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Manuel Varela: Ernst Chain, Howard Florey, and Norman Heatley – On Purifying Penicillin

Jul 6, 2017 by

An Interview with Manuel Varela:  Ernst Chain, Howard Florey, and Norman Heatley – On Purifying Penicillin

Michael F. Shaughnessy –

1) Professor Varela, today we are going to discuss three great scientists, who, apparently working together, “purified” penicillin.  First of all, what do we know about the backgrounds of these three individuals?

You have certainly chosen an excellent topic for this chapter in the series. The fact that the important antibiotic penicillin was discovered by the amazing scientist Sir Alexander Fleming is virtually universally known. Not many people, however, are aware of the amazing biochemists and of their tremendous efforts they took in purifying the penicillin. The individuals who were key to the scientific achievement in which they successfully accomplished the production and isolation of penicillin were Drs. Howard Florey, Ernst Chain, and Norman Heatley. Their efforts represent a critical advancement in the effort towards an efficacious treatment against several serious bacterial infections. Their work resulted in a significant historical first in chemotherapeutic treatment of infectious disease.

Let’s consider first the background of their principal leader of the penicillin isolation project, Dr. Howard Walter Florey. He was born in Malvern, near the suburb of Adelaide and part of the city of Unley, in southern Australia, to parents Joseph and Bertha Mary Wadham Florey, on the 24th of September, just prior to the entry into the 20th century, in 1898. Joseph was a businessman who dealt in the production of boots.

Bertha was the second wife of Joseph after his first wife, Charlotte Ames, died of tuberculosis, frequently called the Great Consumption during the time. Together, Charlotte and Joseph had two daughters, Charlotte and Anne. Bertha and Joseph had three offspring, two older sisters, Hilda and Viletta, plus the youngest, Howard.

As a child, Howard Florey attended Kyre Preparatory College, a private school, in Adelaide, where he was given the nickname “Flos,” and in 1911 he moved to St. Peter’s Collegiate School, also in Adelaide. After graduation in 1916, Florey enrolled in medical school, housed at Adelaide University. As a medical student, Florey’s father Joseph died in September of 1918, leaving the family in dire financial straits. Nonetheless, Florey managed to graduate with his B.A. degree in surgery by December of 1921, earning both excellent grades and a Rhodes scholarship to Oxford, England, where he had moved to in January of 1922.

While at Oxford, Florey studied physiology, which is the study of the function of living systems. At Oxford, Florey studied under the noted neuroscientist Charles Sherrington and earned an M.A. in physiology, in 1924.  Florey received another scholarship to attend Cambridge, England, where in 1924 he entered the laboratory of immunologist and professor of pathology, Henry Dean.

Flourishing in Dean’s laboratory at Cambridge where he studied the pathology of blood circulation in the brain and mucous secretion from the colon, Florey published four papers in prominent scientific journals. In 1926, Florey became a research fellow at London Hospital, and in October of that year, he got married to Mary Ethel Hayter Reed. In 1927, while taking a lectureship post in pathology, Florey earned his Ph.D. degree from Cambridge, where he studied the circulatory flow of lymph and blood.

Dr. Florey spent brief intervals at Oxford and Cambridge before moving to Sheffield to take a professorship in pathology in 1932, and later, in May of 1935, Dr. Florey finally became a professor of pathology at Oxford, where he was to do his penicillin work.

The second member of the penicillin production trio, Ernst Boris Chain, was born on the 19th of June in 1906 in Berlin, Germany. Chain’s mother, Margarete Eisner, was German. His father was Michael Chain, a Russian industrial chemist who worked in a manufacturing plant for chemicals, in Berlin. Chain attended gymnasium called Luisen in Berlin.  The child Chain often visited his father’s laboratory, becoming interested in chemistry, and in 1919 when young Chain was only 13 years old his father passed away, leaving the immediate Chain family in terribly difficult financial circumstances—a situation similar to that seen with Florey.

However, Chain was also able to go to university as an undergraduate, at the Friedrich Wilhelm University, Berlin, Germany, where he learned five languages and was a talented pianist, even seriously considering becoming a concert pianist for a career. Chain took his undergraduate degree in chemistry and physiology from Wilhelm in 1930.

With his undergraduate degree in hand, Chain worked briefly in the physical chemistry department at the Kaiser Wilhelm Institute and at the Charité Hospital, in Berlin, where he is reported to have earned his first Ph.D. degree, in 1930. Being from a Jewish family, Chain left Germany in April of 1933, during the rise of Hitler’s Nazi Germany.

Arriving practically financially destitute in London, England, Chain was barely able to secure tenable work at University College Hospital Medical School under the direction of the J.B.S. Haldane, a distinguished physiological geneticist. Working briefly in the laboratory of Charles Harington in May of 1933, Chain and Harington did not get along well.

Shortly afterwards, in October of 1933, Chain was accepted into graduate school in the Ph.D. program at Cambridge University, working in the biochemistry research laboratory of Prof. Frederick Gowland Hopkins, housed at the Sir William Dunn School of Biochemistry, where Chain took up the study of phospholipids and enzymes. Hopkins was under the impression that earning another advanced degree, at Cambridge, would help keep Chain out of having to return to Germany, where it was considered dangerous.

Chain and Hopkins got along quite well. Chain started his studies on snake venom and its effects on the nervous system, focusing on the biochemistry of the venomous enzyme involved in causing paralysis. Completing his second graduate thesis, Chain took his Ph.D. from Cambridge in 1935.

Hired by Florey, who needed a biochemist, in September of 1935, Chain became demonstrator and lecturer of chemical pathology at Oxford University in the Sir William Dunn School of Pathology, and in his new laboratory he completed his studies on the biochemistry of snake venom. It was towards the end of 1935 when Chain learned that his mother and sister, Hedwig, still in Germany, had been sent to Theresienstadt, a concentration camp in Czechoslovakia. In 1936, Chain suffered a nervous breakdown and during his recovery in Paris he was introduced to Anne Beloff, a prominent biochemist in her own right and who was later to become Chain’s wife in 1948. In 1939, Chain became a citizen of England. In 1942, he had learned that his mother and sister perished in the Nazi holocaust at Theresienstadt.

The third penicillin purifier, Norman George Heatley, frequently referred to as “penicillin’s forgotten man,” was born as an only child to parents Thomas and Grace Heatley on the 10th of January in the year 1911 in Woodbridge, Suffolk, in England. Heatley’s father was a veterinary doctor specializing in farm animals.

Heatley was reported to have accompanied his father on his rounds, and he (Norman) was impressed with his father’s skill with animals; importantly, however, the young Heatley was deeply impressed with his father’s hobby of micro-manipulation skills in fixing broken china tea cups and saucers. This experience was to become a long lasting interest for Heatley. The child Heatley attended St. Felix grammar school in the adjacent town of Ipswich, England and then moved to Folkstone, along the coast of England to attend a boarding school called Westbourne House.

In 1929, Heatley graduated from high school and enrolled in St. John’s College, in Cambridge, England. Concentrating in natural sciences, Heatley took his undergraduate degree in 1933. Heatley then studied biochemistry for an academic year after his college graduation, at which time he became a laboratory assistant to Prof. F.G. Hopkins at Cambridge University. Next, Heatley entered graduate school at Cambridge and studied micro-chemical techniques in the laboratory of Joseph Needham and graduated with his Ph.D. degree in 1936. It is during this time that Heatley’s expertise was noticed by Chain, and upon Chain’s recommendation, Heatley was hired by Florey at Oxford.

2) What brought them together? And where did they work together?

The trio of penicillin makers, Florey, Chain and Heatley, were housed first at the Sir William Dunn School of Pathology, in Oxford, England. Florey had become professor of pathology in May of 1935, and at Oxford he first hired Chain, who arrived in September of that same year.  The following year, in 1936, Florey hired Heatley, who arrived at Oxford almost exactly a year after Chain.

All three individuals were very highly recommended by their immediate superiors, and all of them brought to the table badly needed expertise. Florey supplied facilities, funding, effective leadership, and stability amongst the other two seemingly strong-willed and completely divergent colleagues.

Chain provided expertise and knowledge of biochemistry, metabolism, fermentation, and cell culturing. Heatley, also a biochemist, delivered expertise in the assembly of apparatuses, scaling-up of techniques, bacteriology, testing of penicillin’s efficacy, and especially of micro-manipulation of equipment. In particular Heatley was key to solving a problem with the instability of the penicillin during purification.

Later, Florey, Heatley and other personnel were to move to the U.S. to complete their large-scale production of penicillin, in relative safety from the German blitz bombing. Here, legend has it that Florey, Heatley and others actually smeared the penicillin-producing mold spores on the insides their sewn coats as they travelled to the U.S., in order to remain incognito and, importantly, to keep penicillin out of German hands, should they be captured.

There is a famous quotation by Prof. Henry Harris, Florey’s successor at Oxford in later years.  Harris is reported to have summarized the contributions of the four main penicillin players as such, “Without Fleming, no Chain or Florey; without Chain, no Florey; without Florey, no Heatley; without Heatley, no penicillin.”

3) Now, can you walk us through this process of “purifying penicillin” and review the initial stages of discovery and then the need for this purification process?

The first attempts to purify penicillin from the Penicillium notatum mold were performed by Dr. Alexander Fleming, himself, shortly after his famous discovery, in 1928. Several other groups also tried to isolate the penicillin, and all failed to adequately produce enough for practical purposes. There were essentially three main focuses: growing the mold, extracting and purifying the penicillin from the cultured mold, and the testing of the purified penicillin on bacteria.

Let’s consider the mold growth efforts, first. On Petri plates, the P. notatum mold formed large solid mats, and on top of these mats secreted penicillin emerged from the mats forming little droplets of liquid juice harboring the penicillin. The penicillin juice could be taken up with syringes and tested on bacteria. The problem here was that this method was not practical for large-scale production and the secreted penicillin juice was found to contain over 50 other non-necessary components. Obviously, it was important to have penicillin in a pure state. Efforts turned to large-scale culturing of the P. notatum mold, using a variety of broth medium nutrients, pH, temperatures, time-periods, etc.

As the mold culturing conditions were being optimized, the problems of small- and large-scale penicillin extraction and isolation were addressed, as well. The first step involved filtration of the grown mold, addition of acid and cooling of the filtered acidified mold juice. Next, the chemical ether (or sometimes amyl acetate) was added to the acid mold juice—the penicillin would move into the ether.  Next, using Heatley’s instability problem-solving idea, a mixture of alkaline and water was added to the ether-penicillin mixture, shaken vigorously, allowed to separate and distilled. The penicillin moved to the basic water and in stable form. Next, the penicillin solution was freeze-dried into a powder.

Next, the technique was moved to a large-scale process, using an automated apparatus to filter, add acid, alkali, and other reagents, etc. Interestingly, the large-scale production apparatus was assembled by the trio using make-shift and rudimentary materials, like bathtubs, trash cans, and even milk churns, in order to produce the penicillin.

Lastly, the anti-bacterial nature of the purified penicillin needed to be tested against bacteria, to see if it killed any of them. The first experiments of purified penicillin involved making Petri plates with agar growth medium in them.  The bacteria targets were inoculated onto the Petri agar plates. Next, short hollow cylinders, made out of glass, and resembling extremely short straws, were placed on top of the inoculated agar, and the purified penicillin was poured into the cylinders.

The pure penicillin would then emerge from the cylinder vessels and diffuse into the agar where the bacteria were located. If the purified penicillin killed or inhibited the growth of these bacteria, then a zone of growth inhibition would form about the cylinders in the agar medium. No bacteria were found in these zones where the penicillin moved into around the cylinders. The presence of these bacteria-free zones were good indicators of the effectiveness of the pure penicillin in killing bacteria.

4) Today, in the year 2017- what are the challenges in terms of constructing if you will, this miracle drug we call penicillin?

After the large-scale production of penicillin by Florey, Chain and Heatley, in the mid-1940s, the miracle occurred of actually surviving serious bacterial infections, for the first time in history. Other antibiotics were discovered, purified and used clinically, such as the tetracyclines, streptomycin, etc., and they were effective against bacterial infection, at first.

A major challenge then (as well as today) was finding new ways to chemically modify the penicillin platform so that the new derivatives retained their stability and anti-bacterial effectiveness. Current challenges continue to fall into the category of ensuring that the new derivatives are safe, and this involves extensive cellular and bacteriological testing and clinical trials, all of which constitute a financial burden.

Sadly, the miracles of penicillin and other antimicrobials, have been compromised. A primary challenge has been that the bacteria evolved to resist the antibacterial activities of the penicillin antibiotics and other antimicrobial agents. In fact, new bacterial variants have emerged in which they are resistant to multiple antibiotics. A good example is the penicillin derivative called methicillin, which initially provided an effective treatment of the dread Staphylococcus aureus bacteria, a causer of a wide variety of infections in humans and other animals.

Soon after the clinical implementation of methicillin, variants of the S. aureus emerged from patients who failed to respond to the new medicine. Shortly after the discovery of the methicillin-resistant S. aureus (MRSA) clinical isolates, it was further found that these MRSA bacteria were also resistant to multiple antimicrobial agents. Today, MRSA constitutes a serious public health concern.

Present in virtually all penicillins and derivatives, is the active chemical site, called a β-lactam ring structure, which is necessary for the killing effect of these antibiotics, which are oftentimes called β-lactam antimicrobials. This is because penicillin, its derivatives, and other antimicrobial agents that contain the active β-lactam ring structure. Another challenge is the discovery of bacteria, some from soil, that have super enzymes that biochemically destroy penicillin and its derivatives, like β-lactams, with super high destructive efficiencies. These bacteria have specialized enzymes called extended-spectrum β-lactamases (ESBLs) that may confer upon these bacteria the role of the superbug status.

One challenge has been allergic reactions to penicillin and other β-lactams. Penicillin allergies can be potentially life-threatening if an anaphylaxis reaction occurs. One well-known mechanism of allergy to penicillin involves first the drug binding to a person’s proteins forming conjugates (penicillin bound to host self-proteins), which induce helper T-cells to activate B-cells, causing them to make lots of a specific type of antibodies (their technical term is class E immunoglobulin, or IgE) against penicillin.

These antibody-penicillin complexes stimulate a person’s mast cells to produce a set of mediators that cause a person to experience anaphylaxis, which may be mild, characterized by hives, or severe, as in the case of anaphylactic shock, characterized by a drop in blood pressure, shock, difficulty in breathing due to airway constriction, and a swelling of the epiglottis, causing suffocation. Thus, anaphylactic shock can be lethal.

Furthermore, for every class of antimicrobial agent in use today, there are bacteria that are resistant to each of these antibacterial agents. Interestingly, and perhaps disturbingly, use of an antibacterial agent not only selects for bacteria that harbor resistance to that agent used, it actually selects for bacteria that are multiple drug resistant. What’s more, genes encoding these multidrug resistance elements may be transferred to other species of bacteria, like in the human gut, in soil, or in sewage, etc., making resistance spread around.

Both investigators and clinicians are actively fighting back against multiple antimicrobial resistances from bacteria, by searching for new antibiotics with novel ways of killing bacteria and in invoking prudent and proper stewardship practices in the uses of anti-bacterial agents. These efforts are of primary importance in hospitals, the environment, and in agriculture. Efforts are aimed at modulating these antibacterial resistance mechanisms, in order to restore their antibacterial effectiveness.

Therefore, study of the biochemistry and molecular biology of the antimicrobial resistance mechanisms will continue to be important areas, in order to find new ways to circumvent bacterial resistance. There is so much work that still needs to be done in each of these areas.

5) Were these three men recognized during their lifetimes? What awards did they win, if any?

Of all four players in this story, Alexander Fleming was to receive the vast majority of the accolades. In 1945 the Nobel Prize in Physiology or Medicine for this amazing and important history-changing discovery went to Fleming, for his discovery of the penicillin, and to Florey and Chain, for their production of the penicillin and permitting its clinical use. Others were left out of the Nobel, namely, Heatley, who was instrumental in so many ways for penicillin production, and many others who helped to determine penicillin’s structure, its mode of action, biosynthetic pathways, mechanisms of resistance, etc.

Florey, Chain and Heatley were relatively recognized in various ways, with numerous awards, knighthood (to Fleming, Florey and Chain but not to Heatley), and honorary degrees. There were, apparently, varying degrees of disappointment in the recognition received amongst the trio of penicillin producers. First, Florey and Chain, despite becoming Nobel Laureates, continually felt that they did not get the full credit for their efforts that they felt they had deserved. Heatley received the least recognition and credit of all three. It was to profoundly affect Heatley for many years to come.

6) What have I neglected to ask?

I thought it would be informative to your readers to provide an overview of the various cellular molecular strategies that bacteria have evolved to resist the killing effects of penicillin and β-lactam antibacterial agents. These mechanisms have been problematic in the efforts exerted to treat infections caused by bacteria which harbor these resistance factors. The presence or absence of these resistance factors may quite literally determine the death or life matters in patients with bacterial infections.

One of the first β-lactam resistance mechanisms to be discovered was the enzymatic inactivation mechanism in which an enzyme, typically called β-lactamase, cuts the antibiotic into a form that doesn’t kill bacteria. Thus, the bacteria live in the presence of (i.e., resist) the antibiotic. Some of these enzymes can destroy large quantities of the antibiotics.

Another resistance mechanism involves the alteration of the target of the antibiotic. These targets are known as either penicillin-binding proteins (PBPs) or transpeptidases, which are important components of the bacterial cell wall building machinery. Penicillin will bind to these targets and prevent their function. As a consequence, the bacteria either fail to make cell walls or produce weak cell walls, making the bacteria die by explosion. The altered target mechanism reduces the penicillin binding, and the bacteria live with their cell walls left intact.

Along these lines, the penicillin target may be made in a great abundance, resulting in lots of target molecules left alone by the penicillin. With the drug target left untouched, the bacteria synthesize their cell walls, letting the bacteria live.

Another effective resistance mechanism prevents entry of penicillin and other antibiotics into the insides of the cell; this bacteriological mechanism is called simply reduced permeability or entry, and prevention of access to the drug target. If the antibiotic cannot get to the cytoplasmic insides of a bacterium, where the targets are, then the bacterial transpeptidase targets successfully make their cell walls, and the bacteria live.

One last resistance mechanism includes an export apparatus called an antimicrobial efflux pump, which is embedded in the bacterial membrane and which sends any internal antibiotic to the outside of the cell, where there are no targets. These antimicrobial efflux pumps may send out only one type of antibiotic or send out several different antibiotic types. These so-called multidrug efflux pumps are studied in my own research laboratory.

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