. The structure varies among species--human insulin differs by three amino acids from the bovine form and just one from the porcine form. The resultant similarity in conformation and activity is what, fortuitously, makes animal insulins so efficacious in humans.

FOR 60 YEARS, cattle and pigs were the sources of insulin. Although these products were highly effective, concerns arose about the growing diabetic population, long-term supply, and potential allergic reactions. Scientists had succeeded in synthesizing insulin in the 1960s, but synthesis was not seen as a viable commercial alternative. With the dawn of genetic engineering in the 1970s, new options emerged for making synthetic insulin that is chemically identical to human insulin.

Insulin consists of a 21-amino acid A chain and a 30-amino acid B chain, linked by two disulfide bonds. It can be produced either by generating the chains separately and chemically combining them or by creating a single-chain precursor, human proinsulin, and cleaving out a 35-amino acid connecting peptide. For manufacturing the drug, the proinsulin route is favored because it requires a single fermentation and isolation step.

In 1978, the fledgling biotech company Genentech and City of Hope National Medical Center produced human insulin in the laboratory using recombinant DNA (rDNA) technology. City of Hope scientists had synthesized the genes for the protein's two chains before inserting them into Escherichia coli. It was only the second time a human gene had been expressed in bacteria; Genentech workers had expressed the hormone somatostatin in 1977.

With its expertise in purifying and handling insulin and the desire to remain a leader in the field, Lilly immediately licensed recombinant insulin from Genentech and set about developing it. Fortunately, Lilly also had experience isolating antibiotics from fermentation processes. Because rDNA guidelines at the time allowed the expression of only inactive protein products, Lilly had to use the two-chain method. But, anticipating an eventual easing in restrictions, it simultaneously worked on the proinsulin route.

Clinical studies began in 1980. In 1982, Lilly's Humulin became the first genetically engineered drug approved by the Food & Drug Administration. U.S. approval came just one month after British regulatory authorities allowed its introduction in the U.K. The same year, Novo Nordisk began selling the first semisynthetic human insulin made by enzymatically converting porcine insulin; by 1987, Novo was using recombinant production methods as well.

Although the development of Humulin took just four years, it was a major undertaking for both the developers and regulators, breaking new ground. FDA had quickly expanded its expertise in genetic engineering to respond to new biotechnology products. Still, at a time when approvals often took two years, the agency's quick endorsement of recombinant insulin, a unique and somewhat controversial product, surprised outside observers.

"I had an excellent team that was highly motivated, and we wanted to get this done," explains Henry I. Miller, the FDA medical officer who played a key role in advancing insulin and later human growth hormone, the second rDNA drug, at the agency. Miller is now a research fellow at Hoover Institution.

"The quality of the submission from Lilly was unsurpassed," Miller adds, "and the evidence of safety and efficacy was unequivocal and copious." Like Lilly, FDA had decades of experience approving and certifying insulin products, and the agency took just a few months to approve the rDNA version.

A large part of the success in getting Humulin approved came from Lilly scientists being "constantly in discussion with FDA and European regulators," says Bruce H. Frank, a retired Lilly scientist. "We had to agree on what was going to be necessary to make everyone comfortable with the safety of this product." Frank led the team that advanced the proinsulin route, while Ronald E. Chance, also now retired from Lilly, headed one developing the A/B chain process.

BEING THE FIRST company to produce an rDNA product for human use involved many unknowns and steps, starting with expressing the gene and then characterizing and reproducibly making the drug, Frank and Chance explain. The program involved significant resources and hundreds of scientists across many disciplines and functions within Lilly.

"The thrill of it was that nobody had ever done it before," Frank says about producing and getting approval for the first recombinant drug. "The problem with it was that no one had ever done it before."

Tasks included defining the biological, chemical, and physical characteristics of the recombinant form, as well as addressing issues of immunogenicity, efficacy, and stability. Manufacturing and purity considerations were crucial when employing the entirely new rDNA vectors and production systems.

In addition, the chain combination process was not very efficient, Chance says, and had to be improved to reach the scale and level of cost efficiency needed. A combination of specialized chemistry and purification avoided contaminants from or the occurrence of a potential array of mislinked or misfolded materials.

The researchers established a battery of nearly 20 physicochemical and analytical tests, many of which were complex and not routinely practiced. X-ray crystallography, nuclear magnetic resonance, ultraviolet, and other spectroscopic methods were used to confirm the correct 3-D structure. High-performance liquid chromatography was emerging as a powerful tool and became key in monitoring and controlling the manufacturing process and testing the product for purity and identity.

The company also faced strong public opinion and an extremely cautious scientific and regulatory community, according to Irving S. Johnson, former Lilly vice president for research and front man on the project (Nat. Rev. Drug Discovery 2003, 2, 747). For example, rDNA fermentation volumes were limited to just 10 L, well below the 40,000 L needed for production. And there were strict rDNA hazard-containment requirements in the U.S., although Lilly also had a lab in Europe where the restrictions were not as stringent.

Lilly did get exemptions allowing stepwise increases in fermentation capacity; by mid-1981, the company was investing $80 million for new commercial plants in the U.S. and U.K. In the end, it had to make a product that was at least as good as, if not better than, existing insulins. Up through the 1970s, Lilly, Novo, and others had worked hard to increase the purity of animal-based insulins.

"The fact of the matter is that purified pork insulin was really quite an excellent product, and the human recombinant version did not represent a huge advance," Miller points out. It was "certainly nowhere near the advance that we saw when the animal insulins began to be used and saved millions of lives virtually overnight." Humulin initially was also more expensive.

Since 1986, Lilly has employed a recombinant proinsulin route. More recently, producers have used genetic engineering to create insulin analogs that differ in a few amino acids as a way to control their onset and duration of activity. Today, more than 20 insulin and analog products are available, including a small amount of animal-based materials. In 2004, insulin products generated about $3.4 billion in sales for Novo, $2.1 billion for Lilly, and $1.1 for Sanofi-Aventis.

RECOMBINANT INSULIN may not have been a major medical achievement, but it was a significant step in paving developmental, regulatory, commercial, and even political and societal paths for genetically engineered drugs. For the first time, it married a biotech firm's new technology with the resources of a major pharmaceutical partner. It was a proof of principle that sparked the interest of large drug companies, generating the funding and product revenues to fuel the emerging biotech industry.

The real challenge, however, remains in preventing or curing diabetes (C&EN, Oct. 25, 2004, page 59). In drug therapy, work continues on analogs and delivery methods, such as inhaled insulin. Many drugs are available to treat the more common type 2 diabetes, in which insulin production or the body's response to insulin needs to be increased. Insulin had been the only treatment for type 1 diabetes until March, when FDA approved Amylin Pharmaceuticals' Symlin to control blood sugar levels in combination with the polypeptide hormone.

Insulin

CAS Registry
◾ 9004-10-8

Common Product Names
◾ Porcine and bovine forms: Iletin, Lente
◾ Semisynthetic human insulin: Novolin
◾ Recombinant insulin and analogs: Apidra, Humulin, Humalog, Novolin, NovoLog, Levemir, Lantus, Velosulin

Introduced
1922 in Great Britain via the Medical Research Council, and on a larger scale in 1923 by Eli Lilly & Co.

Sales
$6.75 billion in 2004 (insulin and analogs)