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Biochemical Engineering Solutions to Biotec~nologica! Pro6/ems CHARLES L. COONEY Biotechnology is often defined as the integration of biochemistry, microbiology, and process technology for the purpose of manufacturing or environmental management. Thus, biotechnology is a broad area that encompasses much more than genetic engineering and hybridoma tech- nology. Furthermore, the translation of scientific discovery to commer- cial reality requires tremendous skills in process development using both existing and new technology, and, most importantly, it requires the integration of basic biological sciences with chemical process as well as electronic and mechanical engineering. Several seemingly unrelated developments that have occurred simul- taneously over the past several years have stimulated activity in bio- technology. First, the discoveries in genetic engineering that permit one to move DNA between different organisms and to amplify its expression into proteins allow scientists to do things that could not be done before. Second, volatile pricing in traditional feedstocks used in the chemical process industry (CPI) has catalyzed interest in the use of biotechnology to access inexpensive alternative and renewable raw materials for use in chemicals manufacturing. Third, some of the key patents protecting some major products of the pharmaceutical industry either have expired or will expire soon. As a consequence there is need to develop new technology or to improve existing technology used for manufactur- ing these products. Fourth, changing consumer demands, with greater concern for safety, convenience, and environmental impact, require new or improved products. Insulin is one example highly purified 42

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BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY 43 human insulin rather than traditional products is desired. Sweeteners are another example; for instance, aspartame is replacing some of the traditional and less desirable artificial sweeteners. Fifth, biotechnology provides opportunities for developing new products as well as for im- proving the manufacture of existing ones. New technology provides a vehicle for corporate growth by improving existing processes and de- veloping new markets, as exemplified by companies in the business of building manufacturing plants or process equipment. In the sections that follow, problems of the chemical process industry are discussed first. To help assess whether biotechnology can be used to address some of these problems, the second section examines the current biochemical process industry. The third section discusses the use of genetic engineering in addressing CPI problems. Biochemical process development is then discussed, and the concluding section enum- erates problems in process development that need to be solved in order for the results of the molecular biologist to be translated to goods for the consumer. CHEMICAL PROCESS INDUSTRY Biotechnology can solve problems. That, after all, is what engineering is all about. In this regard it is interesting to examine the problems of the chemical process industry, which are summarized in the following list: High feedstock cost Volatile feedstock pricing Overcapacity Energy-intensive processes Environmental and safety concerns There is a strong dependence on feedstock cost, which typically com- prises 50 to 75 percent of the manufacturing cost for commodity prod- ucts. The feedstock costs are quite volatile, and it is not clear where or how fast they are going to change, except that they will generally in- crease. Overcapacity in the industry, which now operates at about 70 percent of nameplate capacity, makes it difficult to implement new technology unless there are very large savings. A strong dependence on energy costs further increases the concern over petroleum feedstock pricing. Lastly, an increasing awareness of environmental and safety factors surrounding the operation of chemical manufacturing plants makes less hazardous processes attractive. Can biotechnology solve some of these problems? Do these problems

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44 NEW FRONTIERS IN BIOTECHNOLOGY of the CPI represent opportunities for biotechnology? The answer is a definite yes. One important attribute of bioprocessing is the possibility of assessing renewable and possibly cheaper raw materials, which could be important to the CPI. Such feedstocks may be lower in cost and may be readily available in local situations. They may provide swing capacity in cases that involve not displacing an existing chemical industry but picking up growth or taking care of fluctuation in demand. In addition, there may be potential for less hazardous operation or less negative environmental impact. BIOCHEMICAL PROCESS INDUSTRY In order to assess whether or not one can use biotechnology to address some of these problems in the CPI, it is important to examine the current biochemical process industry (BPI), exemplified by processes for pro- duction of enzymes, antibiotics, amino acids, organic acid, and so forth, alongside of the CPI. How do the CPI and BPI compare and what are the differences? Figure 1 plots selling price versus annual production volume for a wide variety of biochemical and chemical products (see Table 1~. The correlation between the price in dollars per pound and production in tons per year is quite good over a wide dynamic range from 1 through lo4 1o2 ~9 101 10 10 Oat 3~0 - 12 103 _ 34 On Ella - DSAL RENNET \ O J ~ ~80T.^ 3~e .~0 0e :'oig\a. -~N~Lum ^3 \ O 0~ LACING 3b 0~ \ 0~7 O ~PART^~ off \ O - 6 3~ 0 Olaf all P em ~ HALOhi ~ ~~R .~AOI.N. O WOOD PULP ~ SOL I I I . I I I 1 1 lol 102 1o3 104 105 106 107 108 PRODUCTION (ton/yr) FIGURE 1 Price versus production volume for selected commodity and specialty chem- icals and biochemicals (see accompanying Table 1 for listing of individual products).

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BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY TABLE 1 Price Versus Production Volume for Selected Commodity and Specialty Chemicals and Biochemicals (See accompanying Figure 1) Chemical or Biochemical Production Price (1,000 tons/yr) ($/lb) 1 Ethylene 2 Propylene 3 Toluene 4 Benzene 5 Ethylene dichloride 6 Methanol 7 Styrene 8 Xylene Formaldehyde Ethylene oxide Ethylene glycol Butadiene Acetic acid Phenol Acetone Pyopylene oxide Isopropanol Adipic acid Ethanol (synthetic) Ethanol (term) Dextrose Citric acid Monosodium glutamate Lysine L-glutamate Fructose Penicillin Glucose isomerase Glucoamylase Bacterial amylase Bacterial protease Fungal protease Microbial rennet Pectinase 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Aspartic acid 36 D-HO-phenylglycine 37 D-phenylglycine 38a D-calcium pantothenate 38b D-calcium pantothenate 39 Penicillin 40 Palm oil 41 Cephalosporin 42 Tetracycline 15,000 7,000 5,000 5,000 4,600 4,200 3,300 3,200 1,100 2,600 2,100 1,600 1,400 1,300 1,100 900 800 600 600 700 430 300 350 43 20 22 7 1 0.35 0.35 0.5 0.0 0.0 0.1 4.5 13 3.6 77,000 45 0.25 0.24 0.21 0.23 0.14 0.11 0.38 0.21 0.29 0.45 0.33 0.38 0.26 0.36 0.32 0.44 0.32 0.57 0.27 0.27 0.45 0.80 1.10 5.20 1.80 1.10 20.00 25 30 35 60 70 500 700 1.75 13.6 7.7 6.4 15 19 0.20 0.55 3.4 _ (Continued)

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46 NEW FRONTIERS IN BIOTECHNOLOGY TABLE 1 (Continued) Chemical or Biochemical Production Price (1,000 tons/yr) ($/lb) 43 L-arginine 0.5 13.6 44 L-aspartic acid 0.6 2.7 45 L-glutamine 270 1.8 46 L-lysine 25 7.3 47 D,L-methionine 100 2.9 48 L-phenylalanine 0.1 38 49 L-tryptophan 0.06 44 50 Gluconate 75 0.48 51 Vitamin BE 0.0 ' NOTE: Numbers in left-hand column correspond to individual points on Figure 1. 100 million (108) tons per year, and from $0.10 to $10,000 per pound. It is interesting that the correlation holds for chemical commodities as well as for specialty biochemicals such as amino acids and antibiotics, and even more complex products, such as vitamins, and enzymes. This plot (Figure 1) shows several things. The slope of the line reflects the economy of scale in manufacturing. Products from the biochemical process industry fall on the same line as those of the chemical industry and take advantage of the same economies of scale experienced in com- modity chemicals production. In addition, the vertical distance from the abscissa reflects value added to the initial feedstock. Most biochemicals are made using inexpensive raw materials, such as sugar, and they offer good potential value added. The profit margin depends on the efficiency in transforming these raw materials into products. It is this biochemical problem that needs to be translated into a biochemical process. At this point one begins to see the need for integrating improved conversion yields, better metabolic pathways, and new reaction mechanisms. This requires integrating bio- chemistry, microbiology, and chemical process technology. A number of things will stimulate success in biotechnical routes in manufacturing. One is higher prices for petrochemical feedstocks, which would make the use of biological routes to access renewable resources more important. Another is market growth for biological and chemical products that would require new manufacturing capacity. Continued development of genetic engineering is another stimulant, because this new technology permits doing things that could not be done before, as described below.

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BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY APPLICATIONS OF GENETIC ENGINEERING 47 Genetic engineering is an important tool for the chemical engineer as well as for the molecular biologist. From a chemical engineer's point of view, recombinant DNA and other techniques of genetic engineering permit several things that previously were not possible. First, the ability to introduce foreign DNA into new places means that microorganisms can be made to do or to produce something they did not do or produce before. Second, the expression of that DNA can be amplified into pro- tein products. Third, biochemical pathways can be altered: for instance, an organism previously not capable of making compound C from com- pound B can be altered by the addition of a new enzyme in that cell to allow it to make compound C. Lastly, plant and animal metabolism can be altered through gene therapy. The discussion here will focus only on the first three capabilities. The ability to introduce foreign DNA into a cell is especially impor- tant. From a process point of view it means several things. For instance, it is now possible to produce interferon, insulin, growth hormone, and other protein pharmaceutical products by fermentation. These are not new products, but microbial fermentation is a new way of making them at potentially much lower cost. There is also opportunity for making better products. When isolated from microorganisms rather than from natural sources, material can be produced that is more pure and that does not contain related proteins having other biological functions. It is also possible to use the technology of protein engineering. DNA contains the code for a unique sequence of amino acids that imparts the unique structure responsible for the catalytic or physiologic activity. By altering the DNA, hence altering the protein sequence and thus the structure and functionality of the resulting protein, it is possible to improve the final product. Several years ago this was a dream. Today it is reality, and several successful examples exist. Thus, one can perform molecular engineering to improve a product for the consumer or to improve a process. To achieve this goal it is important to understand the molecular basis for functions in order to manipulate the DNA struc- ture. This understanding is still weak; nonetheless, protein engineering to make better products is very exciting technology. It is possible to reduce manufacturing costs as well as to improve the end product. The ability to amplify DNA by causing a cell to make multiple copies of genes allows one to obtain more product per cell. This will lower the manufacturing cost because less energy, less labor, and less material are required for production. It is also possible to get higher productivity and higher purity. Thus, recombinant DNAis im-

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48 H C COO 3 \ / CH 1 OH \ I CoASH LACTATE \< NEW FRONTIERS IN BIOTECHNOLOGY H3 C\ CSCoA CH OH LACTY L-CoA H2O \ (B) ~~ O H2C= CHC ACRYLYL-CoA [Hi] ~ (C) N\~(A} O CoASH H C CSCoA~ H3 C COO CH2 CH2 PROPIONYL-CoA o SCoA ' H2C =CH C OH Acrylic Acid PROPIONATE FIGURE 2 Metabolic pathways in Clostridium propionicum. The pathway in Clostr~dium propionicum for synthesis of acrylic acid is shown by the solid lines. If reaction A is blocked or eliminated, acrylic acid could be produced from lactic acid. If reaction B were blocked or eliminated and reaction C requiring a new enzyme (shown by the dotted line) were added, acrylic acid could be made from propionic acid. portent to improving process technology, reducing manufacturing cost, and leading to new opportunities for process development. The ability to alter biochemical pathways is important in terms of both old and new products there are opportunities to develop better ways of making existing antibiotics or other biochemicals and to create new pathways for new products. The ability to alter biosynthetic path- ways is important to manufacturing commodity chemicals that hereto- fore could not be made through biological routes. An example is acrylic acid, which is not a usual biochemical intermediate. However, using the pathway shown in Figure 2, it is possible to make cells excrete it and

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BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY 49 produce it by a variety of mechanisms. Through recombinant DNA one could conceivably improve this production route. In this way, through biotechnology, it is possible to gain access to new and cheaper raw materials for the production of commodity products. By now it should be clear that genetic engineering can be used to address some of the problems of the CPI. It is important to understand this role of genetic engineering in both the existing biochemical process industry and in the chemical process industry because it has catalyzed much interest and investment in biotechnology. Let us now consider the realities of how one translates genetic engineering into a process that will deliver a product to the consumer, which is really the primary objective. In other words, the objective is not how to commercialize biotechnology, but how to use biotechnology to commercialize new processes and products. BIOCHEMICAL PROCESS DEVELOPMENT Figure 3 illustrates a typical biochemical process with its various unit operations. Raw materials are pretreated by heat to be sterilized and are then fed to the bioreactor. The bioreactor may be a traditional, batch fermenter or a novel device designed for a specific product. At this point in the process, value is added to the raw materials in synthesis of the final product. This is where genetically engineered organisms function. The important problems are how to control these reactors optimally; how to build them large enough to obtain economy of scale; how to get high productivity; what the limits in productivity are; and, once there is a product, which is invariably in dilute aqueous solution, how to recover it from the cell and the broth. As a consequence of recombinant DNA, problems have arisen that | Feedstock | | Storage | ~ , Raw Materials Preparation and At Pretreatment Ai r _ | Process I Control T Energy Energy Sterilization _ - BIOREACTOR Product - _ Product Recovery Heat Waste FIGURE 3 Schematic flow sheet for a typical biochemical process showing the major unit operations.

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so NEW FRONTIERS IN BIOTECHNOLOGY several years ago did not exist for example, how does one recover a highly purified protein from a microorganism for use in the therapeutic marketplace? This was not a problem several years ago because phar- maceutical materials were not processed in this way. But today it is a problem, and the technology for protein recovery is not well established, especially for use in large-scale operations. An examination of the biotechnology literature, both journal and patent, over the past 20 years reveals that 90 percent of the literature focuses on fermentation or bioconversion. Yet 90 percent of the prob- lems and costs that limit the translation of scale from the test tube to the manufacturing plant exists downstream from the bioreactor and not in the bioreactor itself. While biocatalysis is enabling technology, re- covery of product is needed for realizing a process. What are the limits for biocatalytic processes? Some insight into this question can be gained from Figure 4, which shows productivity Qp as )I / A' > o P max o ___ ! /// Xmax CATALYST CONCENTRATION, X FIGURE 4 Volumetric productivity. Qp (g/liter-h) as a function of catalyst concentration x (g/liter). Sa is the specific activity of the catalyst. r

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BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY 51 a function of catalyst concentration X. It is really the biocatalyst and its exquisite selectivity on which the potential of the biochemical process industry is based. There are two productivity limits. The first is that the maximum productivity is limited by transport phenomena, by the rate at which one can process biological materials. This is a fundamental heat and/or mass transfer limitation. Clearly, there are opportunities for the chemical engineer who has been dealing with these problems in chemical reactors for many years. This problem can also be considered as one of dealing with equipment limitations. The second boundary is a limit on the amount of catalyst that can be put into the bioreactor. By increasing the specific activity, Sa, of the catalyst, which is the role of the molecular biologist and the microbiol- ogist, productivity can be improved and cost can often be reduced. Improvement in productivity clearly requires cooperation between the engineer and the molecular biologist. CONCLUSION Finally, a series of problems in process development need to be solved in order to translate in scale the results of the molecular biologist to a process operating to deliver goods to the consumer. These bottlenecks in the development of biocatalytic processes are as follows: Limitations in productivity Biocatalytic rate Biocatalyst concentration High capital investment System stability New product routes Recovery processes The problems include fundamentals of biocatalysis, heat and mass trans- fer, efficient conversion, and product recovery. Solving them requires integration in process development and integration between disciplines, and presents some very important challenges to those in engineering working closely with chemists and molecular biologists.