We can now manipulate life at its most basic level—the genetic. For thousands of years, people have practiced genetic engineering at the level of selection and breeding. But now it can be done in a purposeful, predetermined manner with the molecular-level manipulation of DNA. We now have tools to probe the mysteries of life in a way unimaginable even a few decades ago. We are at the doorstep of being able to chemically synthesize whole genomes and transfer them into a host bacterium to create semisynthetic life. We now have a whole host of new techniques to rationally design novel organisms.
With this intellectual revolution emerged new visions and new hopes: new medicines, semisynthetic organs grown in large vessels, abundant and nutritious foods, computers based on biological molecules rather than silicon chips, superorganisms to degrade pollutants, a wide array of consumer products, and potentially new sources of energy.
These dreams will remain dreams without hard work. Engineers will play an essential role in converting these visions into reality. Biological systems are very complex and beautifully constructed, but they obey the rules of chemistry and physics and are susceptible to engineering analysis. Living cells are predictable, and the processes to use them can be rationally constructed on commercial scales. Doing this is the job of the bioprocess engineer.
Probably the reason you are reading this book is your desire to participate in this intellectual revolution and to make an important contribution to society. You can do it, but it is not easy to combine the skills of the engineer with those of the biologist. This book’s intent is to help you begin to develop these skills. This book and a one-term course are not enough to make you a complete bioprocess engineer, but it will help you form the necessary foundation.
In this chapter, we discuss the origins of bioprocess engineering, how bioprocess engineering evolved, and the complementary roles of biologists and engineers. We then consider the regulatory constraints on bioprocesses and how they impact the engineer.
When new fields emerge from new ideas, old terminology is usually not adequate to describe these fields. Biotechnology and what constitutes engineering in this field are best described with examples rather than single words or short phrases. However, it is critical to understand the cell’s inner workings to harness the cellular machinery and engineer the cell to perform a designated task.
Biotechnology usually implies the use or development of methods of direct genetic manipulation for a socially desirable goal. Such goals might be the production of a particular chemical, but they may also involve the production of better plants or seeds, or gene therapy, or the use of specially designed organisms to degrade wastes. The key element for many professionals is the use of sophisticated techniques outside the cell for genetic manipulation. Others interpret biotechnology in a much broader sense and equate it with the technical use of biology for production of pharmaceuticals, chemicals, food, and diagnostics; they may include engineering as a subcomponent of biotechnology.
Many terms are used to describe engineers working with biotechnology. Bioengineering is a broad title and includes work on medical, bioprocess, agricultural, and environmental systems; its practitioners include agricultural, electrical, mechanical, industrial, environmental, and chemical engineers as well as others. Biological engineering is similar but emphasizes applications to plants and animals. Metabolic engineering is the design of cells with genetically altered pathways to make small molecules that are often novel for that cell. This term stresses human design of cells for manufacture of specialty chemicals. Biochemical engineering has usually meant the extension of chemical engineering principles to systems using a biological catalyst to bring about desired chemical transformations. It is often subdivided into bioreaction engineering and bioseparations, particularly for production of biologics, chemicals, and fuels. Biomedical engineering has been considered to be separate from biochemical engineering, although the boundary between the two is increasingly vague, particularly in the areas of cell-surface receptors and animal cell culture. It focuses on the human body with an emphasis on application of engineering principles from a variety of disciplines to design medical devices, synthetic organs, and novel methods for drug delivery and to develop diagnostics and instrumentation. Another relevant term is biomolecular engineering, which has been defined by the National Institutes of Health as “research at the interface of biology and chemical engineering and is focused at the molecular level.”
There is a difference between bioprocess engineering and biochemical engineering. In addition to chemical engineering, bioprocess engineering includes the work of mechanical, electrical, environmental, and industrial engineers to apply the principles of their disciplines to processes based on using living cells or subcomponents of such cells. The problems of detailed equipment design, sensor development, control algorithms, and manufacturing strategies can utilize principles from these disciplines. Biochemical engineering is more limited in the sense that it draws primarily from chemical engineering principles and broader in the sense that it is not restricted to well-defined, artificially constructed processes but can be applied to natural systems.
This book focuses primarily on the application of chemical engineering principles to systems containing biological catalysts, but with an emphasis on those systems making use of biotechnology. The rapidly increasing ability to determine the complete sequence of genes in an organism offers new opportunities for bioprocess engineers in the design and monitoring of bioprocesses. The cell itself is now a designable component of the overall process.
The fundamental trainings of biologists and engineers are distinctly different. In the development of knowledge in the life sciences, unlike chemistry and physics, mathematical theories and quantitative methods (except statistics) have played a secondary role. Most progress has been due to improvements in experimental tools. Results are qualitative, and descriptive models are formulated and tested. Consequently, biologists often have incomplete backgrounds in mathematics but are very strong with respect to laboratory tools and, more importantly, with respect to the interpretation of laboratory data from complex systems.
Engineers usually possess a solid background in the physical and mathematical sciences. Often, a theory leads to mathematical formulations, and the validity of the theory is tested by comparing predicted responses to those in experiments. Quantitative models and approaches, even to complex systems, are strengths. Biologists are usually adept at the formation of testable hypotheses, experimental design, and data interpretation from complex systems. Engineers quite often are unfamiliar with the experimental techniques and strategies used by life scientists.
The skills of the engineer and life scientist are complementary. To convert the promises of molecular biology into new processes to make new products requires the integration of these skills. To function at this level, the engineer needs a solid understanding of biology and its experimental tools. This book provides sufficient biological background for you to understand how to apply engineering principles to biosystems. However, if you are serious about becoming a bioprocess engineer, you will need to take further courses in microbiology, biochemistry, and cell biology, as well as more advanced work in bioengineering. If you already have these courses, this book can be used for review and to provide a coherent framework for applications to bioprocessing.
In September 1928, Alexander Fleming at St. Mary’s Hospital in London was trying to isolate the bacterium Staphylococcus aureus, which causes boils. The technique in use was to grow the bacterium on the surface of a nutrient solution. One of the dishes had been contaminated inadvertently with a foreign particle. Normally, such a contaminated plate would be tossed out. However, Fleming noticed that no bacteria grew near the invading substance (see Figure 1.1).
Fleming’s genius was to realize that this observation was meaningful and not a “failed” experiment. Fleming recognized that the cell killing must be due to an antibacterial agent. He recovered the foreign particle and found that it was a common mold of the Penicillium genus (later identified as Penicillium notatum). Fleming nurtured the mold to grow and, using the crude extraction methods then available, managed to obtain a tiny quantity of secreted material. He then demonstrated that this material had powerful antimicrobial properties and named the product penicillin. Fleming carefully preserved the culture, but the discovery lay essentially dormant for over a decade.
World War II provided the impetus to resurrect the discovery. Sulfa drugs have a rather restricted range of activity, and an antibiotic with minimal side effects and broader applicability was desperately needed. Howard Florey and Ernst Chain of Oxford decided to build on Fleming’s observations. Norman Heatley played the key role in producing sufficient material for Chain and Florey to test the effectiveness of penicillin. Heatley, trained as a biochemist, performed as a bioprocess engineer. He developed an assay to monitor the amount of penicillin made so as to determine the kinetics of the fermentation, developed a culture technique that could be implemented easily, and devised a novel back-extraction process to recover the very delicate product. After months of immense effort, they produced enough penicillin to treat some laboratory animals.
Eighteen months after starting on the project, they began to treat a London bobby for a blood infection. The penicillin worked wonders initially and brought the patient to the point of recovery. Most unfortunately, the supply of penicillin was exhausted, and the man relapsed and died. Nonetheless, Florey and Chain had demonstrated the great potential for penicillin, if it could be made in sufficient amount. To make large amounts of penicillin would require a process, and for such a process development, engineers, in addition to microbial physiologists and other life scientists, would be needed.
The war further complicated the situation. Great Britain’s industrial facilities were already totally devoted to the war. Florey and his associates approached pharmaceutical firms in the United States to persuade them to develop the capacity to produce penicillin, since the United States was not at war at that time.
Many companies and government laboratories, assisted by many universities, took up the challenge. Particularly prominent were Merck, Pfizer, Squibb, and the USDA Northern Regional Research Laboratory in Peoria, Illinois.
The first efforts with fermentation were modest. A large effort went into attempts to chemically synthesize penicillin. This effort involved hundreds of chemists. Consequently, many companies were at first reluctant to commit to the fermentation process beyond the pilot-plant stage. It was thought that the pilot-plant fermentation system could produce sufficient penicillin to meet the needs of clinical testing, but large-scale production would soon be done by chemical synthesis. At that time, U.S. companies had achieved a great deal of success with chemical synthesis of other drugs, which gave the companies significant control over the drug’s production. The chemical synthesis of penicillin proved to be exceedingly difficult. (It was accomplished in the 1950s, and the synthesis route is still not competitive with fermentation.) However, in 1940 fermentation for the production of a pharmaceutical was an unproved approach, and most companies were betting on chemical synthesis to ultimately dominate.
The early clinical successes were so dramatic that in 1943 the War Production Board appointed A. L. Elder to coordinate the activities of producers to greatly increase the supply of penicillin. The fermentation route was chosen. As Elder recalls, “I was ridiculed by some of my closest scientific friends for allowing myself to become associated with what obviously was to be a flop—namely, the commercial production of penicillin by a fermentation process” (from Elder, 1970). The problems facing the fermentation process were indeed very formidable.
The problem was typical of most new fermentation processes: a valuable product made at very low levels. The low rate of production per unit volume would necessitate very large and inefficient reactors, and the low concentration (titer) made product recovery and purification very difficult. In 1939 the final concentration in a typical penicillin fermentation broth was one part per million (ca. 0.001 g/l). Furthermore, penicillin is a fragile and unstable product, which places significant constraints on the approaches used for recovery and purification.
Life scientists at the Northern Regional Research Laboratory made many major contributions to the penicillin program. One was the development of a corn steep liquor–lactose-based medium. This medium increased productivity about tenfold. A worldwide search by the laboratory for better producer strains of Penicillium led to the isolation of a Penicillium chrysogenum strain. This strain, isolated from a moldy cantaloupe at a Peoria fruit market, proved superior to hundreds of other isolates tested. Its progeny have been used in almost all commercial penicillin fermentations.
The other hurdle was to decide on a manufacturing process. One method involved the growth of the mold on the surface of moist bran. This bran method was discarded because of difficulties in temperature control, sterilization, and equipment size. The surface method involved growth of the mold on top of a quiescent liquid medium. The surface method used a variety of containers, including milk bottles, and the term “bottle plant” indicated such a manufacturing technique. The surface method gave relatively high yields but had a long growing cycle and was very labor intensive. The first manufacturing plants were bottle plants because the method worked and could be implemented quickly.
However, it was clear that the surface method would not meet the full need for penicillin. If the goal of the War Production Board was met by bottle plants, it was estimated that the necessary bottles would fill a row stretching from New York City to San Francisco. Engineers generally favored a submerged tank process. The submerged process presented challenges in terms of both mold physiology and tank design and operation. Large volumes of absolutely clean, oil- and dirt-free sterile air were required. What were then very large agitators were required, and the mechanical seal for the agitator shaft had to be designed to prevent the entry of organisms. Even today, problems of oxygen supply and heat removal are important constraints on antibiotic fermenter design. Contamination by foreign organisms could degrade the product as fast as it was formed, consume nutrients before they were converted to penicillin, or produce toxins.
In addition to these challenges in reactor design, there were similar hurdles in product recovery and purification. The very fragile nature of penicillin required the development of special techniques. A combination of pH shifts and rapid liquid–liquid extraction proved useful.
Soon, processes using tanks of about 10,000 gal were built. Pfizer completed in less than 6 months the first plant for commercial production of penicillin by submerged fermentation (Hobby, 1985). The plant had 14 tanks, each of 7000-gal capacity. Large-scale fermenters (typically 50,000 to 100,000 l) now used in antibiotic fermentations are shown in Figures 1.2 and 1.3.
By a combination of good luck and hard work, the United States had the capacity by the end of World War II to produce enough penicillin for almost 100,000 patients per year. The impact of penicillin was immediate, and the advent of antibiotics saved many lives. Although the first impact was in war zones, the overall effect was critical globally to fighting many diseases (see Figure 1.4).
This accomplishment required a high level of multidisciplinary work. For example, Merck realized that men who understood both engineering and biology were not available. Merck assigned a chemical engineer and microbiologist together to each aspect of the problem. They planned, executed, and analyzed the experimental program jointly, “almost as if they were one man” (Silcox in Elder, 1970).
Progress with penicillin fermentation has continued, as has the need for the interaction of biologists and engineers. From 1939 to now, the yield of penicillin has gone from 0.001 g/l to over 50 g/l of fermentation broth. Progress has involved better understanding of mold physiology, metabolic pathways, penicillin structure, methods of mutation and selection of mold genetics, process control, and reactor design (see Figure 1.5).
Before the penicillin process, almost no chemical engineers sought specialized training in the life sciences. With the advent of modern antibiotics, the concept of a bioprocess engineer was born. The penicillin process also established a paradigm for bioprocess development and biochemical engineering. This paradigm still guides much of our profession’s thinking. The mindset of bioprocess engineers was cast with the penicillin experience. It is for this reason that we have focused on the penicillin story rather than on an example for production of a protein from a genetically engineered organism. Although many parallels can be made between the penicillin process and our efforts to use recombinant DNA, no similar paradigm has yet emerged from our experience with genetically engineered cells. We must continually reexamine the prejudices the field has inherited from the penicillin experience.
It is you, the student, who will best be able to challenge these prejudices.
To understand the mindset of a bioprocess engineer, you must understand the regulatory climate in which many bioprocess engineers work. The U.S. FDA (Food and Drug Administration) and its equivalents in other countries must insure the safety and efficacy of medicines. For bioprocess engineers working in the pharmaceutical or biotechnology industry, the primary concern is not reduction of manufacturing cost (although that is still a very desirable goal), but the production of a product of consistently high quality in amounts to satisfy the medical needs of the population.
Consider briefly the process by which a drug obtains FDA approval. A typical drug undergoes 6.5 years of development from the discovery stage through preclinical testing in animals. Human clinical trials are conducted in three phases. Phase 1 clinical trials (about 1 year) are used to test safety; typically 20 to 80 volunteers are used. Phase II clinical trials (about 2 years) use 100 to 300 patients, and the emphasis is on efficacy (i.e., whether it helps the patient) as well as further determining which side effects exist. Compounds that are still promising enter phase III clinical trials (about 3 years) with 1000 to 3000 patients. Since individuals vary in body chemistry, it is important to test the range of responses in terms of both side effects and efficacy by using a representative cross section of the population. Data from clinical trials is presented to the FDA for review (about 18 months). If the clinical trials are well designed and demonstrate statistically significant improvements in health with acceptable side effects, the drug is likely to be approved. Even after this point, there is continued monitoring of the drug for adverse effects. The whole drug discovery-through-approval process takes 12 to 15 years on the average and costs up to about $5 billion (in 2013). Only one in ten drugs that enter human clinical trials receives approval. Recent FDA reforms have decreased the time to obtain approval for life-saving drugs in treatment of diseases such as cancer and AIDS, but the overall process is still lengthy.
This process greatly affects a bioprocess engineer. FDA approval is for the product and the process together. There are tragic examples in which a small process change has allowed a toxic trace compound to form or become incorporated in the final product, resulting in severe side effects, including death. For example, heparin contaminated with oversulfated chondroitin sulfate caused significant problems. Thus, process changes may require new clinical trials to test the safety of the resulting product. Since clinical trials are very expensive, process improvements are made under a limited set of circumstances. Even during clinical trials it is difficult to make major process changes.
Drugs sold on the market or used in clinical trials must come from facilities that are certified as good manufacturing practice (GMP). GMP concerns the actual manufacturing facility design and layout, the equipment and procedures, training of production personnel, control of process inputs (e.g., raw materials and cultures), and handling of product. The plant layout and design must prevent contamination of the product and dictates the flow of material, personnel, and air. Equipment and procedures must be validated. Procedures include not only operation of a piece of equipment but also cleaning and sterilization. Computer software used to monitor and control the process must be validated. Off-line assays done in laboratories must satisfy good laboratory practices (GLP). Procedures are documented by standard operating procedures (SOP).
The GMP guidelines stress the need for documented procedures to validate performance: “Process validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics” and “There shall be written procedures for production and process-control to assure that products have the identity, strength, quality, and purity they purport or are represented to possess.”†
† Guidelines on General Principles of Process Validation, Federal Register of May 11, 1987 (52 FR 17638).
The actual process of doing validation is often complex, particularly when a whole facility design is considered. The FDA provides extensive information and guidelines, which are updated regularly. Bioprocess engineers involved in biomanufacturing for pharmaceuticals need to consult these sources. For example, the recent introduction of disposable bioreactors requires considerations of how FDA regulations can be applied. However, certain key concepts do not change. These concepts are written documentation, consistency of procedures, consistency of product, and demonstrable measures of product quality, particularly purity and safety. These tasks are demanding and require careful attention to detail. Bioprocess engineers often find that much of their effort is to satisfy these regulatory requirements.
The key point is that process changes cannot be made without considering their significant regulatory impact.
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History of Penicillin
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1.1. What is GMP, and how does it relate to the regulatory process for pharmaceuticals?
1.2. When the FDA approves a process, it requires validation of the process. Explain what validation means in the FDA context.
1.3. Why does the FDA approve the process and product together?
1.4. Carefully specify the differences among biotechnologist, bioprocess engineer, and bioengineer.
1.5. What is achieved by submerged fermentation in penicillin production versus surface fermentation?