Making Insulin

Figuring out how to make pure and potent insulin consistently, safely and affordably on a large scale was a unique biotechnology challenge in the early 1920s. The discovery that insulin effectively and safely controlled the effects of Type I diabetes in Leonard Thompson using a pancreatic extract prepared in Dr. J. Bertram Collip’s small lab was really only the first chapter of this drug development story. Leonard required injections of the extract indefinitely, as would every other Type I diabetic. But if a consistently pure, potent and ultimately affordable supply could not be prepared on a large scale, the discovery of insulin would have very limited value.

The January 25, 1922, agreement between the University of Toronto’s Connaught Antitoxin Laboratories and the discovery team of Banting, Best, Collip and Macleod, was especially exciting for Collip. That day, shortly after signing this critical agreement, Collip wrote to the president of the University of Alberta, Dr. H.M. Tory. He described how he had “finally unearthed” a method to isolate the internal secretion on January 19th. After trying the extract on one clinical case (Leonard Thompson), the results were so encouraging that, Collip wrote, “today $5,000 has been placed at our disposal to secure apparatus, four assistants, etc. to rush the work for the next four months in the hope we may establish a block of clinical evidence which will prove either the value or the worthlessness of this substance in treating diabetes in the human.” 

Charles Best was also quite excited. Even before the first human trials of the extract took place, Best secured an appointment at Connaught, as of January 1, 1922, as director of production of the pancreatic extract, for which he would earn $2,000 per year. The appointment was also designed to support Best’s work towards his MA thesis, which was focused on respiratory quotient tests with Dr. John Hepburn. Its title was, “The role of pancreatic extracts in the utilization of carbohydrates by diabetic animals.” During January and February, however, Best’s time, as he later reported, was mostly spent “superintending the collection and initial concentration of material which was then handed over to Dr. Collip for completion.”

The initial extract production work at Connaught took place in a room in the basement of the south wing of the Medical Building. “We have our new Lab, fairly well completed,” Best wrote in a February 12th letter to his father, Dr. H.H. Best. “We began work yesterday p.m. but one of the stills blew up at the first trial so we are held up again temporarily.” It was a disconcerting start, amidst news that Dr. Allen in New York and Dr. L.F. Barker at Johns Hopkins University were both working on pancreatic extracts. “We will have to hurry up to keep ahead of them,” Best said to his father. “I hope we get our stills working well soon.” However, as he added, “We don’t work thru the night now, but the days are from 9:00 a.m. to 11 p.m. quite often.” 

Photograph of an insulin still, c. 1931. University of Toronto, The Discovery and Early Development of Insulin.

The initial approach to pancreatic extract production was to apply Collip’s laboratory purification method to larger volumes. Starting with freshly minced beef pancreas, the extract was prepared through a series of steps that involved adding solutions of alcohol, followed by filtration, concentration, and centrifugation (spinning at a high speed to separate components of the solution). Collip had discovered that the active principle (i.e. the internal secretion) was found entirely in the alcohol, which constituted the upper-most layer in the centrifuge tube. This alcoholic solution was removed from the tube and added to several volumes of 95% or absolute alcohol, but the active principle was practically insoluble in alcohol of this concentration. The precipitate thus obtained was removed by filtration, then dissolved in distilled water and concentrated to remove any traces of alcohol and to secure the desired concentration of the active ingredient. 

Under Collip’s direction, this method, applied to small-scale production, worked out satisfactorily for the first few weeks. However, in mid-to-late February, when Collip attempted larger scale experiments, they were unsuccessful. Most frustratingly, it soon proved impossible for Collip to consistently duplicate the earlier results on any scale. The result was little to no extract available to the first diabetic patients, including Leonard Thompson, who was soon sent home to return to a strict dietary treatment regime. Indeed, despite frantic efforts led by Collip, a pancreatic extract famine persisted for some two months. 

Banting suspected, incorrectly, this situation arose out of Collip’s desire to keep his process a secret, thus his failure to keep careful records. For Collip, however, the production crisis was very trying: he’d gone from the joy of discovery in mid-January to dark nights of despair a month later. What’s more, the sudden halt in extract production expedited a total breakdown in his relationship with Banting. He spent long hours in the lab, frantically trying to reconstruct the process for making the extract. Adding to his misery, his family endured an attack of influenza. Collip certainly was not the first biochemist to experience this type of failure with extractions of an unknown substance using crude and unreliable equipment. Still, from Banting’s perspective, he knew Collip’s record-keeping habits were less precise than his and thus felt less sympathetic to his plight.

As the extract famine persisted through March, Banting struggled with his own sense of despair, frustration and isolation, not only from Collip, but also Macleod and Best, who were all focused on resolving the production crisis. Banting felt he had little to contribute, nor could he treat any diabetic patients — he still had no access to Toronto General Hospital’s diabetic clinic — with the meagre amounts of extract that could be produced, much of it of low potency. Such extract, in fact, would soon contribute to the death of a young girl, who was a friend of Best’s. She had fallen into a coma and was revived, but ultimately died when the extract ran out.

However, Best was able to re-invigorate Banting. On March 31st, he visited Banting’s boarding-house room and found him depressed and half-drunk. Banting had spent most evenings during March drinking himself to sleep to get his mind off his troubles. Frustrated and impatient with Banting’s mood, Best proceeded to give him a “bawling-out.” He also reminded Banting of the situation in the lab and the opportunity it presented for them to work together again trying to make an effective extract. But Banting wasn’t interested. He didn’t seem to care and planned to finish up his teaching, get out of Toronto and find some decent people to live with. 

As Banting later recalled, “Then Best said probably the only thing that would have changed my attitude.”

 “What will happen to me?’” Best asked. 

Banting said, “Your friend Macleod will look after you.” 

 “If you get out,” Best replied, “I get out.” 

After a few moments of silence, as Banting later recounted, “I thought of all the joy of the early experiments which we had known together. Here was loyalty. I emptied my glass. That is the last drink which I will ever take until insulin circulates in diabetic veins. Shake on it, Charley. We start in tomorrow morning at nine o’clock where we left off. Best was pleased. We sat down and as we had done hundreds of times, planned experiments.”

According to Banting, for the next few months “it was back to the old grind.” He and Best worked day and night in Connaught’s pancreatic extraction room. “Time, meals, sleep, all were of secondary consideration. We had to get insulin into a form that was refined enough for continued clinical use. The atmosphere was different. We were happy. To Best must be given the greatest amount of credit for this phase of the development. It was he more than anyone else who bridged the gap between the test tube and the beaker and later large-scale production.”

David A. Scott. Sanofi Pasteur Canada Archives, University of Toronto, Connaught Laboratories & The Making of Insulin, By Dr. Christopher Rutty.

A key figure who joined the Connaught Labs team that were focused on restoring pancreatic extract production was chemist, David A. Scott. “Scotty,” as he was affectionately known, was a quiet, modest and friendly man, happiest working at his lab bench and directing other scientists. He demonstrated unusual abilities in math and science at an early age and, starting in 1914, furthered this passion at the University of Toronto, where he specialized in chemistry and mineralogy. The First World War interrupted his studies in 1917, but he applied his skills to the production of acetone in an explosives plant. After graduating in 1920, Scott worked as a research chemist in a paper mill and then went to Queen’s University for further research related to the paper industry. In the spring of 1922, Scott received a letter from Connaught’s director, Dr. J.G. FitzGerald, inviting him to join the Labs. Scott’s skills and experience, especially with acetone, seemed ideal for helping figure out how to produce insulin on a larger scale and he worked closely with Best. 

During April and early May, Banting, Best, Macleod and Collip worked long hours together, and more than ever as a team, along with considerable help from Scott, focused on restoring a supply of the extract. It soon became apparent that the crux of the problem were variables with heating and water pressure that the extract experienced during the process of evaporating off the alcohol. There were also significant variations in the pressure of the water that was supplied to the crude vacuum pumps. As a result, increased heat seemed to neutralize some of the proteins in the solution, resulting in a less potent and effective extract. The best strategy to restoring extract production, as Macleod suggested, was to go back to using a warm air current for alcohol evaporation rather than a vacuum pump, and to follow Scott’s suggestion for using acetone as a solution rather than alcohol during most of the extraction process.

The revised method combined elements of Banting and Best’s original method, with Collip’s, but began with minced beef pancreas mixed with an equal volume of 95% acetone, to which a small amount of 0.1% formic or acetic acid was added. The pancreas acetone mixture was allowed to stand for several hours and was then filtered. The resultant filtrate was placed in a set of 500 c.c. enamel lined trays, which were placed in a tunnel through which a current of hot air was drawn. The 500 cc. of filtrate was evaporated down to about 50 c.c. over a period of one hour, the temperature of the liquid never exceeding 35° C. The residue was removed from each of the trays, chilled to a temperature of 0° C, and then filtered. This step removed any fatty material. The resultant filtrate was treated with 95% ethyl alcohol until a concentration of 80% alcohol was secured. This mixture was then filtered, and the filtrate added to at least 5 volumes of 95% alcohol (a 5% alcohol in water solution). The precipitate was allowed to settle to the bottom of the alcoholic solution over a period of 24 to 48 hours. The alcohol was then decanted, the precipitate dissolved in distilled water and any remaining traces of alcohol removed by vacuum distillation. This precipitate was the internal secretion. Suspended in the distilled water solution, it was ready for testing and use.

Although considerable progress was made in restoring production, led primarily by Best, by May 10th, the sense of teamwork within the group had broken down, at least as far as Collip was concerned. As Best outlined that day in a letter to his father, “I will have a lot to tell you when I see you about our work. There has been a lot of trouble, quarrels, etc., but we are getting on. Collip has not played fair. He was in charge of making the extract. Banting and I were going along with the clinical and physiological, respectively. He [Collip] was kicked out yesterday and I am in charge of making the dope. It means a lot of work, long hours, etc., but I hope to get it standardized before July.” 

It is unclear what happened that prompted the team to oust Collip. He knew that the term of his sabbatical at U of T was ending at the end of May and he planned to return to Edmonton and a position as chair of physiology at the University of Alberta. Tensions with Banting may well have flared up again and Collip was more comfortable working on his own in a laboratory.

Beginning on May 10th, Best was placed in charge of all aspects of the development, purification and production of insulin at Connaught. Best had the full support of FitzGerald and Connaught’s assistant director, Dr. Robert D. Defries. Besides Scott, Best also had the assistance of technicians Jessie Ridout and Arthur Wall, and the additional scientific expertise of a pair of chemists, Donald Fraser and Peter J. Moloney. At about this time the group began referring to the extract as “insulin,” which had been suggested by Macleod based on the Latin root word for “island.”

Moloney was Connaught’s first research chemist, originally recruited by FitzGerald in 1919 to focus on improving the purification of diphtheria antitoxin. Before pH meters were available (the first was constructed in 1934), Moloney developed a quick-acting hydrogen electrode (known as the “Moloney Electrode”) that was designed to more accurately and rapidly determine the acidity of the bacterial culture broths used in antitoxin and vaccine production. The Moloney Electrode also proved useful for measuring pH levels (i.e., the measures of acidity and/or alkalinity of a substance) during insulin production and purification. 

Moloney wasn’t involved with Connaught’s initial insulin production work, but he was a witness to it. His first direct involvement came about accidently, when he heard an explosion coming from the lab that was being used to make the acetone-based concentration of insulin. A bottle of picric acid had fallen off a shelf in the lab. As he later recalled, “there was a most terrific explosion.” The blast could well have been much larger and “blown the room to pieces” and set off a major fire. But fortunately for Moloney and the lab, only a small dry piece of picric acid exploded. It had been stuck between a cork-stoppered glass bottle and a bottle containing about half a kilo of the dry acid. 

Christie Street Hospital, c. 1940. Toronto Star Archives.

Events moved quickly on several fronts during the rest of May as Connaught’s production of insulin was restored and steadily increased. Banting could finally administer insulin to diabetic patients at either his new private practice, established at 160 Bloor Street West in Toronto, as well as through an appointment as head of a new diabetes clinic established at the Christie Street Military Hospital in Toronto (it was overseen by the Canadian government’s Department of Soldiers Civil Re-Establishment). Dr. Joe Gilchrist, a friend of Banting’s who was a diabetic and had received an ineffective version of the extract in December 1921, served as both a physician at the clinic and a patient; indeed, he tested each new batch on himself after they were tried on rabbits. 

An arrangement led by Macleod, Banting and Duncan Graham, of the Toronto General Hospital, was then made with Connaught for the distribution of insulin for clinical use. Banting would secure a third for use in his private practice. Another third went to the Christie Street clinic, and the remainder split between Toronto General Hospital and the Hospital for Sick Children. Macleod, especially following his well-received presentation on May 3rd about the Toronto work before a large conference in Washington, D.C., served as the intermediary between Banting and U.S. and Canadian diabetic specialists making inquiries about the extract. One such request, from Rochester, N.Y., led to the first use of insulin in the U.S. Jim Havens, the 22-year-old son of the vice-president of Eastman Kodak, was in desperate condition. An appeal from his physician to Banting prompted Connaught to ship a package of insulin on May 21st. Banting followed up on May 26th, personally delivering more insulin and consulting with Haven’s doctor.

Connaught’s insulin output, however, remained limited and subject to unforeseen technical complications while Best and his team struggled to develop methods to increase production. They clearly needed help to expand the clinical use of insulin, especially in the U.S., and to prepare for full commercial production to ultimately meet global demand. 

Macleod’s May 3rd presentation in Washington was followed by a meeting with Dr. George H.A. Clowes, director of research at Eli Lilly & Co. of Indianapolis. He had been closely following the developments in Toronto, especially after Banting’s presentation in New Haven on December 30, 1921. Impressed with what he heard, Clowes left Banting and Macleod with an open offer of Eli Lilly’s collaboration with the University of Toronto to help produce the pancreatic extract. Macleod was appreciative of the offer, but at the time hesitant to work with a pharmaceutical company. However, the situation was quite different in May. As Clowes noted in a May 11th letter to Macleod, his paper in Washington had created an extraordinary amount of interest in the U.S. and Lilly was anxious to duplicate Connaught’s results. Clowes was invited to Toronto on May 22nd, along with several other Lilly personnel, including a chemist, a patent attorney and the vice-president of the company, Mr. Eli Lilly. Over the next few days of meetings, the University of Toronto and its Connaught Labs agreed to a unique cross-border partnership with Eli Lilly, facilitating the next critical steps in the development of insulin.

Insulin finishing line at Eli Lily, c. 1923. Image courtesy of Breakthrough: Elizabeth Hughes, the Discovery of Insulin, and the Making of a Medical Miracle by Thea Cooper and Arthur Ainsberg (2010).