An Introduction to Bioprocess Engineering

{Dr. Michael L. Shuler & Fikret Kargi, 3rd edition, Draft—not for further

distribution}

C H A P T E R

1 What Is a Bioprocess Engineer?

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 dacades ago. We are at the doorstep of being able to chemically synthesize whole genomes and transfer them into a host of bacterium to create semi-synthetic life. We now have a whole host of new techniques to rationally design novel organisms.

With this intellectual revolution emerge new visions and new hopes: new medicines, semisynthetic organs grown in large vats, abundant and nutritious foods, computers based on biological molecules rather than silicon chips, superorganisms to degrade pollutants, and a wide array of consumer products effective 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 beautiful-ly constructed, but they obey the rules of chemistry and physics and they are susceptible to en-gineering 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 intellec-tual 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 this will help you form the necessary foundation.

1.1 BIOTECHNOLOGY AND BIOPROCESS ENGINEERING 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 ma-nipulation 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 profes-sionals 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 engi-neering as a subcomponent of biotechnology.

Many terms have been used to describe engineers working with biotechnology. Bioengi-neering is a broad title and includes work on medical, bioprocess, agricultural and environmen-tal systems; its practitioners include agricultural, electrical, mechanical, industrial, environmen-tal and chemical engineers, and others. Biological engineering is similar but emphasizes appli-cations to plants and animals. Metabolic engineering is the design of cells with genetically al-tered 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, alt-hough the boundary between the two is increasingly vague, particularly in the areas of cell sur-face receptors and animal cell culture. It focuses on the human body with an emphasis on ap-plication of engineering principles from a variety of disciplines to design medical devices, syn-thetic organs, novel methods for drug delivery and development of diagnostics and instrumen-tation. Another relevant term is biomolecular engineering, which has been defined by the Na-tional 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 sys-tems containing biological catalysts, but with an emphasis on those systems making use of bio-technology. 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.

1.2 DIFFERING APPROACHES TO RESEARCH FOR BIOLOGISTS AND ENGINEERS The fundamental trainings of biologists and engineers are distinctly different. In the develop-ment 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 very good background in the physical and mathematical sci-ences. Often a theory leads to mathematical formulations, and the validity of the theory is test-ed by comparing predicted responses to those in experiments. Quantitative models and ap-proaches, even to complex systems, are strengths. Biologists are usually better at the formation of testable hypotheses, experimental design, and data interpretation from complex systems. Engineers are typically 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 ex-perimental 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 coherent framework for applications to bioprocessing.

1.3 THE STORY OF PENICILLIN: HOW BIOLOGISTS AND ENGINEERS WORK TOGETHER 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.

FIGURE 1.1 Alexander Fleming’s original plate showing the growth of the mold Penicillium notatum and its inhibitory action on bacterial growth. (With permission, from Corbis Corporation.)

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 Flem-ing’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, per-formed 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 pro-cess, and for such a process development, engineers would be needed, in addition to microbial physiologists and other life scientists.

The war further complicated the situation. Great Britain’s industrial facilities were al-ready 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 Re-gional 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 a great deal of 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 fermenta-tion.) 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 ap-pointed 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 contribu-tions 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 labora-tory 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 penicil-lin 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 in-volved 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 manu-facturing 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 nec-essary 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 in tank design and operation. Large volumes of absolutely clean, oil- and dirt-free sterile air were required. What were then very large agitators were re-quired, 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 re-covery and purification. The very fragile nature of penicillin required the development of spe-cial 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 six months the first plant for commercial production of penicillin by submerged fermentation (Hobby, 1985). The plant had 14 tanks each of 7000-gal capacity (see Figures 1.2 and 1.3).

FIGURE 1.2 Series of large-scale antibiotic fermenters. (With permission, from T.D. Brock, K.M. Brock, and D.M. Ward. Basic Microbiology with Applications, 3d ed., Pearson Education, Upper Saddle River, NJ, 1986, p. 507.)

FIGURE 1.3 Inside view of a large antibiotic fermenter. (From Trends in Biotechnology 3 (6), 1985. Used with permission of Elsevier Science Publishers.)

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, while the first impact was in war zones, the overall effect was critical globally to fighting many diseases (see Figure 1.4).

FIGURE 1.4 First impact of penicillin in war zones.

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” (see the chapter by 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 physiol-ogy, metabolic pathways, penicillin structure, methods of mutation and selection of mold ge-netics, process control, and reactor design (see Figure 1.5).

FIGURE 1.5 Schematic of penicillin production process.

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 mind set 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 pro-tein 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 reex-amine the prejudices the field has inherited from the penicillin experience.

It is you, the student, who will best be able to challenge these prejudices.

1.4 BIOPROCESSES: REGULATORY CONSTRAINTS To understand the mind set of a bioprocess engineer you must understand the regulatory cli-mate in which many bioprocess engineers work. The U.S. FDA (Food and Drug Administra-tion) 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 un-dergoes 6.5 years of development from the discovery stage through preclinical testing in ani-mals. 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., does it help 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 about up to 5 billion dollars (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 have been tragic examples where a small process change has al-lowed 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 clini-cal trials to test the safety of the resulting product. Since clinical trials are very expensive, pro-cess 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 certi-fied 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 proce-dures (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 as-surance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics” and “There shall be written procedures for produc-tion and process-control to assure that products have the identity, strength, quality, and purity they purport or are represented to possess.”

The actual process of doing validation is often complex, particularly when a whole facili-ty 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, con-sistency of product, and demonstrable measures of product quality, particularly purity and safe-ty. These tasks are demanding and require careful attention to detail. Bioprocess engineers of-ten 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 consider-able regulatory impact.

SUGGESTIONS FOR FURTHER READING

A. Synthetic Biology

GIBSON, D.G., ET AL., Creation of a bacterial cell controlled by a chemically synthesized genome, Science 329: 52-56, 2010.

B. History of Penicillin

ELDER, A. L., ed., The History of Penicillin Production, Chem. Eng. Prog. Symp. Ser. 66 (#100), Ameri-can Institute of Chemical Engineers, New York, 1970.

HOBBY, G. L., Penicillin. Meeting the Challenge, Yale University Press, New Haven, CT, 1985. LAX, E., The Mold in Dr. Florey’s Coat: The Story of the Pneicillin Miracle, Henry Holt & Company,

New York, New York, 2004. MOBERG, C. L., Penicillin’s Forgotten Man: Norman Heatley, Science 253: 734–735, 1991.

C. Regulatory Issues

AGALLOCO, J.P., AND CARLETON, F.J., Validation of Phramceutical Processes, 3rd Edition, CRC Press, Boca Raton, FL, 2007.

KISHIMOTO, T.K., ET AL., Contaminated heparin associated with adverse clinical events and activation of the contact system, N. Engl. J. Med. 358: 2457-2567, 2008.

MULLARD, A. New drugs cost US $2.6 billion to develop. Nature Reviews Drug Discovery 13: 877, 2014. NAGLAK, T. J., KEITH, M. G. AND OMSTEAD, D. R., Validation of fermentation processes, Biopharm (Ju-

ly/Aug.):28–36, 1994. RAO, G., MOREIRA, A., AND BRONSON, K., Disposable bioprocessing: the future has arrive, Biotechnol.

Bioeng. 102: 348-356, 2009.

D. Education

LEE-PARSONS, C.W.T., Biochemical engineering taught in the context of drug discovery to manufacturing. Chemical Engineering Education, Summer 2005: 208-221, 2005.

QUESTIONS 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?