4
Current Bioprocess Technology, Products, and Opportunities

Products and services that depend on bioprocessing can be grouped broadly into

  • Biopharmaceuticals. Therapeutic proteins, polysaccharides, vaccines, and diagnostics.

  • Specialty products and industrial chemicals. Antibiotics, value-added food and agricultural products, and fuels, chemicals, and fiber from renewable resources.

  • Environmental-management aids. Bioprocessing products and services used to control or remediate toxic wastes.

This chapter reviews the status of bioprocessing for manufacture of products in categories that are relevant for the next 10 years. Much of the relevent background is derived from an Office of Technology Assessment report Biotechnology in a Global Economy (OTA, 1991).

4.1 BIOPHARMACEUTICALS

The success of biotechnology is seen in the impact of new products and processes. The products include biotherapeutics, specialty chemicals, and reagents. Such as diagnostics, biochemicals for research and enzymes for the food and consumer markets. The purpose of this section is to exmine the state of bioprocessing of biopharmaceuticals , including the status of current research and the needs and opportunities form innovation in bioprocessing for manufacturing of biotherapeutic products. Biotherapeutics include therapeutic proteins, vaccines, therapeutic polysaccharides, diagnostics, and low-molecular-weight pharmaceutical chemicals.



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Putting Biotechnology to Work Bioprocess Engineering 4 Current Bioprocess Technology, Products, and Opportunities Products and services that depend on bioprocessing can be grouped broadly into Biopharmaceuticals. Therapeutic proteins, polysaccharides, vaccines, and diagnostics. Specialty products and industrial chemicals. Antibiotics, value-added food and agricultural products, and fuels, chemicals, and fiber from renewable resources. Environmental-management aids. Bioprocessing products and services used to control or remediate toxic wastes. This chapter reviews the status of bioprocessing for manufacture of products in categories that are relevant for the next 10 years. Much of the relevent background is derived from an Office of Technology Assessment report Biotechnology in a Global Economy (OTA, 1991). 4.1 BIOPHARMACEUTICALS The success of biotechnology is seen in the impact of new products and processes. The products include biotherapeutics, specialty chemicals, and reagents. Such as diagnostics, biochemicals for research and enzymes for the food and consumer markets. The purpose of this section is to exmine the state of bioprocessing of biopharmaceuticals , including the status of current research and the needs and opportunities form innovation in bioprocessing for manufacturing of biotherapeutic products. Biotherapeutics include therapeutic proteins, vaccines, therapeutic polysaccharides, diagnostics, and low-molecular-weight pharmaceutical chemicals.

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Putting Biotechnology to Work Bioprocess Engineering The development of recombinant-DNA and hybridoma technologies has revolutionized the array of pharmaceutical products available. Unlike traditional therapeutics, the pharmaceuticals produced by the new technologies are primarily protein products; they include insulin, growth hormone, &x97; -interferon, OKT-3 monoclonal antibody, tissue plasminogen activator, hepatitis vaccine, and erythropoietin. With the availability of large amounts of those products, new clinical applications are being discovered. For example, it has been discovered that growth hormone is effective in wound healing, in addition to the treatment of pituitary dwarfism. Although regulatory requirements for safety and efficacy lead to long delays in the approval of biotherapeutic products for sale, 15 products had been approved in the United States by the end of 1991 (Table 4.1). Estimates of annual sales range from $3 to 5 billion for 1991 and constitute about 7–10% of total U.S. pharmaceutical sales. Most noteworthy are the increase of more than 10% in annual sales of existing biotherapeutics and the large number (158) of products in the clinical-trial pipeline (Tables 4.2 and 4.3) with an expectation that a substantial number will be approved for therapeutic use. Clearly, biotherapeutics have an important role in improving human health care. The important question to be addressed here is: What are the technological needs in the next decade related to facilitating manufacturing and commercialization of products evolving from biotechnology? To address that question, we first need to examine and understand the current state of bioprocessing. 4.1.1 Proteins from Recombinant Microorganisms Extensive research on eukaryotic gene expression in bacteria, yeasts, plants, insects, and mammals has resulted in many options for producing proteins in recombinant hosts. In spite of the numerous options, most of the products manufactured today are made either in recombinant E. coli or in animal cells, i.e., Chinese hamster ovary (CHO) cells or hybridoma cells. E. coli is the microbial system of choice for the expression of heterologous proteins. No other microorganism is used to produce so large a number of products at high level. Typical levels of foreign protein expressed represent 10–30% of total cellular protein. Rapid progress in the development of E. coli as a host for foreign-gene expression is due mainly to E. coli's having been the focus of intense study over the last 50 years in academic laboratories. The body of knowledge that has accumulated has facilitated the adaptation of this bacterium for foreign-protein expression. Sophisticated cloning vectors, tools for regulated gene expression, and knowledge about the process of protein secretion and the physiology of growth were available in E. coli, and it became the logical choice for heterologous-gene expression.

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Putting Biotechnology to Work Bioprocess Engineering Table 4.1 Approved Biotechnology Drugs and Vaccines Product name Company Indication U.S. Approval Revenuesa 1989 Revenuesa 1990 Epogen TMb Epoetin Alfa Amgen Thousand Oaks, CA Dialysis anemia June 1989 95 300 Neupogenb Granulocyte colony stimulating factor G-CSF Amgen Thousand Oaks, CA Chemotherapy effects February 1991 NA NA Humatrope ®b Somatotropin rDNA origin for injection Eli Lilly Indianapolis, IN Human growth hormone deficiency in children March 1987 40 50 Humulin ® Human insulin rDNA origin Eli Lilly Indianapolis, IN Diabetes October 1982 200 250 Actimmuneb Interferon gamma 1-b Genentech San Francisco, CA Infection/chronic granulomatous disease December 1990 NA NA Activase ® Alteplase, rDNA origin Genentech San Francisco, CA Acute myocardial infarction November 1987 175 200 Protropin ®b Somatrem for injection Genentech San Francisco, CA Human growth hormone deficiency in children October 1985 100 120 Roferon ®-Ab Interferon alfa-2a (recombinant/Roche) Hoffmann-La Roche Nutley, NJ Hairy cell leukemia AIDS-related Kaposi's sarcoma June 1986 November 1988 40 60

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Putting Biotechnology to Work Bioprocess Engineering Product name Company Indication U.S. Approval Revenuesa 1989 Revenuesa 1990 Leukineb Granulocyte macrophage colony stimulating factor GM-CSF Immunex Seattle, WA Infection related to bone marrow transplant March 1991 NA NA Recombivax HB ® Hepatitis B vaccine (recombinant MSD) Merck Rahway, NJ Hepatitis B prevention July 1986 100 110 Orthoclone OKT ® 3 Muromonab CD3 Ortho Biotech Raritan, NJ Kidney transplant rejection June 1986 30 35 Procritb Erythropoietin Ortho Biotech Raritan, NJ AIDS-related anemia Pre-dialysis anemia December 1990 NA NA Hib Titer TM Haemophilus B conjugate vaccine Praxis Biologics Rochester, NY Haemophilus Influenza type B December 1988 10 30 Intron ® Ab Interferon-alpha2b Schering-Plough Madison, NJ Hairy cell leukemia June 1986 60 80     Genital warts AIDS-related Kaposi's sarcoma June 1988 November 1988         Hepatitis C February 1991 NA NA Energix-B Hepatitis B vaccine (recombinant) SmithKline Beecham Philadelphia, PA Hepatitis B September 1989 20 30 a Estimated U.S. revenues in millions of dollars. b Orphan drug. NA = not applicable. SOURCE: OTA, 1991, p. 77.

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Putting Biotechnology to Work Bioprocess Engineering Table 4.2 Conditions for Which Biotechnology-Derived Drugs are Under Development AIDS and AIDS-related complex (ARC) Chemotherapy effects Leukemia Aplastic anemia Cancer Bone marrow transplant Hematological neoplasms Neutropenia Myelodysplastic syndrome Infectious diseases Thermal injury Reperfusion injury related to myocardial infarction and renal transplantation Anemia secondary to kidney disease, AIDS, premature infants, chemotherapy, rheumatoid arthritis Autologous transfusion Hemophilia Corneal transplants Wound healing Chronic soft tissue ulcers Diabetes Wasting syndromes Nutritional and growth disorders Venous stasis Turner's stasis Burns Venereal warts Herpes simplex 2 Hepatitis-B, non-A non-B hepatitis Hypertension Platelet deficiencies Septic shock Pseudomonas infections Heart and liver transplant rejection Malaria Need for cervical ripening to facilitate childbirth Myocardial infarction Deep vein thrombosis Acute stroke Pulmonary embolism   SOURCE: OTA, 1991, p. 77.

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Putting Biotechnology to Work Bioprocess Engineering Table 4.3 Additional Indications for Approved Drugs Drug Approved Indications Additional Indications EPO Dialysis anemia, AIDS-Related anemia Autologous transfusion, prematurity, rheumatoid arthritis, chemotherapy Tissue plasminogen activator Acute myocardial infarction Deep vein thrombosis, acute stroke, pulmonary embolism Interferon &x97; -2a Hairy cell leukemia, AIDS-related Kaposi's sarcoma, hepatitis C Cancer, infectious disease, genital herpes, colorectal cancer, chronic and acute hepatitis B, chronic myelogenous leukemia, gastric malignancies, HIV-positive ARC, AIDS Interferon &x103; -2b Hairy cell leukemia, genital warts, AIDS-related Kaposi's sarcoma Genital herpes, superficial bladder cancer, basal cell carcinoma, chronic and acute hepatitis B, non-A non-B hepatitis, delta hepatitis, chronic myelogenous leukemia, HIV   SOURCE: OTA, 1991, p. 77. Expression in E. coli can now be designed for either intracellular accumulation of the heterologous protein in the cytoplasmic space or translocation of the protein across the cytoplasmic membrane from the cytoplasmic space into the periplasmic space. After translocation, the protein can accumulate within the periplasmic space or might be released to the surrounding medium. If the protein is secreted and accumulated within the cytoplasmic space, it normally aggregates into large inclusion bodies visible with a light microscope. These must be isolated, solubilized, and folded to obtain an active molecule. Isolation and solubilization are routine, but folding to an active form is difficult with present technology. Intracellular accumulation often has the additional disadvantage of producing a substance with an extra amino acid on the N terminus of the protein. Several signal sequences are now available to drive the secretion of eukaryotic proteins across the bacterial cytoplasmic membrane. Occasionally, that results in the formation of properly folded, bioactive proteins. More often, however, the secreted proteins also accumulate as aggregates in

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Putting Biotechnology to Work Bioprocess Engineering the periplasmic space; again, it is necessary to isolate, solubilize, and fold the proteins to their proper conformation. Perhaps the most exciting application for secretion of proteins from E. coli has been the demonstrated ability to produce active fab fragments (the binding portion of antibodies) directly; these will have a variety of uses as assay reagents, immunoaffinity ligands, and therapeutics. For both intracellular and secreted eukaryotic proteins, proteolytic degradation in E. coli is a problem. Several approaches have been taken to reduce undesirable proteolysis, including the expression of fusion proteins and the elimination of specific proteases by host-cell mutation. The latter approach has been useful, but continued removal of proteases can be expected to affect general cellular metabolism adversely. Mistranslation has also been an occasional problem, but published technology now exists to minimize it. In summary, E. coli expression of eukaryotic proteins has been an important ''workhorse'' for the production of rDNA proteins. The cells grow and express rDNA proteins rapidly and in high quantities. They also are easily modified genetically and generally require inexpensive growth media. However, the system is often limited by its inability to produce intact, properly folded proteins and by a limited ability to yield posttranslational modifications, such as glycosylation and specific proteolytic modification. Nonetheless, the system has enabled the commercialization of such products as human insulin, human growth hormone, human α-interferon, and human γ-interferon. 4.1.2 Inclusion Bodies High levels of protein synthesis have been obtained with several intracellular expression systems, particularly in E. coli. High expression of a foreign protein in the cytoplasm of E. coli often results in the accumulation of nonnative aggregates called inclusion bodies. Isolation of inclusion bodies by centrifugation has become an important first step in the purification and recovery of recombinant proteins. Extensive protein-chemistry studies have revealed substantial fundamental information on the mechanism of inclusion-body formation. Various solubilization agents have been defined (strong chaotropes, detergents, and organic solvents) for use in recovery of active proteins; the process requires unfolding the protein with strong denaturants and refolding to an active monomer. Studies of the refolding of denatured proteins both in vitro and in vivo indicate that aggregates derive from specific partially folded intermediates and not from mature native or fully unfolded proteins (Mitraki and King, 1989). Those discoveries focused attention on the properties of intermedi-

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Putting Biotechnology to Work Bioprocess Engineering ates (as distinct from native states) and the factors interacting with them, such as the intracellular cytoplasmic environment, cofactors, and molecular chaperones. Molecular chaperones were first identified as host proteins needed for phage morphogenesis and have recently been identified as heat-shock proteins (Goloubinoff et al., 1989). In a recent review (Pelham, 1986), it was proposed that heat-shock proteins can act as molecular chaperones and prevent aggregation by binding to hydrophobic regions of partially unfolded polypeptide chains. On the basis of those fundamental discoveries, studies are under way to mimic the mechanics of mammalian protein synthesis (compartmentation, interprotein interactions, and posttranslational modifications) in bacteria. With rational selection of the characteristics necessary for correct maturation, it might be possible to direct the fate of the intermediates toward the native conformation. Alternatively, it might be possible to use molecular chaperones to repair and disaggregate proteins outside the cell before releasing them for refolding to the active monomer. 4.1.3 Mammalian Host Systems Production of heterologous proteins by mammalian cells has usually used CHO cells or hybridoma cells. Initially, hybridoma cells were the only hosts used for antibody production. More recently, CHO cells and mouse myeloma cells have also been used. CHO cells are generally able to produce bioactive mammalian proteins that are glycosylated and properly folded. As yet, the system is often not able to effect specific proteolytic maturation, except to remove the secretion-signal sequence. Although bioactive molecules are usually formed by CHO cells, the product is a mixture of many subforms that differ in degree of glycosylation, electrostatic charge, the presence of proteolytic clips, and other possible modifications. The modifications do not necessarily compromise the potency or safety of the product, but it is essential that the process be carefully controlled to ensure that the same profile of molecular variants is produced from each batch. Mammalian cells have the advantage of being able to produce complex, bioactive molecules. However, they grow and express proteins at approximately one-twentieth the rate of E. coli. That has the effect of increasing capital and labor costs for protein production. The cells also require expensive media (although efforts are under way to reduce these costs) and have additional, although tractable, regulatory and safety concerns, such as concern about undetected viral contamination. In spite of those limitations, CHO-cell production of biopharmaceuticals is an established and important technology that has enabled the delivery of such important therapeutics as tissue plasminogen activator and erythropoietin.

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Putting Biotechnology to Work Bioprocess Engineering 4.1.4 Other Hosts for Heterologous Gene Expression Several new systems for the production of heterologous proteins are under development. They include such new bacterial systems as Bacillus and Streptomyces, the filamentous fungi, insect cell lines of Drosophila , and systems that rely on the baculovirus expression system, Xenopus oo-cytes, and yeast. Although none of these is as developed or has been studied as extensively as E. coli, each has advantages and disadvantages. In Bacillus, for example, strains that lack most of the usual proteases have been generated. Streptomyces does not compete with E. coli in level of expression, but is useful for making small quantities of soluble, nature-identical product. Filamentous fungi, such as Neurospora crassa and Aspergillus nidulans, can secrete copious quantities of protein and have long been used in the pharmaceutical industry to make natural products. Yeast has been used to produce rDNA proteins, such as IGF-1 and human serum albumin; in spite of substantial effort, it has not been used as extensively as E. coli or CHO cells. 4.1.5 Isolation and Purification Isolation generally denotes the separation of the product from the bulk of the producing organism. The disposition and state of the expressed protein affect the isolation procedure. For mammalian cells and some E. coli, Streptomyces, Bacillus, and yeast products, the protein is released from the cell into the surrounding medium, and isolation is effected by a solid-liquid separation step, usually centrifugation or microfiltration or ultrafiltration. If the product has aggregated either in the cytoplasmic or periplasmic space, isolation is more involved. Generally, the cell is first lysed by mechanical, chemical, or enzymatic treatment (or a combination). In some cases, the more dense aggregate can be separated by centrifugation from most of the soluble and insoluble cell components; in other cases, the aggregate is first solubilized while still in the soluble protein mixture. Purification of the protein is a critical and often expensive part of the process. It might account for 50% or more of the total production cost. Purification has several objectives: to remove contaminating components from the host organism, i.e., other proteins, DNA, and lipids; to separate the desired protein (or family of proteins) from undesired variants of the desired protein; to remove and avoid the introduction of endotoxin; to inactivate viruses; to obtain required yields at acceptable cost; to avoid chemical or biochemical modification of the protein; and to make the process consistent and reliable. In some cases, the first and additional objective is to fold the protein into its desired conformation. Much of the accumulated knowledge about protein purification is the

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Putting Biotechnology to Work Bioprocess Engineering property of individual companies. However, the available information suggests a general consistency in the type and order of process steps. The most common individual operations are centrifugation, filtration, membrane separation, adsorption separation, and chromatography. Regulatory and safety concerns have combined with the desire for stable liquid formulations to motivate the removal of host-organism proteins to a maximal degree. Measurement of those contaminants requires sophisticated assays capable of detecting a spectrum of possible contaminants at a few parts per million of the product protein. The presence of undesired variants of the target protein has motivated the development of techniques to detect and separate (on a large scale) proteins modified at one of several hundred amino acids. The difficulty of separation can often be decreased by changing the organism or culture conditions to produce a more uniform protein. However, it is still necessary to combine a series of purification steps each of which separates according to a different principle. Ultrafiltration steps are often used between separation steps to concentrate the protein solution or to make the buffer solution compatible with the next separation step. The final steps are designed to place the purified protein in the solution used for the product form. The complexity of the individual purification steps and the need to be able to integrate them into a manufacturing system translate into a major opportunity for bioprocessing engineering as the process moves from the bench to the plant. Research and development in purification, scaleup integration, and system design will continue to have high priority. 4.1.6 Protein Engineering Advances in molecular biology have provided researchers with the opportunity to develop increasingly rational approaches to the design of therapeutic drugs. This technology, when used with computer-assisted molecular modeling, is called protein engineering. Protein engineering combines many techniques, including gene cloning, site-directed mutagenesis, protein expression, structural characterization of the product, and bioactivity analyses; it can be used to modify the primary sequence of a protein at selected sites to improve stability, pharmacokinetics, bioactivity, and serum half-life. A second application of protein engineering is the design of hybrid proteins that contain regions that aid separation and purification. That is achieved by introducing, next to the structural gene for the desired product, a DNA sequence that encodes for a specific polypeptide "tail." The tails can be inserted at the N or C terminal of the protein to yield a fusion protein with special properties that facilitate separation. Such genetic modifications

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Putting Biotechnology to Work Bioprocess Engineering can be designed to take advantage of affinity, ion-exchange, hydrophobic, metal-chelate, and covalent separations. Examples of affinity tails and the corresponding ligands are given in Table 4.4. The special properties of fusion proteins allow crude microbial extracts to be passed over an adsorbent that binds specifically to the tail, so that the desired product is retained and contaminants pass through. After elution and treatment to remove the tail, the product is purified further by standard methods, such as size-exclusion chromatography or high-performance liquid chromatography (HPLC). 4.1.7 Glycobiology Recent studies of receptor biology have resulted in fundamental discoveries about the role of complex oligosaccharides in disease, in modulation of protein function, and as anchors for integral membrane glycoproteins. As additional glycoproteins are identified and cloned, there is an increasing need for more effective chromatographic methods, production systems that mimic mammalian glycosylation patterns, and fast, reproducible analytical methods to minimize microheterogeneity during manufacture. Variability in oligosaccharide biosynthesis has been found to be an important source of heterogeneity for glycoproteins produced by eukaryotic cells (Marino, 1989). Glycoprotein oligosaccharides are covalently attached to proteins through the amino acid serine (O-linked) or asparagine (N-linked). If a selected carbohydrate type and site are required for bioactivity of a candidate glycoprotein, the expression system must be carefully selected. Table 4.4 Examples of Affinity Tails Tail Ligand &x98; -Galactosidase β-D-Thiogalactoside Protein A IgG Protein G IgG or albumin Chloramphenicol acetyl transferase β-Amino chloramphenicol Glutathione S-transferase Glutathione Avidin Biotin Streptavidin Biotin Arginine Anion exchange Glutamate or aspartame Cation exchange Cysteine Thiol Histidine Ni2+, Cu2+, Zn2+ Phenylalanine Phenyl Antigenic peptide Monoclonal antibody SOURCE: Derived from Hammond et al., 1991.

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Putting Biotechnology to Work Bioprocess Engineering ment of efficient, integrated manufacturing systems will be more important in bioprocess engineering. All the issues now discussed in other types of manufacturing—design for manufacturing, intrinsic manufacturability, integrated systems of sensors and controls, and integrated information-processing systems for manufacturing—will become relevant for bioprocessing. Development of the appropriate, sensored systems is probably the key element in the early stages of efficient computer-managed biological manufacturing systems. 4.2 SPECIALTY BIOPRODUCTS AND INDUSTRIAL CHEMICALS Specialty products are chemicals, proteins, microbial substances, and other biologically derived materials whose volume of annual domestic or worldwide use is measured in tons. Specialty products tend to have sale prices less than 3 times their manufacturing costs. Human therapeutics, in comparison, have sale prices 10–12 times their manufacturing costs and annual volumes of use measured in kilograms to hundreds of kilograms. Specialty chemicals and biologicals are further defined to include antibiotics; food products, additives, and processing aids; oxygenated chemicals and fuel additives; biological agents used in agricultural and environmental applications; value-added products derived from agricultural commodities and other renewable resources; and energy-related products (OTA, 1991). Biotechnology will affect the formulation of products in agriculture, energy, the environment, and human health and has potential to surpass the computer industry in size and importance, because of the pervasive role of biologically produced substances in everyday life (Council on Competitiveness, 1991). Bioprocess engineering is an essential component for rapid transition of bioproducts from the laboratory to a manufacturing scale able to provide the benefits of biotechnology on a large scale at a reasonable cost. The opportunities for biotechnology products to affect the U.S. chemical industry are substantial. In 1990, chemical shipments were estimated at about $300 billion, and the chemical industry employed a million people and produced more than 50,000 chemicals and formulations (OTA, 1991). According to the Office of Technology Assessment (OTA), biotechnology will likely be used in the chemical industry in the production of fermentation-derived chemicals and synthesis of complex chemicals. It is envisioned that improvement of production processes used by major chemical companies will ''be introduced without the fanfare that has accompanied other biotechnology developments.'' The impact of biotechnology is expected to be incremental and unheralded and result in improvements in productivity when, for example, enzymes are used to replace difficult or expensive steps in chemical synthesis (OTA, 1991).

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Putting Biotechnology to Work Bioprocess Engineering 4.2.1 Enzyme Technology and Specialty Bioproducts Specialty products derived from biotechnology processing range from low-value commodity materials, such as fuel ethanol, to catalytic enzymes derived from recombinant Bacillus species for use in detergents. The worldwide enzyme market is estimated to be about $650 million, of which 50% is attributed to enzymes used in making detergents (Layman, 1992). That group of products is sensitive to processing costs, and manufacture is carried out at a scale in which bioprocess engineering is an important component of technology and design. Specialty products also include flavor enhancers, such as monosodium glutamate, and amino acids. In some cases, recombinant technology has been used to improve the productivity of microorganisms in what is otherwise a mature art of the fermentation industry. 4.2.2 Biopesticides The search for biodegradable and environmentally compatible pesticides will affect the markets dominated by synthetic insecticides, of which $2 billion worth is sold annually in the United States. Recent examples are Bacillus thuringiensis (BT) insect toxins (produced by Pfizer for Ecogen's biopesticide products) and azadirachtin derived from the oil of neen tree seeds (Anonymous, 1992; Stone, 1992). Alternative strategies for BT toxins are to introduce the genes for the toxins (proteins) into other microorganisms that are found in parts of plants attacked by pests and to introduce an insect-resistance gene directly into a plant (OTA, 1991; Crawford, 1988). Sales of BT products have grown from $2.4 million in 1980 to $10.7 million in 1989 and are expected to grow at 11%/year (Feitelson et al., 1992). Another important biopesticide will result from the use of baculovirus; this technology has not been realized, but is clearly imminent (see Chapter 6). 4.2.3 Microalgae and New Chemicals Although over 50,000 microalgal species are known, fewer than 10 have been studied intensively or commercially exploited (Behrens and Delente, 1991). Recent efforts to cultivate phototropic microalgae in bench-and pilot-scale photobioreactors have yielded an exciting new group of organisms to screen for novel chemical entities. The National Cancer Institute has awarded screening contracts for cyanobacteria and protozoal microalgae; more than 5% of microalgal strains have shown initial activity in primary screens. Microalgae hold promise as an untapped source of new chemicals and will certainly be used in future manufacturing processes.

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Putting Biotechnology to Work Bioprocess Engineering 4.2.4 Plant-Cell Tissue Culture Plant-cell tissue culture is used commercially in Japan to produce the pigment shikonin and ginseng as a health food. In Germany, a 75,000-liter facility has been built and used to produce an immunoactive polysaccharide, although it is not a commercial process. In Israel, bioreactors form an integral part of a micropropagation process used for commercial production of 14 plant species. In the United States, there are no commercial plant-cell culture processes (Payne et al., 1991). However, the production of taxol, a chemotherapeutic, by plant-cell culture is being actively pursued by at least two companies. Taxol is in clinical trials against a wide range of cancers. Taxol is critically scarce, because the current source is the bark of the Pacific yew tree-an uncommon and very slow-growing tree. Alternative sources are essential, and plant-cell culture is one of the potential solutions to the supply problem. 4.2.5 Research Needs and Opportunities The examples of current manufacturing technology for bioproducts just cited illustrate the wide array of processing steps that are involved in their manufacture. The economics of the individual steps are reflected in product quality, cost, and final application. A wide array of biosciences are required to develop transformed microorganisms, new biocatalysts, and a fundamental understanding of interactions of proteins with their environments; these all go into the final synthesis of ideas and techniques for production of new molecules or for new methods for producing existing bioproducts. The diversity of bioprocess-engineering skills that must be applied to such a wide array of bioproducts is often overlooked. Challenges that bioprocess engineering faces include specialty-equipment design that meets regulatory, biological, and economic constraints; integration of manufacturing processes into environmentally acceptable and economically feasible process concepts; and rapid purification and monitoring of purification processes to obtain high quality, high purity, and consistent output. Many specialty products are obtained through fermentation in dilute solution. There is an important opportunity to improve the economics of their production by improving the energy efficiency and selectivity of removal of water. Energy-efficient methods for recovering these products from dilute aqueous solutions are needed to reduce the cost of their synthesis and handling and the volume of downstream processing. Improvements in the design of bioreactors and in conditions for cultivating microorganisms and obtaining products are also needed. Both solid-substrate and submerged fermentations present major opportunities for biomanufacturing and improvement through bioprocess engineering. In the

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Putting Biotechnology to Work Bioprocess Engineering case of solid-substrate fermentations, the application of enzymes such as would be used in biopulping requires efficient solid-liquid contact. Bioprocess engineering solutions are needed to enable the penetration of enzyme solutions or microbial inoculations into solid material, which must be rapidly processed on a relatively large scale. For submerged fermentations, product inhibition presents an important challenge to bioprocess engineers, because it limits both the rate and the extent of product formation. Novel bioreactor designs, membrane-separation technologies, and processing aids could all improve the economics of producing specialty bioproducts. Another approach is through cell engineering to alter the fundamental physiology of microbial membranes and to moderate the effects of otherwise toxic extracellular fermentation products. The challenges and opportunities of bioprocessing technology for the manufacture of specialty bioproducts are similar to those related to biopharmaceuticals, except for the special hazards of pathogens and the specialty-product emphasis on economics of production—the lower the value of the product, the more intense the economic focus. 4.3 ENVIRONMENTAL APPLICATIONS The application of biotechnology on an environmental scale encompasses subjects ranging from pollution control and bioremediation to mining. Inexpensive yet effective methods are being sought for cleaning up hazardous wastes and other contaminants. Present technologies used are land-filling and incineration, but these are becoming increasingly unpopular, expensive, and difficult to institute, because of stricter regulations and public objections. The magnitude of the problem is seen in the amount of national expenditures for environmental cleanup, estimated to be $115 billion for 1990 (Carlin, 1990). Forecasts for the future are even higher. There is a need for more effective technology that uses such organisms as bacteria or such constituents as enzymes (Hinchee and Olfenbuttel, 1991 a,b). Biological systems have been used to degrade or transform objectionable chemicals and materials into more environmentally benign substances for years on a very large scale. For example, today's municipal wastewater-treatment plants use bioprocess-engineering principles to dispose of sewage and to provide clean and safe drinking water. Composting is a practice known to many, including weekend gardeners, and is a use of microorganisms to degrade gardening and other wastes. The recent attention to the environment has focused some bioprocessing technology on the transformation of hazardous wastes and the use of biological processes that produce desired products but little or no waste byproduct. Current bioproducts with markets of environmental importance can be grouped into reagents for pollution control, agriculture, mining, and oil

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Putting Biotechnology to Work Bioprocess Engineering recovery. An excellent summary is found in the recent OTA report Biotechnology in a Global Economy (OTA, 1991), and we quote from that document later. 4.3.1 Bioremediation Bioremediation refers to the use of entire organisms (mostly soil microorganisms) or selected constituents of microbial cells (mostly enzymes) for chemical transformations. Bioremediation transforms a toxic substance into a harmless or less toxic substance. Ideally, the toxic substance is transformed into carbon dioxide and water. If the toxic substance contains a metal or a halogen, such as chlorine or fluorine, there will be additional side-products (perhaps the free metal atom or its ion or a halide ion). Mineralization is the term used to describe the complete degradation of a chemical substance to water and carbon dioxide. Bioaugmentation, another frequently used term, involves the deliberate addition of microorganisms that have been cultured, adapted, and enhanced for specific contaminants and conditions at the site. Microorganisms used in bioremediation include aerobic (which use free oxygen) and anaerobic (which live only in the absence of free oxygen). Aerobic microbes have been the organisms of choice for degrading hazardous wastes. Bioremediation is practiced in two modes—in situ and ex situ. In situ bioremediation involves the use of microorganisms to degrade wastes at the site (both on and below the surface) and avoid excavation of contaminated soil and transfer to different locations. Surface remediation is used to treat the top parts of the soil through aeration by the addition of microorganisms, nutrients, and water. Subsurface bioremediation uses microorganisms already in the soil and groundwater and adds oxygen and nutrients. Ex situ treatment involves the excavation of contaminated soil and its transfer to appropriate treatment sites, i.e., bioreactors. The contaminated soil is aerated and treated with nutrients to provide an active environment for the microorganisms of choice. Treatment continues until the soil is sufficiently clean and can be returned to the site. Ex situ techniques are varied but can involve slurry-phase treatments that combine contaminated soil or sludge in bioreactors or solid-phase treatments that involve placing contaminated soils in lined treatment beds. Bioremediation of water or leachate includes treatment with special bioreactors or filters that contain an active film of microorganisms. The choice of method involves many factors, including the contaminant, the site, and the costs that can be borne. Ex situ treatment is usually very expensive (e.g., $100–1,000 per cubic meter of soil). Most often, the microorganisms are expected to reproduce in situ. Encouraging in situ reproduction is a challenge that is being addressed in small experiments. The only major attempts have been for oil spills, partic-

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Putting Biotechnology to Work Bioprocess Engineering ularly the combined Environmental Protection Agency-Exxon tests with fertilizers in Prince William Sound to clean beaches of the Valdez oil spill. The results were encouraging, although not spectacular (OTA, 1991, p. 134). The OTA report concludes (p. 140): Although bioremediation offers several advantages over conventional waste treatment technologies, several factors hinder widespread use of biotechnology for waste cleanup. Relatively little is known about the scientific effects of micro-organisms in various ecosystems. Research data are not disseminated as well as with research affecting other industrial sectors. This is caused by limited Federal funding of basic research and the proprietary nature of the business relationships under which bioremediation is usually used. Regulations provide a market for bioremediation by dictating what must be cleaned up, how clean it must be, and which cleanup methods may be used; but regulations also hinder commercial development due to their sheer volume and the lack of standards for biological waste treatment. Although some research is being conducted on the use of genetically engineered organisms for use in bioremediation, today's bioremediation sector relies on naturally occurring micro-organisms. Scientific, economic, regulatory, and public perception limitations that were viewed as barriers to the development of bioremediation a decade ago still exist. Thus, the commercial use of bioengineered micro-organisms for environmental cleanup is not likely in the near future. The report also summarizes the current prospects for genetically engineered micro-organisms in bioremediation (OTA, 1991, p. 139): Some basic research is underway on the use of genetically engineered microbes for waste cleanup. The first out-of-laboratory applications of genetically engineered microbes for waste cleanup will be done in bioreactors, because conditions for microbial survival and monitoring are easier to control in a closed system than in an open field. Today's bioremediation sector continues to rely on naturally occurring micro-organisms. Due to scientific, economic, regulatory, and public perception reasons, the imminent use of bioengineered micro-organisms for environmental cleanup is not likely to happen in the near future. More needs to be learned about naturally occurring microbes—much less those that are genetically engineered. The lack of a strong research infrastructure, the predominance of small companies, the lack of data sharing, and the existence of regulatory hurdles all serve as dominant barriers to commercial use of genetically engineered organisms. Bioremediation promises lower costs than other types of technology for cleaning up the environment. No academic or regulatory agency has

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Putting Biotechnology to Work Bioprocess Engineering published an analysis of the costs of biological treatment compared with other technologies, such as incineration. The only available information is in individual companies' marketing materials. The present committee recommends analysis of that type. 4.3.2 Point-of-Source Biocontrol A worldwide industry of pollution biocontrol in sewage treatment has existed since the nineteenth century. The various clean-water acts in the United States have stimulated technology advances in the industry since World War II. We will not cover that special application further here, except to the extent that modern biotechnology and transfer of new bioprocessing technology might affect it. A perhaps-attractive application of biotechnology and bioprocess engineering is in point-of-origin control of pollutants before they disperse into the environment. Many industries—such as the medical industry, electronics, and polymers—are important sources of waste solvents. Their hazardous disposal has caused much groundwater pollution and is a major market for in situ bioremediation. An appealing concept is to offer those sources a biocontrol process at their own sites that could eliminate the environmental hazard. No such processes are on the market, except the traditional sewage-treatment facility. Because of the characteristic biochemical variability of the influx, such systems often fail. Given the importance of the subject to both the environment and the industries, the committee recommends a study of it. 4.3.3 Agriculture Potential environmental applications of genetically engineered organisms in agriculture are varied (see Table 4.5). Genes have been introduced into several plant species to confer resistance to or tolerance of particular herbicides. Plants have also been better engineered to resist disease and pests. Most DNA work on animals focuses on altering livestock, poultry, or fish to improve reproductive performance, weight gain, or disease resistance. Planned introduction of genetically engineered organisms into the environment, often called deliberate release, was the focus of an earlier OTA report. 4.3.4 Mining Natural microorganisms have been used for mineral leaching and metal concentration. No federal funding directly supports microbiological mining, however, and commercial activity is sparse. Some international research in biohydrometallurgy is proceeding in Canada, South Africa, the United Kingdom, and the United States. The Canadi-

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Putting Biotechnology to Work Bioprocess Engineering Table 4.5 Some Potential Uses of Biotechnology in Agriculture (a) Microorganisms Bacteria as pesticides: "Ice-minus" bacteria to reduce frost damage to agricultural crops Bacteria carrying Bacillus thuringiensis toxin to reduce loss of crops to dozens of insects Mycorrhizal fungi to increase plant growth rates by improving efficiency of root uptake of nutrients Nitrogen-fixing bacteria to increase nitrogen available to plants and decrease the need for fertilizers Viruses as pesticides: Insect viruses with narrowed host specificity or increased virulence for use against specific agricultural insect pests, including cabbage looper, pine beauty moth, cutworms, and other pests Vaccines against animal diseases: Swine pseudorabies Swine rotavirus Vesicular stomatitis (cattle) Foot and mouth disease (cattle) Bovine rotavirus Rabies Sheep foot rot Infectious bronchitis virus (chickens) Avian erythroblastosis Sindbis virus (sheep, cattle, chickens) (b) Plants Herbicide resistance or tolerance to: Glycophosphate Atrazine Imidazolinone Bromoxynil Phosphinotricin Disease resistance to: Crown gall disease (tobacco) Tobacco mosaic virus Pest resistance: BT-toxin protected crops, including tobacco (principally as research tool) and tomato Seeds with enhanced antifeedant content to reduce losses to insects while in storage Enhanced tolerance to environmental factors, including: Salt Drought Temperature Heavy metals

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Putting Biotechnology to Work Bioprocess Engineering Enhanced marine algae: Algae enhanced to increase production of such compounds as B-carotene and agar or to enhance ability to sequester heavy metals (e.g., gold and cobalt) from seawater Forestry: Trees engineered to be resistant to disease or herbicides, to grow faster, or to be more tolerant to environmental stresses (c) Animals Livestock and poultry: Livestock species engineered to enhance weight gain or growth rates, reproductive performance, disease resistance, or coat characteristics Livestock animals engineered to function as producers for pharmaceutical drugs Fish: Triploid salmon produced by heat shock for use as game fish in lakes and streams Fish with enhanced growth rates, cold tolerance, or disease resistance for use in aquaculture Triploid grass carp for use as aquatic weed control agents   SOURCE: OTA, 1991. an Center for Mineral and Energy Technology is the leading government research agency in the field. One focus for the Canadians is uranium bioleaching; one mine is now bioleaching 90,000 lb of uranium per month. The biological mitigation of acidic mine drainage is another Canadian project. Research is slow, however, because of the economics of the mineral market. As long as metals are plentiful and easily mined, no economic advantage is realized by microbiological mining. 4.3.5 Microbial-Enhanced Oil Recovery* It has been estimated that more than 300 billion barrels of U.S. oil cannot be recovered by conventional technology and might be accessible through enhanced oil production. That volume is 2.5 times as large as the amount of oil produced by the United States since 1983. The actual enhanced oil recovery has been low—no greater than 5% of total U.S. production, even though various Department of Energy incentives have been available. Other countries, such as Canada, have projected that by the year 2010 one-third of its oil recovery will use enhanced techniques. In recent years, *   Adapted from the OTA (1991) report.

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Putting Biotechnology to Work Bioprocess Engineering advanced oil-drilling techniques have enhanced overall yield, and it is expected that these techniques, not microorganisms, will satisfy oil companies' needs for greater yield in the short term. Although most of the major oil companies have in-house staff investigating and perfecting microbial-enhanced oil recovery (MEOR), the methods' low cost might appeal more to small-field operators, who have already pumped and sold the easy-to-get component of their fields. MEOR is not predictable; like the use of microorganisms for hazardous-waste remediation, the use of microorganisms for oil recovery is site-specific. Individual oil deposits have unique characteristics that affect the ability of microorganisms to mobilize and displace oil. An understanding of the microbial ecology of petroleum reservoirs is a prerequisite to the development of any MEOR process, whether microbial or not, because an inappropriate design might accelerate the detrimental activities of microorganisms (e.g., corrosion, reservoir souring, and microbial degradation of crude oil). Basic environmental-biotechnology research under way for contaminated soil and groundwater will provide much needed information to those working on MEOR, who face several serious challenges (OTA, 1991): Better biochemical and physiological understanding of microorganisms already present in oil reservoirs. Development of microorganisms that degrade only less-useful components of oil. Screening of microorganisms for production of surfactants and viscosity enhancers and decreasers. 4.3.6 Research Needs and Opportunities The committee concurs with the OTA report (OTA, 1991) that immediate opportunities for bioprocessing, particularly those which would use genetically engineered microorganisms, exist. The impact of bioprocessing on environmental remediation and industrial waste control could be tremendous over the longer term. The technical aspects of environmental issues are broad and complex and the technical elements of the opportunities ill-defined, and the committee recommends that a study be carried out to set priorities. 4.4 REFERENCES Anonymous. 1992. Ecogen, Pfizer sign production agreement. Chem. Eng. News 70(3):7. Bailey, J. E. 1991. Toward a science of metabolic engineering. Science 252:1668–1681. Behrens, P. W., and J. J. Delente. 1991. Microalgae in the pharmaceutical industry. BioPharm 4(6):54–58.

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