394 Biotechnology Chap.9 Add - -, Recombinam-c-c , nansf~rmed ~:.~l DNA~' £.'0""," l - Agar : n ., :::~~ tj ,,,,~,,, _~ DNA fragments Transformrecombinant Growcells joined DNA into E. coli cells rn 0 ~ Agar contains ampicillin I<J Agar contains Two pure cultures containing tetI1Bcline cloned lab;;: DNA F1gure 9.24 Procedures for obtaining pure clones containing rabbit DNA: (8) plasmid DNA with resistance to tetracycline and ampicillin is mixed with rabbit eDNA; (b) DNA ligase is added to join the DNA fragments; (e) E. coli cells act as host; (d) plasmid-containing cells are selected by growth on agar containing tetracycline, and cells with plasmids joined to rabbit eDNA are identified by screening on ampicillin-containing agar; (e) and (f) these cells grow only on tetracycline; (g) pure cultures containing cloned genes (from Understanding DNA and Gene Claning by Drlica, Copyright © 1992, Reprinted by permission of John Wiley & Sons, Inc.} Rabbit DNA '~::~~~~':l ~(~\\'.'~~p':[f~ ~ IIncubate 37"9 IHeatto60°C) then cool @] Mix DNAs Plasmid DNA cut Endonuclease I> and enzyme m amp" gene inactivated ( . Replica plate 3 Replica plate "No colony I-No colony 9.9 Bioprocess Engineering 39. The eDNA could then be isolated, made single-stranded (typically by boiling), and radioactively labeled. 9.8.7 Step 5: Final Screening by Nucleic Acid Hybridization to Isolate Genomic Clones (Bottom of Figure 9.231 The final step is to screen the colonies grown from the phages containing the orig- inal fragmented rabbit DNA and to find which ones have the desired hemoglobin genes (recall this is on the right side of Figure 9.23).The DNA in each plaque is tested to see if it will hybridize with the radioactive hemoglobin eDNA nucleic acid com- plement to the hemoglobin genes being sought. The technique relies on the principle of complementary base pairing between the cDNA probes and the desired hemo- globin genes in those very few plaques described in Step 2. Practical Techniques After the phages were used to grow E. coli colonies on an agar plate, a piece of filter paper was placed on the agar and removed. The cells were thus transferred to the paper, which was placed in a dilute solution of sodium hydroxide (lye or caustic soda). The first key feature was that the sodium hydroxide caused the hemoglobin DNA to become single-stranded. The second key feature was that when the single- stranded,radioactive eDNA was next added, complementary base pairs formed only if the filter-paper-bound DNA contained the likewise single-stranded hemoglobin DNA gene being sought. The radioactive cDNA bound to the filter paper, identifying the locations of the rabbit hemoglobin gene being sought. The filter paper was next washed to remove any radioactive probes that were not base paired. X-ray film was then used to find bacterial colonies containing the hemoglobin gene of interest. This procedure resulted in a pure culture of E. coli in which each cell contained a phage into which the hemoglobin gene of interest had been cloned. In summary, the cDNA radioactive probes derived from mRNA were used to probe the plaques formed from the bacteria-containing phage, which contained rabbit DNA, and eventually isolate the desired genomic clones of hemoglobin. 9.9 BIOPROCESS ENGINEERING 9.9.1 Bloreactors A bioreactor is a container or vessel in which a biological reaction occurs. For example, fermentation takes place in a bioreactor: it is a process of growing (incu- bating) microorganisms on a substrate containing carbon and nitrogen (the "food" for the organisms). A natural example of a bioreactor is a pond, which "manufac- tures" algae or pond scum. Bioreactors can range from small bench-top fermentors holding 11iter to larger l-miltion-liter production units. In addition to producing various food products, bioreactors are used to generate industrial chemicals, enzymes, and biofuels. Using microorganisms to generate fuel is of particular interest to researchers in energy- poor countries. In Finland, for example, baker's yeast has been used in a bioelectro- chemical device in which the chemical process of substrate oxidation-reduction generates electrical energy. 396 Biotechnology Chap. 9 The biosynthesis process, as well as the resulting relationship between cell growth and bioproduct formation, differs according to the type of bioreactor. Two conventional methods are batch and continuous culture fermentation. Bioconver- sions can also take place on moist but solid substrates. A primary task in biotechnology processing is to design, operate, and control bioreactors such that conversion rates and yields are economically feasible. Another challenge is keeping cells and catalysts alive as they are put through various mashing, mixing, heating, and other processes. For this reason, bioprocess engineers must not only be conversant with process development, equipment design,and scale-up but also understand what is needed to keep organisms viable and growing at an optimal rate. A common type of bioreactor is a mechanically stirred tank, which utilizes a three-phase (gas-solid-liquid) reaction. In this type of device, gas is sparged into the bottom of the vessel, then mixed with the liquid phase of the fermentation process by a mechanical stirrer (Figure 9.25). There are strict constraints in su.ch a proceS& For example, a steady supply of oxygen gas bubbles for aerobic fermentations is crit- ical. Stirring must be rapid enough to disperse gas bubbles, develop a homogenous liquid, and ensure a solid suspension. Overstirring tends to shear cells, while under- stirring may asphyxiate them. Another challenge is optimizing heat removal rates; faster fermentation creates a faster rate of heat production. During scale-up, surface- to-volume ratios decrease, reducing the rate of heat removal. Sterility is another major challenge. Processes must be absolutely aseptic. Elim- ination of unwanted organisms is required to ensure product quality and to prevent contaminating organisms from displacing the desired production strain. This creates significant design and operation difficulties, particularly in combination with other process requirements. For example, a big problem has been designing high-quality temperature sensors that can stand up to repeated sterilizations. The molecular reactions inside the bioreactor govern the growth charecteris- tics of cells. Cell growth patterns depend on a variety of factors, including oxygen availability, nutrient supply, pH, temperature, and population density. In a typical batch fermentation, cell growth follows four distinct phases: lag, exponential, sta- tionary, and decline. Little observable growth occurs in the lag phase, during which the cell restructures its biosynthetic mechanisms to take account of the environment. In the exponential phase, the cell grows as quickly as possible given these factors. (In industrial bioprocessing, this phase is typically measured in terms of the time required to double the concentration of cells, known as a biomass.) Exponential growth ceases for a variety of reasons, including nutrient depletion, physical over- crowding, or buildup of by-products of the metabolic process. There follows a sta- tionary phase in which the enzymes that catalyzed rapid growth, along with excess ribosomes, are degraded to create other enzymes or supply fuel for cell maintenance. When internal energy sources are depleted, the cell is unable to carry out basic func- tions. The result may be cell lysis (breakage) or inviability (inability to reproduce). In the depletion phase, the biomass reduces (Figure 9.26). Optimizing bioreactor functionality involves a number of discrete process variables, including aeration, agitation, mass and heat transfer, measurement and control, cell metabolism, and product expression and preparation of inoculates. Development of expert systems for real-time monitoring and adjustment of the 9.9 Bioprocess Engineering 397 Motor drive Foam breaker Cooling coils Baffle plates Figure 9.25 A mechanically stirred bioreactor (adapted from Shuler, 1992). Gearbox Aseptic seal Bearing ~ssemblies Gas exit Flat blade turbines Sterile air inlet Sparger 398 Biotechnology Chap. 9 Time (hours) Figure 9.26 Batch fermentation growth curve. fermentation process is one area of research. One key goal is to be able to correctly identify process problems and correct them online. For a fermentation process, this includes sensor, equipment, and process failure monitoring. Control and batch maintenance are also important candidates for automation. 9.9.2 Postprocessing The bioreactor phase is the heart of the bioprocess. However, the recovery and purification of a fermentation product are essential to any commercial process. The degree of difficulty in the recovery and purification process depends heavily on the nature of the product. Typical downstream product recovery and enrichment pro- cessing includes filtration, crystallization, and drying techniques. Packaging and ship- ping the product are also important postprocessing activities. 9.'0 MANAGEMENT OF TECHNOLOGY 9.10.1 Present Trends Significant advances have been made in a very short time, and these will continue to foster industrial growth in biotechnology. For example, the Human Genome Project has been under way since 1990,jointly funded by the National Institutes of Health and the Department of Energy. Also in May 1998, a collaboration was announced between The Institute of Genomic Research (TlGR)-a private, nonprofit genetics laboratory-and Perkin-Elmer-the main manufacturer of DNA sequencing instru- ments. These projects are focusing on sequencing the 3 billion base pairs of human DNA and identifying approximately 60,000 to 80,000 human genes. Naturally enough, these rival projects and enterprises like the Celera Genomics Group, are creating a highly competitive business environment as they race for completion." The early phases of such research are devoted to mapping each human chro- mosome as a step toward ultimately determining all the genes in the DNA sequence. "ror popular press reviews, see "The Race 10Cash in on the Genetic Code," The New York 1l'mel; August29, 1999,Section 3.A1so,Sciou:e News, VoL 154, October 10, 1998,p,239;and The New Yorker, June 12, 2000 p. 66.The Human Genome Project plans to finish sequencing the human genome by 2003 and have a working draft in 2001. Stationary growth pha.,,\ Exponential phase , I Log phase 9.10 Management of Technology 399 In the process, researchers are developing methods to automate and optimize genetic mapping and sequencing. Subsequent phases will focus on the development of "molecular medicine" based on early detection of disease, effective preventive medicine, efficient drug develop- ment, and, possibly, gene therapy or gene replacement. Research efforts are also under way to sequence the genomes of bacteria, yeast, plants, farm animals, and other organ- isms. Much of the technology developed during genome research, particularly the automation and optimization routines, will greatly benefit the biotech industry. 9.10.2 Manufacturing Despite these advances in knowledge, the path from the laboratory to the market is full of obstacles. Biotech research is expensive, time-consuming, and frequently fruitless. The scale-up to economically feasible production levels is also a tremendous challenge. Biotechnology is in many ways related to one of its parent disciplines, chemical engineering. However, it is far more difficult because its raw materials, catalysts, and products are living organisms, which are inherently more fragile and temperamental than petrochemicals and other substances. Stringent product safety requirements, especially for therapeutics, create special problems for commercial production. Equipment and facilities must meet strict safety and quality control standards to ensure product purity. To date, standards for critical processing components (such as valve design and function) are still being established, making it difficult to design and build biotechnology equipment and systems. Lacking manufacturing expertise, many companies arc sticking to research and licensing their technologies to biotech firms that have already developed production capabilities. This is similar to the "fabless- IC" model presented in the management of technology section of Chapter 5. The long approval process and other product development risks make it espe- cially important to shorten the critical path for bringing a new product to market. With the inherent challenges associated with scale-up of industrial bioprocesses, it is important to begin developing synthesis at the bench and pilot plant scales well before clinical trials are concluded (Figure 9.27). This is similar to the general push for concurrent engineering described in several of the earlier chapters. In addition, there are significant regulatory barriers. New medical compounds must be rigorously tested in numerous clinical trials using strict Food and Drug Administration (FDA) regulations. The approval process typically takes from five to seven years, and the likelihood that a product will fail ishigh. Agricultural regulation is less rigorous; the federal government recently relaxed its regulations of field testing and marketing of genetically engineered crops. 9.10.3 Investment Given the potential range and impact of its commercial applications (Figure 9.28), it is not surprising that biotech attracts much attention on Wall Street and with venture capitalists. Throughout the 1980s,hundreds of cash-infused new companies sprouted up. Each vied to beat the others to market with a breakthrough product. Beginning in the 19905, the well-known pharmaceutical companies also became involved in biotech. In many cases, these larger pharmaceuticals bought, or acquired a major 400 Biotechnology Chap. 9 Design issues Manufacturing Issues F1IUre 9.27 Critical path for biotech design for planning to large-scale production. (Adapted from O'Connor, 1995 © 1995 IEEE. Reprinted, with permission, from iEEE Engineering ill Medicine and Biology, vot. 14, no. 2, p.2rf7,March-ApriI1995.) interest in, the smaller start-up companies. As a result, today's picture is one in which a wide range of company types exists. At one extreme, individuals at universities with molecular and cell biology departments continue to start private research-oriented companies. The integrity of such practices is discussed in Kenney (1986). At the other extreme, the large pharmaceuticals have set up production lines for well-established materials. In between, the industry pioneers such as Chiron and Genentech continue with a balance of basic research into new products and the production of well- established products. 9.10.4 The Future As can be seen in the popular press, biotechnology research creates more ethical debates than the industries reviewed in Chapters 5 through 8.Part of the controversy stems from popular fascination and fears about the potential dangers of "messing with nature." Michael Crichton and Hollywood have helped fuel such concerns with reconstituted dinosaurs wreaking havoc in tropical islands. However, beyond the fanciful terrors of science fiction, biotech does indeed provoke a host of real ethical as well as practical concerns. Selective breeding was once the only method available to develop desirable plant and animal characteristics over several generations. By contrast, genetic engineering can clone precisely defined species. This may be less threatening for the well-known cloning of sheep. But it is clear from the recent government bans that society feels threatened by the same possibilities for humans. Does society have the right to intervene so forcefully in the evolutionary processes? What are the implications for biodiversity? Privacy and equity are also a concern. Suppose an insurance company decides to do a DNA test on all its potential clients, and it finds that one of the clients inher- ited part of some DNA sequence from his or her grandmother that makes the client a likely candidate for a heart attack at 45.Although the cause is clear, a cure is not Software fm drug design I ~olecular biology Generate compounds Molecular targeting Bench-scale synthesis Pilot-scale production Large-scale manufacturing 9.10 Management of Technology 401 F1gure9.28 The future of biotech. available yet. As a result, the company refuses to insure the potential client and informs other insurance companies of the risk factor. Is this ethical'? 9.10.5 Summary This brief chapter on biotechnology has been included because the field will experi- ence rapid growth and provide many future career opportunities for people inter- ested in manufacturing engineering. It can be seen that the issues range from ••• Biotechnology Chap. 9 interesting scientific principles that are fundamental to life itself, to the engineering of gene splicing, to the operation of bioreactors, and finally to the ethical issues men- tioned. 9.11 GLOSSARY 9.11.1 Amino Acids The building blocks of proteins. There are 20 different amino acids that link together via peptide bonds during the process of protein synthesis on the surface of the ribo- some (see Transcription and Translation) according to the genetic information on mRNA. 9.11.2 Biar.actors Vessels for biological reaction through fermentation or other transformation processes. Interferon, for example, is manufactured in a genetically engineered fer- mentation process. 9.11.3 Biosensors Combining biology, Ie-design, and IC-microfabrication technologies, biosensors are devices that use a biological element in a sensor. Biosensors work via (a) a biological molecular recognition element and (b) a physical detector such as optical devices, quartz crystals, or electrodes. 9.11.4 Cen The smallest unit of living matter capeblc of self-perpetuation. 9.11.5 Central Dogma The concept in molecular biology in which genetic information passes unidirec- tionally from DNA to RNA to protein during the processes of transcription and translation. 9.11.6 Chromosome A subcellular structure consisting of discrete DNA molecules, plus the proteins that organize and compact the DNA. 9.11.7 Codon A set of three nucleotide bases on the mRNA molecule that code for a specific amino acid. For example, the codon C·G-Urepresents arginine. There are 64 unique codon combinations. Some amino acids can be specified by more than one codon sequence. 9.11.8 DNA (Deoxyribonucleic Acid) The genetic material in every organism. It is a long, chainlike molecule, usually formed in two complementary strands in a helical shape. 9.11 Glossary 403 9.11.9 Enzymes Enzymes are proteins that catalyze reactions. 9.11.10 Fermentation A process of growing (incubating) microorganisms in a substrate containing carbon and nitrogen that provide "food" for the organisms. 9.11.11 Gene The basic unit of heredity, a gene is a sequence of DNA containing the code to con- struct a protein molecule. 9.11.12 GeneCloning One of the most common techniques of genetic engineering, gene cloning is a way to use microorganisms to mass-produce exact replicas of a specific DNA sequence. The cloned genes are often used to synthesize proteins. The basic technique is to construct recombinant DNA molecules, insert the resulting DNA sequences into a carrier mole- cule or vector, and introduce that vector into a host cell so that it propagates and grows. 9.11,13 GeneticCode Figure 9.14 shows the 64 possible codons and the amino acids specified by each. 9.11.14 Genetic Engineering The practice of manipulating genetic information encoded in a DNA fragment to con- duct basic research or to generate a medical or scientific product. Selective breeding is one of the oldest examples of genetic engineering. The much newer gene cloning technology is now fairly common practice. Genetic engineering isused in three areas: to aid basic scientific research into the structure and function of genes, to produce pro- teins for medical and other applications, and to create transgenic plants or animals. 9.11.15 Genome The total genetic information of an organism. 9.11.16 Host A cell used to propagate recombinant DNA molecules. 9,11,17 Natural Selection The process by which a species becomes better adapted to its environment-the mechanism behind the evolution of the species. The process depends on genetic variations produced through sexual reproduction, mutation, or recombinant DNA. Variant furms that are best adapted tend to survive and reproduce, ensuring that their genes will be passed on. Over hundreds and thousands of generations, a species may develop a whole set of features that have enhanced its survival in a particular environment. [...]... scientists in pharmaceuticals Science 26 1 (5 125 ): 197 Watson, 1 D 1968 The double helix Atheneum Focus on biotech- beginners Wrilens and Readers, and biotechnology CHAPTER FUTURE ASPECTS OF MANUFACTURING 10.1 RESTATEMENT OF GOALS AND CONTEXT The goals of this book are to: • Illustrate general principles of manufacturing (Chapters 1 and 2) •Review some of the main manufacturing techniques needed during... new DNA 9 .11 .22 Transcription and Translation The processes by which genetic information on a DNA molecule is used to synthesize proteins During transcription, a strand of mRNA is synthesized from a DNA template, During translation, the genetic information on mRNA is read by tRNA on a ribosome (a cell's protein factory) in order to build the chain of amino acids that form a protein 9 .11 .23 Vector A... commercial enterprise Future Aspects of Manufacturing 408 Chap 10 10.3 FROM THE PAST TO THE PRESENT 10.3.1 Mass Production and Taylorism Chapter 1 reviewed the history of manufacturing It included some details of the industrial revolution (1780 1 820 ),the importance of interchangeable parts (Colt and Whitney), and organized mass production with a division between design and manufacturing (Taylor and Ford) In... to stay on top of manufacturing developments in their field and to pursue distance learning/continuing education opportunities in manufacturing processes that have been outsourced Even though the physical manufacturing process may be outsourced, the internal designers must still keep pace with the knowledge in that field In summary, for all firms,the manufacturing process is always a part of a larger... (7903) I., E Ziff, and B Van Loon 19B3 DNAfor Shuler,M L.19 92 Bioprocess engineering 2 San Diego, CA; Academic Press in Med- Press animals." Science News 140 (10): 148 The university-industrial complex New Haven; Yale Univer- O'Connor, G M.1995 From new drug discovery to bioprocess operations: nology.IEEE Engineering in Medicine and Biology 14 (2) : 2m Rosenfield, Oxford: Engineering In Encyclopedia Press... Biotechnology: Baker, A 1994 Engineers From A to Z Oxford University further biotechnology's Berger, S A, W Goldsmith, Oxford University Press reach Design News 29 : 29 and E R Lewis 1996 Introduction Bruley,D.EI995.An emerging icine and Biology 14 (2) : 20 1 Bud, R 1993 The uses of life: A history of biotechnology Economist 1995 A survey of biotechnology Ezzell, C 1991 Milking engineered Kenney, M 1986 Biotechnology:... study projects or consulting projects, requires a certain amount of flexibility and compromise on everyone's part The book and its related lectures are deliberately a survey of each manufacturing topic and also focus on issues in today's business environment This has two obvious limitations: 406 10 .2 Management of Technology 407 First, as far as depth is concerned, each of the chapters deals with technical...Biotechnology 404 Chap 9 9 .11. 18 Nucleotide A building block for DNA and RNA A nucleotide is composed of three parts: a sugar, a phosphate, and a chemical base In DNA, the bases are adenine (A), guanine (G), thymine (T), and cytosine (C) RNA contains A, G, and C, hut has uracil (U) in place of thymine 9 .11. 19 Proteins Proteins are long-chained molecules containing... a necessary condition for sustained corporate success, and (c) sets performance standards and pay raises based on quality achievement 4 '2 Future Aspects of Manufacturing Chap 10 a service industry to the world.' Even though companies might subcontract specialized manufacturing functions (such as rapid prototyping by SLA), it is still crucial to be working as an integrated team with the subsuppliers... 19 92 presidential campaign, one of then-President George Bush's advisers announced that "it doesn't matter if the United States is making computer chips or potato chips." Luckily, this atntude does not seem to have prevailed into the next century 10.7 Layer II: Compressing Time-to-Market 10.7 LAYER II: COMPRESSING 413 TIME·TO·MARKET For many years, companies focused on product design and treated manufacturing . York 1l'mel; August29, 1999,Section 3.A1so,Sciou:e News, VoL 154, October 10, 1998,p ,23 9;and The New Yorker, June 12, 20 00 p. 66.The Human Genome Project plans to finish sequencing the human genome by 20 03. "food" for the organisms. 9 .11. 11 Gene The basic unit of heredity, a gene is a sequence of DNA containing the code to con- struct a protein molecule. 9 .11. 12 GeneCloning One of the most common. ASPECTS OF MANUFACTURING 10.1 RESTATEMENT OF GOALS AND CONTEXT The goals of this book are to: • Illustrate general principles of manufacturing (Chapters 1and 2) . •Review some of the main manufacturing