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Orville May, Director of the NRRL, agreed to have the Laboratory undertake a vigorous program to increase penicillin yields under the direction of

Robert

Corn-steep liquor was a by-product of the corn wetmilling process, and the NRRL, in an attempt to find a use for it, tried it in essentially all of its fermentation work.

Later, the Peoria laboratory increased the yield of penicillin still further by the addition of penicillin precursors, such as phenylacetic acid, to the fermentation medium.

It was recognized that the Oxford group's method of growing the mold on the surface of a nutrient medium was inefficient, and that growth in submerged culture would be a superior process. In submerged culture fermentation, the mold is grown in large tanks in a constantly agitated and aerated mixture, rather than just on the surface of the medium. Florey's Penicillium culture, however, produced only traces of penicillin when grown in submerged culture. Under the direction of Kenneth Raper, staff at the NRRL screened various Penicillium strains and found one that produced acceptable yields of penicillin in submerged culture.

Soon a global search was underway for better penicillin producing strains, with soil samples being sent to the NRRL from around the world.

Ironically, the most productive strain came from a moldy cantaloupe from a Peoria fruit market.

A more productive mutant of the so-called cantaloupe strain was produced with the use of X-rays at the Carnegie Institution. When this strain was exposed to ultraviolet radiation at the University of Wisconsin, its productivity was increased still further.

While Norman Heatley remained in Peoria helping the NRRL staff to get the penicillin work started, Howard Florey visited various pharmaceutical companies to try to interest them in the drug. Although Florey was disappointed in the immediate results of his trip, three of the companies (Merck, Squibb and Lilly) had actually conducted some penicillin research before Florey's arrival and Pfizer seemed on the verge of investigating the drug as well.

At this time, however, the promise of penicillin was still based on only limited clinical trials.

Florey next visited his old friend Alfred Newton Richards, then vice president for medical affairs at the University of Pennsylvania. More importantly, Richards was chair of the Committee on Medical Research (CMR) of the Office of Scientific Research and Development (OSRD). The OSRD had been created in June, 1941, to assure that adequate attention was given to research on scientific and medical problems relating to national defense.

Richards had great respect for Florey and trusted his judgment about the potential value of penicillin. He approached the four

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Department of Agriculture, participants included research directors Randolph T. Harrop of the Squibb Institute for Medical Research; Jasper Kane of Pfizer; and Y. The next CMR penicillin conference, held in New York in December, ten days after Pearl Harbor and U.S. entry into the Second World War, was more decisive.

At this meeting, which was attended by the heads of Merck, Squibb, Pfizer and Lederle, as well as the company research directors, Robert Coghill's report on the success at the NRRL with corn steep liquor was encouraging to the industry leaders present. Merck, who had been pessimistic about the possibility of producing adequate quantities of penicillin given the constraints of available fermentation techniques and yields,".

immediately spoke up, saying that if these results could be confirmed in their laboratories, it was possible to produce the kilo of material for Florey, and industry would do it!". It was agreed that although the companies would pursue their research activities independently, they would keep the CMR informed of developments, and the Committee could make the information more widely available (with the permission of the company involved) if that were deemed in the public interest. Although there was some concern that investments in fermentation processes might be wasted if a commercially-viable synthesis of penicillin were developed, other companies also began to show an interest in the drug. Some firms worked out collaborative agreements of their own (e.g., Merck and Squibb in February 1942, joined by Pfizer in September). Merck's pilot plant continued to produce several hundred liters of penicillin culture per week using both flasks and tray, and in December, Heatley joined the Merck research staff for several months, where he introduced the Oxford cup plate method of penicillin assay, which soon became a standard method industry-wide. By March 1942 enough penicillin had been produced under OSRD auspices to treat the first patient (Mrs.

Ann Miller, in New Haven, Connecticut); a further ten cases were treated by June 1942, all with penicillin supplied by Merck & Co., Inc.

Pharmaceutical and chemical companies played an especially important role in solving the problems inherent in scaling up submerged fermentation from a pilot plant to a manufacturing scale. As the scale of production increased, the scientists at Merck, Pfizer, Squibb and other companies faced new engineering challenges.

Smith captured the complexity and uncertainty facing these companies during the scale-up process: "The mold is as temperamental as an opera singer, the yields are low, the isolation is difficult, the extraction is murder, the purification invites disaster, and the assay is unsatisfactory." Because penicillin needs air to grow, aerating the fermentation mixture in deep tanks presented a problem.

When corn steep liquor was used as the culture medium, bubbling sterile air through the mixture caused severe foaming.

Squibb solved this problem by introducing glyceryl monoricinolate as an anti-foaming agent.

Submerged fermentation also required the design of new cooling systems for the vats and new mixing technology to stir the penicillin mash efficiently.

Lilly was particularly successful in making the mold synthesize new types of penicillin by feeding precursors of different structure. Once the fermentation was complete, recovery was also difficult; as much as two-thirds of the penicillin present could be lost during purification because of its instability and heat sensitivity.

Methods of freeze-drying under vacuum eventually gave the best results in purifying the penicillin to a stable, sterile, and usable final form. The steps of fermentation, recovery and purification and packaging quickly yielded to the cooperative efforts of the chemical scientists and engineers working on pilot production of penicillin.

On March 1, 1944, Pfizer opened the first commercial plant for large-scale production of penicillin by submerged culture in Brooklyn, New York.

Meanwhile, clinical studies in the military and civilian sectors were confirming the therapeutic promise of penicillin.

The drug was shown to be effective in the treatment of a wide variety of infections, including streptococcal, staphylococcal and gonococcal infections.

The United States Army established the value of penicillin in the treatment of surgical and wound infections.

Clinical studies also demonstrated its effectiveness against syphilis, and by 1944, it was the primary treatment for this disease in the armed forces of Britain and the United States.

The increasingly obvious value of penicillin in the war effort led the War Production Board (WPB) in 1943 to take responsibility for increased production of the drug. The WPB investigated more than 175 companies before selecting 21 to participate in a penicillin program under the direction of Albert Elder; in addition to Lederle, Merck, Pfizer and Squibb, Abbott Laboratories (which had also been among the major producers of clinical supplies of penicillin to mid-1943) was one of the first companies to begin large-scale production.

These firms received top priority on construction materials and other supplies necessary to meet the production goals. The WPB controlled the disposition of all of the penicillin produced. One of the major goals was to have an adequate supply of the drug on hand for the proposed D-Day invasion of Europe. Feelings of wartime patriotism greatly stimulated work on penicillin in the United Kingdom and the United States.

For example, Albert Elder wrote to manufacturers in 1943: "You are urged to impress upon every worker in your plant that penicillin produced today will be saving the life of someone in a few days or curing the disease of someone now incapacitated. Create an enthusiasm for the job down to the lowest worker in your plant." As publicity concerning this new "miracle drug" began to reach the public, the demand for penicillin increased. But supplies at first were limited, and priority was given to military use. Chester Keefer of Boston, Chairman of the National Research Council's Committee on Chemotherapy, had the unenviable task of rationing supplies of the drug for civilian use.

Keefer had to restrict the use of the drug to cases where other methods of treatment had failed.

Part of his job was also to collect detailed clinical information about the use of the drug so that a fuller understanding of its potential and limitations could be developed.

Not surprisingly, Keefer was besieged with pleas for penicillin. A newspaper account in the New York Herald Tribune for October 17, 1943, stated: "Many laymen - husbands, wives, parents, brothers, sisters, friends - beg Dr.

In every case the petitioner is told to arrange that a full dossier on the patient's condition be sent by the doctor in charge.

When this is received, the decision is made on a medical, not an emotional basis." Fortunately, penicillin production began to increase dramatically by early 1944. Production of the drug in the United States jumped from 21 billion units in 1943, to 1,663 billion units in 1944, to more than 6.8 trillion units in 1945, and manufacturing techniques had changed in scale and sophistication from one-liter flasks with less than 1% yield to 10,000-gallon tanks at 80-90% yield.

The American government was eventually able to remove all restrictions on its availability, and as of March 15, 1945, penicillin was distributed through the usual channels and was available to the consumer in his or her corner pharmacy. By 1949, the annual production of penicillin in the United States was 133,229 billion units, and the price had dropped from twenty dollars per 100,000 units in 1943 to less than ten cents. Most British companies moved over to the deep tank fermentation production of penicillin, pioneered in the United States, after the end of the war to meet civilian needs. In the United Kingdom, penicillin first went on sale to the general public, as a prescription only drug, on June 1, 1946.

In Britain, Chain and Abraham continued to work on the structure of the penicillin molecule, aided by the X-ray crystallographic work of Dorothy Hodgkin, also at Oxford.