Insulin Research & Innovation Legacies: The Best in Biotechnology
By Christopher J. Rutty, Ph.D
Lead Historian, Defining Moments Canada, “Insulin 100”.
On August 25, 1923, Frederick Banting and Charles Best were the guests of honour at the official opening ceremonies for the Canadian National Exhibition in Toronto. Their presence was associated with a special Insulin exhibit from the University of Toronto for the CNE’s “Science and International Year” theme. As described in news reports, the exhibit was quite detailed in illustrating “the process of manufacturing the curative fluid,” and included several of “the original utensils used by Dr. Banting and Mr. Best in making their world-renowned discovery.” By all accounts, it was a popular exhibit, with all 3,000 copies of the special bulletin about insulin, prepared by Best, distributed on the first day.
In his opening address, Banting stated that “Canada’s greatest monument is the Connaught Anti-toxin Laboratory of the University of Toronto.” “Canada,” he continued, “needs more such monuments” so that scientists interested in pursuing problems in medical research did not have to leave the country. Best was one of those scientists who could have gone abroad to pursue his research interests, but he was quite content to remain at Connaught in charge of insulin production, especially in the newly expanded production facility in the university’s former Y.M.C.A. building.
Best remained quiet during Banting’s opening address, and also at the special luncheon that preceded it, hosted by the CNE’s directors and attended by some of Toronto’s leading citizens. As the luncheon began, Banting called for Best, who had modestly hidden himself among the guests (which included his father as well as Banting’s) to take his place next to him. Countering all the public attention he had received, Banting emphasized that the work on insulin was “not the product of any one man.” He pointed out Best’s assistance from the beginning, explaining how he had been in charge of the large-scale production of insulin at Connaught from its commencement. In establishing, stabilizing and expanding Connaught’s insulin production, Best was greatly assisted by two chemists, David A. Scott and Peter J. Moloney. As the immediate practical problems of insulin production were resolved, Best, Scott and Moloney were soon able to apply their knowledge and experience with insulin towards developing new biotechnology tools to better manage, treat, or prevent other health problems. Ironically, for Best and Scott, building on their insulin biotechnology work would soon involve leaving Canada for a while.
Charles Best: Travelling Man
Best had been especially busy during August, 1923, starting with a move into Connaught’s refurbished building for insulin production. He had also been travelling around the U.S. to help inspect the facilities of several pharmaceutical firms applying for insulin production licenses from the University of Toronto Insulin Committee. Best, moreover, was working towards completing medical school while directing Connaught’s insulin production operations and serving as research associate for the Banting and Best Chair of Medical Research. In 1924, Best was also promoted to Assistant Director of Connaught Laboratories, and on September 4, 1924, married his long-time girlfriend, Margaret Mahon.
By the end of 1924, ideas were also developing for Best to pursue new research opportunities elsewhere. Two years earlier, Best had met Sir Henry Dale, of Britain’s Institute for Medical Research, during a visit to Toronto to explore establishing insulin production in the U.K. Dale had suggested to Best that after he completed his medical degree, he should consider leaving the “hot house atmosphere of the insulin research” to obtain fresh perspectives and broaden his experience in other areas of scientific research. In December, 1924, Connaught’s director, Dr. John FitzGerald, a member of the Rockefeller Foundation’s International Health Board, initiated a process to enable Best to be given a Rockefeller Fellowship to study abroad in London with Dale, and then in Copenhagen, with August Krogh, who had established insulin production there. However, Best had to first graduate from medical school, which he did in June 1925. As he graduated at the top of his class, Best also received additional awards and a grant from the Connaught Research Fund to support him for 18 moths. On July 9th, Dr. Charles Best and Margaret Best left for London. He was anxious to work on anything except insulin.
However, one of the first projects Best worked on in Dale’s lab involved the use of insulin in glucose metabolism studies of animals from which the liver had been removed. Best’s next work did not involve insulin and was focused on examination of the metabolism of histamine in lung tissue and his development of a method for the assay (analytical tests) of histamine in different types of tissues. Histamine is found in certain cells and is involved in local immune responses that cause many of the symptoms of allergies.
Charles Best & David Scott: Developing Heparin
In developing and applying the method he used for the assay of histamines, as well as for choline (an essential nutrient for humans and many animals), Best often ran into problems with blood clotting during the tests. He first encountered this reaction while doing research on turtles as an undergraduate student; blood often clotted during the experiments. A potential solution involved using a blood anticoagulant known as heparin, which was discovered in 1916 by Jay McLean and William Henry Howell at Johns Hopkins University. Heparin is an organic compound, found in various animal and human tissues, that regulates the process of blood coagulation. McLean was able extract heparin from dog liver, but did little further work with it, leaving Howell to continue the research. The McLean-Howell heparin could only be made on a small scale. It proved to be toxic and of minimal value. Best thought that heparin could perhaps be purified, not unlike insulin, but he could do little until he got back to Toronto.
Best returned to Toronto for Christmas, 1926, but it took a while before he was able to think much more about heparin. While he was away, construction of a new Rockefeller Foundation-funded public health building was proceeding. This new “Hygiene Building” would provide much larger research and production facilities for Connaught’s biological products. These included an even larger insulin production facility, along with the expanded public health teaching and research infrastructure for a new School of Hygiene. The building formally opened in June, 1927, and Best was appointed as head of the School’s new Department of Physiological Hygiene. The focus of this department was studying the impact on human physiology and health of ventilation, muscular exercise, ultra-violet light, and variations in atmospheric pressure, among other influences. Best’s research work was interrupted when he returned to England in March, 1928, to complete his Doctor of Science degree during the summer. Afterwards, he came back in Toronto to add another position to his list of responsibilities: head of the Department of Physiology, succeeding Professor J.J.R. Macleod, who had returned to Scotland.
By late October, 1928, Best could finally begin the heparin development project, which would focus on 1) purifying heparin and then producing it in large but affordable quantities; and 2) studying heparin’s effects in animals and then humans. To tackle the first part of the first goal, Best turned to his graduate student Arthur Charles, a specialist in organic chemistry. Best also planned to work with David Scott, who had assumed responsibility for Connaught’s insulin production while Best had been pursuing his postgraduate studies; Scott also designed the new insulin plant in the Hygiene building. However, soon after Best returned to Toronto, Scott left to pursue fresh research opportunities of his own in London, England, in 1928-1929, in the biochemical lab of Sir Charles Harington. Harrington was most noted for his work on the biochemistry of the thyroid gland.
Scott found himself working with insulin in Harington’s lab, focusing on insulin purification and verifying the earlier work of J.J. Abel, who had succeeded in crystallizing insulin in minute amounts. The outstanding problem for Scott was how to crystallize insulin on a large scale. By 1931, and working with Arthur Charles, Scott discovered the conditions that were essential for the preparation of crystals of insulin. The key was the presence of small amounts of metals, such as zinc, nickel, cobalt or cadmium, although zinc chloride proved best suited for insulin production.
Scott used his experience with insulin production to develop a method to extract heparin from not only beef liver, but also lung and intestine tissue on a larger scale. He and Charles took advantage of Connaught’s ongoing arrangement with Canadian meat processors to secure pancreas tissues for insulin production in order to gain access to large amounts of otherwise discarded beef organs for the heparin research. In 1933, Charles and Scott published their first papers on the heparin research, which described how the yield of extracted heparin could be doubled if the beef tissues, especially lung, were left to autolyze for 24 hours before extraction with ammonia. Autolysis is more commonly known as self-digestion and refers to destruction of cells through the work of their own enzymes. However, the potent smell emanating from the autolysis and ammoniacal extraction process, which expedited the spoiling or rotting of the tissues, was so offensive that production had to be moved from the Hygiene Building to the more open environment of Connaught’s farm site north of the city. By 1936, while studying heparin’s elusive and mysterious chemistry, Charles and Scott developed a method to purify and crystalize it into a standardized dry form that could be administered in a salt solution.
Following encouraging results seen in animal testing, heparin was next tested through clinical studies in a surgical setting led by Toronto surgeon, Dr. Gordon Murray. Murray’s initial experimental surgery with various animals demonstrated that heparin effectively cleared up internal blood clots, and it also seemed useful during and after dangerous vascular and cardiac operations, where blood otherwise coagulated quickly. The first human tests with purified heparin were conducted in April, 1937. The initial trials involved hundreds of complex surgical cases, in which heparin played an essential and often dramatic life-saving role. It quickly became clear that Connaught’s heparin was a safe, easily available and effective anticoagulant. Best’s heparin team had opened the door to such life-saving operations as organ transplants and open-heart surgery, as well as the artificial kidney, a key piece of medical technology pioneered by Murray, beginning in the mid-1930s, and with success in the late 1940s. The heparin production method developed by Scott and Charles was used until 1946, when Dr. Peter Moloney and Dr. Edith Taylor devised a new method that allowed much larger amounts of heparin to be obtained from beef tissue.
Peter Moloney: Defeating Diphtheria
Besides Peter Moloney’s critical role in establishing a new method for larger scale heparin production, he had already applied his unique skills as a chemist, biochemist and immunochemist to increasing the purity, potency and production scale of several other biological products at Connaught. Moloney was Connaught’s first research chemist and joined the Labs in 1919, with his initial work focusing on the purification of diphtheria antitoxin. Moloney had observed Connaught’s early insulin production development work and, in the summer of 1922, was asked by FitzGerald and Best to directly apply his expertise to overcoming challenges in insulin purification and production levels. By the fall, Moloney’s initial method, based on the use of benzoic acid adsorption and precipitation, enabled the production of some 250,000 units of insulin, with “very satisfactory results,” according to Best. Moloney’s insulin research caught the attention of the press. A Toronto Star article in early January, 1924, heralded the discovery of a “New System for Purifying Insulin.” This phase of Moloney’s research was focused on a new charcoal purification method that built upon his earlier work with insulin using benzoic acid.
A few months later, Moloney received a telegram from FitzGerald in France asking him to shift his focus to help expedite the development, production and clinical evaluation of another new biotechnology discovery. During the summer of 1924, FitzGerald travelled extensively in Europe to investigate a variety of developments on behalf of Connaught, including those related to insulin and recent research with diphtheria prevention. Although diphtheria antitoxin was freely available in Canada to those stricken by the disease, “the strangler,” as diphtheria was known, as it led to the development of a blockage in the throat that impaired breathing, remained one of the leading public health threats to children under 14. The antitoxin minimized deaths, but it did not stop diphtheria’s spread. FitzGerald had heard that Dr. Gaston Ramon of the Pasteur Institute in Paris had recently discovered that treating diphtheria toxin with formaldehyde and heat rendered the toxin non-toxic. When injected, this “toxoid” could safely stimulate active immunity in humans just like a vaccine. Ramon had only tested the effectiveness of the toxoid in his lab on a small scale. FitzGerald’s visit with Ramon was opportune since Connaught was ideally prepared to facilitate further development and testing of diphtheria toxoid in a much more expeditious way than Ramon could at the time.
FitzGerald described Ramon’s methods to Moloney and asked him to drop everything and immediately begin preparing and improving the toxoid. After encouraging initial studies of the toxoid given to a small group of Connaught staff members that showed strong antigenic responses, and similar results among a larger group of children, field trials were launched in several cities in September, 1925. The trials started in the Essex Border Municipalities (which encompassed Ford, Walkerville, Windsor, and Sandwich), where some 9,000 pre-school and school-age children each received two doses of toxoid. A similar trial took place in Saskatchewan during 1925–26, and by February, 1927, a total of 120,000 individuals in nine provinces had received diphtheria toxoid. During the initial use of the substance, some allergic reactions were reported, prompting Moloney to develop a simple intradermal “reaction test” using a diluted toxoid to detect potential reactors — usually older children or adults already immune to diphtheria. This test came to be known as the “Moloney Reaction Test” and was universally applied.
One of the most dramatic and widely recognized early uses of diphtheria toxoid occurred in Hamilton, Ont. Thanks to the dedicated leadership of the city’s Medical Officer of Health, Dr. James Roberts, the results in Hamilton were particularly significant, with the toxoid sharply reducing both diphtheria incidence and the number of related deaths. The rapid industrial and urban growth of Hamilton left the city quite vulnerable to diphtheria and thus a useful place to test the effectiveness of the toxoid. In 1922, there were 742 cases and 32 deaths due to diphtheria in Hamilton. By 1925–27, once the toxoid was in use, the number of cases fell to 368, with 18 deaths; between November 1, 1926, and September 30, 1927, there were only 10 diphtheria cases and one death. “Hamilton Slays a Dragon” was the message of a dramatic and popular public health exhibit that showed there were only five cases and no deaths in 1931. By this time, the results of the most sophisticated diphtheria toxoid field trial were published. It took place in Toronto between December, 1926, and June, 1929, and involved approximately 36,000 children. The trial conclusively demonstrated that the toxoid reduced diphtheria incidence by at least 90% among those given three doses. This remarkable rate of effectiveness was maintained in Toronto and across Canada into the 1930s, when diphtheria toxoid was widely used. There were, however, examples of when toxoid use lapsed, outbreaks persisted. The Canadian work preceded efforts in the U.S., the U.K. and elsewhere to utilize the toxoid to bring diphtheria under control. Diphtheria toxoid was the first modern vaccine and ushered in a new era of effective prevention of formerly devastating pediatric infectious diseases.
Charles Best: World War II and Blood Serum Biotechnology
Although Canada was in the grips of the Great Depression during the 1930s, Connaught Laboratories was growing and attracting international attention because of its significant innovations in biotechnology, especially successes with diphtheria toxoid, heparin, insulin crystallization, and the development of Protamine Zinc Insulin. While Best and Scott sought research and education opportunities outside Canada in the late 1920s, there were now many tempting offers of academic positions coming over the transom. Banting had received and declined many such offers. During the early 1930s, international universities courted Best, based not only on his insulin work, but also his work on heparin and histamine.
One tempting offer came in March, 1933, from Scotland’s University of Edinburgh, which invited Best to assume the prestigious position of chair of physiology. The current holder, Sir Edward Sharpey-Shäfer, one of the founders of endocrinology, had been chair since 1899. Macleod had become the chair of physiology at the University of Aberdeen in 1928, but it is unclear if he played any role in the Edinburgh offer to Best. The department did have some limitations, and it needed fresh leadership. While Best was honoured to be asked, he declined. “You will perhaps understand that I could not be happy teaching physiology unless facilities for investigations of a variety of physiological problems were available,” he wrote. “These facilities are available to a rather extraordinary degree in Toronto.” There was no reason for Best to leave the University of Toronto. Indeed, Toronto was now one of the best places to be in the world for physiology research and for biotechnology research, innovation and production.
The next phase of biotechnological innovation to draw on the insulin legacy was driven by the medical and health demands generated by World War II, and by Best’s own leadership. When the war began, Best realized that the Canadian military would need a larger, safer and better-preserved supply of blood at the front than had been the case during World War I. While blood transfusions saved the lives of many soldiers during WWI, there were better alternatives to using whole blood in the treatment of wounded soldiers by the 1930s. As head of the Department of Physiological Hygiene at the School of Hygiene, and an associate director at Connaught, Best led an initiative to prepare dried blood serum in collaboration with the National Research Council, the Department of National Defense, and the Canadian Red Cross. Beginning modestly in September, 1939, with the recruitment of blood donors among School of Hygiene and Connaught staff and university students, the effort grew quickly. After the blood was drawn, it was typed and allowed to clot. The serum was then separated from the clotted blood and the various samples combined into groups based on blood type. After passing sterility tests, the freeze-drying process was conducted in a small room in the School of Hygiene.
By October, 1940, a larger coordinated effort for blood processing was required. In January, 1941, in partnership with the federal government through the Department of Pensions and National Health, Connaught assumed responsibility for processing blood collected through a national Red Cross donor program and then preparing the freeze-dried serum. Larger facilities were soon required, forcing some of the Labs’ peacetime operations to stop or be crowded together to make sufficient room. With more than 11,000 blood donations received monthly by March, 1942, even more processing space was needed. By October, this increased to over 57,000 donations received monthly and further expansion was urgently needed. In August, 1943, the large vacant building on Spadina Circle at the west end of the university campus was acquired by the Labs and quickly renovated to accommodate expanded dried blood serum production. By the end of the war, Connaught had received over 2.25 million blood donations, which made it possible to prepare about 500,000 bottles of dried serum. Most of the dried blood serum was sent to England, where it was packed with bottles of distilled water necessary for dissolving the dried serum prior to its use to treat wounded soldiers.
While Connaught’s extensive contributions to the Canadian and Allied war effort continued and expanded in several areas — most notably with a major penicillin production effort and the development and production of several more vaccines — Best’s direct involvement ended in February, 1941. The change was prompted by Banting’s tragic death in a plane crash. Best succeeded him as director of the Banting and Best Department of Medical Research. At the same time, Best resigned as an associate director at Connaught, although he remained a consultant to the Labs. Best focused more on physiological research in support of the war effort.
Banting’s death also followed soon after the sudden death of FitzGerald in June 1940, due to a mental collapse and suicide. FitzGerald’s personal tribulations were not publicly acknowledged at the time. These two deaths, in the midst of World War II, led to a significant transition in the nature and scope of biotechnology research and innovation at Connaught, which had become less scientifically and personally driven by insulin. The war years, and then the early post-war period, would bring a variety of new public health challenges and generate new opportunities at Connaught Medical Research Laboratories, as it became known in 1946, for biotechnology innovation to better manage and ultimately prevent illness.