National Academies Press: OpenBook

Biotechnology: An Industry Comes of Age (1986)

Chapter: The Molecular and Microbial Products of Biotechnology

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Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
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Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
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Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
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Page 16
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 17
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 18
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 19
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 20
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 21
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 22
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 23
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 24
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 25
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 26
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 27
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 28
Suggested Citation:"The Molecular and Microbial Products of Biotechnology." National Academy of Sciences, National Academy of Engineering, and Institute of Medicine. 1986. Biotechnology: An Industry Comes of Age. Washington, DC: The National Academies Press. doi: 10.17226/18677.
×
Page 29

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

2 The Molecular and Microbial Products of Biotechnology THE USE OF BIOTECHNOLOGY in industry often entails a fundamen- tal shift in manufacturing procedures. Biotechnology is based on biological synthesis, usually in water-based solutions at close to room temperature, rather than on chemical synthesis, which often takes place at high temperatures and pressures. This basic attribute of biotechnology gives rise to much of its promise as well as to many of the problems encountered in its large-scale applications. The molecular products of biotechnology fall into three overlapping categories: new substances that have never before been available, rare substances that have not been widely available, and existing sub- stances that can be made more cheaply through biotechnology. Many of these substances are targeted at human health care: genetically engineered microorganisms can be used to produce hormones, immune regulators, vaccines, blood products, antibodies, antibiotics, and many other biologically active molecules. Other commercial sectors, such as the food additive and specialty chemicals industries, are also investigating the use of genetically engineered organisms to make new or scarce products or to make This chapter includes material from the presentations by Philip Leder and William E. Paul at the Symposium on Biotechnology: Creating an Environment for Technological Growth. 14

MOLECULAR AND MICROBIAL PRODUCTS 15 existing products more cheaply. Enzymes, amino acids, vitamins, high-grade oils, adhesives, and dyes are all examples of substances that could be manufactured through biotechnology. As the science evolves and production costs drop, even some industrial chemicals now made from petroleum and natural gas feedstocks might be produced by microorganisms. The molecular products of biotechnology are made through fermen- tation processes, and the design of cost-efficient fermentors and asso- ciated production techniques is a major concern in the industry. But biotechnology will also yield genetically engineered microorganisms that have more direct applications. Many of these will be in agricul- ture, but genetically engineered microorganisms might also be used to decompose sludge at wastewater treatment plants, to leach minerals from low-grade concentrations of ore, or to decrease the viscosity of oil deep underground to allow it to be pumped to the surface. In some of these cases, naturally occurring microorganisms already contribute to these processes. These are possible examples, therefore, of how genetic engineering could be used to expand and improve upon the traditional uses of microorganisms in industry. The Molecular Machinery of the Cell The feature of life on earth that makes genetic engineering possible is the universality of the genetic code. Every living organism uses virtually the same system to translate the information contained in its DNA into proteins, the workhorses of biochemistry. It is this common genetic language that enables researchers to reproduce a gene from a human cell, insert it into bacteria, and have those bacteria manufac- ture the protein encoded by that gene. Proteins are composed of 20 different, relatively simple molecules known as amino acids, strung together in chains of widely varying lengths. The sequence of amino acids in a protein determines how the amino acid chain will fold, resulting in a characteristic shape that enables a protein to carry out its function. In addition, some proteins consist of two or more amino acid chains bound together; some amino acids are chemically modified once they become part of certain pro- teins; and some proteins must have other molecules, such as sugars, attached to them before they can function. By far the largest category of proteins is made up of the enzymes— large, globular proteins that catalyze individual chemical reactions, generally making them occur at least a million times faster than they would in the absence of the enzyme. Other, smaller proteins are

16 BIOTECHNOLOGY hormones, chemical messengers that modify and coordinate the activ- ities of cells. Proteins give bone and skin their tensile strength, and they are involved in the transport and storage of essential molecules within the body. Various proteins produce the movement of muscles, provide immune protection as antibodies, generate and alter nerve impulses, and control growth and differentiation. Clearly, to under- stand the molecular machinery of the cell, it is necessary to understand the construction and function of proteins. The basic process by which the information encoded in the genes of an organism's DNA directs the synthesis of proteins was worked out during the 1950s and 1960s. (The books listed in the section Additional Readings at the end of this chapter all describe this process.) But as late as 1970, molecular biologists faced serious difficulties in trying to investigate that process in specific organisms. They had no way of directly manipulating the DNA within higher organisms to determine the details of its structure or function. This problem was exacerbated by the complexity of the DNA in higher organisms, which almost guaranteed that progress would be arduous. "If we took the DNA from a single set of chromosomes from a single human cell and laid it out, it would be about one meter in length," explains Philip Leder of Harvard Medical School. "If we could stretch that one meter into one kilometer, a single gene would be represented in a millimeter's worth of DNA. That demonstrates the enormous degree of complexity that is repre- sented in the collection of genes from a higher organism." The development that cut through this complexity was the discovery of enzymes that could slice DNA in specific locations. With these enzymes, researchers became able to isolate specific segments of DNA and reinsert them into other segments of DNA. "By the application of this technology, we can reduce this enormous complexity to relative simplicity," says Leder. "We can reach in through these thousands and thousands of genes and pick out the ones that we are interested in." The basic technique of recombining DNA is now fairly well estab- lished, although its application in the laboratory still entails consider- able technical difficulties. First researchers isolate one or more seg- ments of DNA from a living organism, or they chemically synthesize small strands of DNA from its basic constituents. This DNA is usually then spliced into the DNA of a vector, which is most often DNA from a virus; small, independently replicating loops of DNA known as plas- mids, which occur in most bacteria and yeast; or genetic combinations of the two, known as cosmids. This genetically engineered vector is introduced into a host cell, which can then reproduce the DNA many

MOLECULAR AND MICROBIAL PRODUCTS 17 THE GENETIC ENGINEERING OF BACTERIA Bacterium Plant or Animal Cell f t Cleaved Plasmid DNA Segment Containing Gene of Interest Recombinant Plasmid Transformed Bacterium One common way to genetically engineer bacteria involves the use of small, independently replicating loops of DNA known as plasmids. Certain enzymes can cleave these plasmids at specific sequences in their genetic codes. DNA from other organisms that has been treated with the same enzymes can then be spliced into the plasmids with enzymes that join the cut ends of DNA. These recombinant plasmids are reinserted into bacteria, where they can reproduce themselves many times over. At the same time, the bacteria can divide, creating millions of copies of the introduced DNA. This DNA can then be studied through analytical techniques, or, if a gene within the introduced DNA can be made to produce the same protein it did in its original location, the genetically engineered bacteria can be used as microbial factories to make large quantities of the protein. times over, either for further study or for the production of bioengi- neered products. A central concern of researchers has been what causes a gene to produce, or express, the protein it encodes. Unlike the genetic code, the signals that regulate the expression of a protein, which are also encoded in DNA, vary from species to species. Thus, if a human gene is to function in a bacterium, the regulatory signals appropriate to the bacterium must somehow be associated with that gene. This is an

18 BIOTECHNOLOGY important consideration in the industrial application of genetic engi- neering, since many of the prospective products of biotechnology are proteins. Researchers are working on ways to enhance the expression of a protein by increasing the number of copies of a gene in a cell or by controlling the regulation of the gene. Research is also being conducted on ways to alter the DNA within genes to yield proteins with improved properties. For instance, en- zymes might be modified so that they will catalyze reactions over a broader range of temperatures or chemical conditions. With the use of computers it may even be possible to design enzymes that catalyze entirely new kinds of reactions. This so-called protein engineering could also lead to such advances as storage proteins in plants with more nutritious combinations of amino acids, or new kinds of fibers, plastics, and other materials. The Molecular Products of Recombinant DNA Technology The commercial sector that has been most affected by biotechnology is the pharmaceuticals industry. Most of the pharmaceutical products that can be made by genetic engineering are high-value-added sub- stances, which offers an incentive for the large amounts of research and development required to bring them to market. Also, the pharmaceu- ticals industry has considerable experience with biological processing, since about a fifth of its sales are of products manufactured wholly or in part by microorganisms. The first therapeutic agent produced through recombinant DNA techniques to be approved by the Food and Drug Administration and to be marketed was human insulin—a protein hormone 51 amino acids long. Human insulin differs from porcine and bovine insulin, which most diabetics use, by only one and three amino acids, respectively. But researchers are hopeful that the use of human insulin will eliminate some of the problems associated with regular injections of animal insulin, including occasional allergic reactions and long-term medical complications; these advantages have not yet been demonstrated in clinical tests. The use of insulin also demonstrates a problem common to all proteins: they must generally be injected under the skin, because proteins taken orally are broken down in the digestive system before they can reach the bloodstream. Work on new kinds of drug delivery systems is therefore an important adjunct to developments in biotechnology. Another hormone that has been produced through genetic engineer- ing is human growth hormone, a 191-amino-acid protein normally

MOLECULAR AND MICROBIAL PRODUCTS 19 secreted by the pituitary gland. An underproduction of human growth hormone can cause certain kinds of dwarfism, which regular injections of the hormone can at least partially prevent. Clinical trials are being considered to determine if the hormone has additional therapeutic uses. A number of other protein hormones are potential candidates for production by biotechnology, such as several calcium regulators that may be useful in treating bone disorders, various reproductive hor- mones, and a number of growth factors that stimulate the development of specific kinds of cells. A group of hormonelike molecules that have received considerable attention from the biotechnology industry are the interferons, a class of lymphokines. The interferons are glycoproteins—proteins bound to sugar groups—that regulate the body's immune response. They have shown some promise of preventing viral infections, and some evidence suggests that they may be effective in checking certain kinds of infections and cancers. However, it has not been possible to conduct clinical tests to substantiate these claims until recently, when large amounts of interferons became available through genetic engineering. Even if tests do not demonstrate an effective preventive or therapeutic role for interferon, the production of lymphokines through biotechnol- ogy promises to reveal much about the functioning of the immune system, which may in turn point the way toward other therapeutic agents. Proteins fractionated from the human blood represent a substantial market for the pharmaceuticals industry, and several of these have been targeted by the biotechnology industry. Human serum albumin, a protein of 585 amino acids, is used during surgery and to treat shock, burns, and other physical trauma. Antihemophilic factors, specifically factors VIII and IX, are used by the approximately 14,000 hemophiliacs in the United States to control bleeding. And tissue plasminogen activators have demonstrated a remarkable ability to dissolve blood clots in the moments after a heart attack. The production of large quantities of previously scarce proteins may, in the long run, have a much greater impact than their direct therapeutic uses would indicate. Once these proteins are abundantly available, researchers can study their structure and determine how they function in the body. This information can in turn lead to the design and production of new drugs—whether proteins or nonpro- teins—to combat disease. This is one example of how genetic engineer- ing can be used as a research tool to probe life's basic processes, with striking implications for health care.

20 BIOTECHNOLOGY Another important role for proteins in health care may be as vaccines. Today many vaccines consist of the disease-causing orga- nisms in a weakened, or attenuated, state. Once these invaders are injected into the body, the immune system generates antibodies against them and primes itself for future infections of the organisms. However, because the vaccines contain the entire genetic material of the virulent organism, there is a slim chance of contracting the disease from the vaccine. Also, vaccines do not always immunize a person against all strains of a pathogen, and they often need to be refrigerated, making them difficult to use in some parts of the world. The use of subunit vaccines may solve many of these problems, while also offering the possibility of vaccinating people against a much broader range of diseases. Subunit vaccines consist of just part of a virulent organism, such as part or all of a surface protein. If less than about 50 amino acids long, subunit protein vaccines can be chemically synthesized from their constituent amino acids; proteins of these lengths and longer can also be biologically synthesized by genetically engineered microorganisms. If properly delivered to the body, subunit vaccines can generate an immune response powerful enough to protect against infections by the organism itself. In addition, they can be purer, more stable, and less dangerous than existing vaccines. A number of viral diseases, including influenza types A and B, herpes, polio, hepatitis, and acquired immune deficiency syndrome, are currently being investigated to determine whether they can be pre- vented by subunit vaccines. However, it may not be possible to make vaccines for all these diseases. The surfaces of some viruses frequently change, so that previously effective vaccines lose their punch. Ways must also be found to strengthen the immune response that subunit vaccines generate. But even partial successes could have a dramatic effect on health. There are 80,000 to 100,000 cases of hepatitis B in the United States each year, causing about 1,000 deaths, and the incidence of the disease is much greater in other parts of the world. AIDS has already killed thousands of people in the United States, and the incidence of the disease is increasing rapidly. The development of vaccines against bacterial and parasitic patho- gens is more difficult than the development of vaccines for viruses because of the complex and varying surfaces and involved lifecycles of these organisms. For instance, malaria, the most common infectious disease in the world, is caused by a parasite that exists in three different forms in the human body, complicating its prevention by a vaccine. But it may be possible to reproduce surface proteins that occur on some parasites and bacteria and use them as vaccines. It may also

MOLECULAR AND MICROBIAL PRODUCTS 21 be possible to genetically engineer nonpathogenic forms of these organisms that would generate an immune response when injected into the body. Another use of biotechnology in the pharmaceuticals industry is in the manufacture of metabolites and other nonprotein substances whose reactions are catalyzed by enzymes. For instance, genetic engineering could lead to the production of new antibiotics or make the production of known antibiotics more efficient. Similarly, enzymatic processes could be developed to conduct an increasing number of the chemical steps involved in the synthesis of a wide variety of useful drugs. Enzymes are important tools in commercial sectors other than the pharmaceuticals industry. Proteases, amylases, and glucose isomerase, for example, are used in food processing and in the manufacture of textiles, detergents, and leather. Two amylases and glucose isomerase are used to convert starch to high-fructose corn syrup, a substance that has increasingly replaced table sugar in processed foods since the late 1960s. Biotechnology may be used both to improve the properties of these and other enzymes and to increase their production from the microorganisms that make them. The constituents of proteins—amino acids—are another potential product of biotechnology. Amino acids are used as additives in animal feed and human food and for enteral and intravenous feeding. Glutamic acid, whose sodium salt is monosodium glutamate (MSG), and most commercial lysine are now manufactured by strains of Corynebacterium. Researchers are applying genetic engineering to this bacterium in an attempt to increase its productivity or give it other desirable characteristics. Tryptophan and phenylalanine are two more amino acids whose economics of production may favor biotechnology. A number of other metabolites and related high-value compounds may eventually move away from synthetic processing and toward biological processing. Fatty acids and alcohols, vitamins, high-grade oils, flavors and fragrances, adhesives, water-soluble gums, dyes, cosmetics, and many other substances are candidates for production by genetic engineering. As experience with biotechnology accumulates and production meth- ods get cheaper, less expensive chemicals may also be made through biological methods. Today almost all the commodity chemicals used in industry, serving as precursors for products ranging from solvents to plastics, are synthesized from petroleum and natural gas feedstocks. Essentially all these chemicals could be made from biomass, such as starch or cellulose, and most of them could be produced with microor- ganisms. Biological processes cannot yet compete with synthetic meth-

22 BIOTECHNOLOGY ods for these chemicals, but this may change as biotechnology advances and the cost of fossil fuels rises. Fermentation Technologies All the products mentioned in the previous section are made by what is known as a fermentation process. In this process, living cells or enzymes are combined with nutrients and/or the substance to be chemically transformed in some sort of reactor vessel. The nutrients may consist of sugars, starches, vegetable oil, or even petroleum fractions, and cells may also need additional nitrogen, phosphorus, oxygen, vitamins, metals, or other compounds to grow. Once the desired conversion has taken place, the products of the reaction are removed from the vessel, and the specific compound desired is sepa- rated from wastes and by-products and purified for use. The kind of fermentation technology that now dominates the phar- maceuticals and specialty chemicals industries is batch processing, in which the necessary ingredients are combined in a bioreactor, the conversion takes place, the vessel is emptied, and the entire process begins again. However, continuous processing, in which nutrients and feedstocks enter a bioreactor and spent medium and products leave it on a continuous basis, offers significant advantages for many fermen- tation products. Continuous processing can have higher productivities and lower costs, because the cells or enzymes are continuously reused and the product is often easier to separate from the outflow. It generally requires, however, that the cells or enzymes be immobilized within the reactor so that they are not swept out with the product. A number of methods have been devised to do this, including bonding the cells or enzymes to a solid support, trapping them in a polymer matrix, or encapsulating them within semipermeable membranous spheres. The scaling-up to industrial levels of fermentation processes using genetically engineered cells involves a number of difficulties. Main- taining a homogenous mixture of nutrients and dissipating the large quantities of heat generated during fermentation are much more difficult in a full-scale industrial bioreactor than in a small benchtop flask. Also, the bioreactor and incoming nutrients usually have to be thoroughly sterilized, since contaminants can destroy the cells or enzymes or introduce impurities into the final product. This require- ment complicates the monitoring of the ongoing reaction, since sensors of many useful measures are disabled by steam sterilization (by the same token, biotechnology may be used to produce sensors that overcome this limitation). Genetically engineered cells can also mutate

MOLECULAR AND MICROBIAL PRODUCTS 23 Once genetically engineered microorganisms have been grown in a fermentation process, their components are separated using the centrifuges shown here. Further separation is then required to isolate the one protein that is desired from the thousands of other proteins produced by the microorganism. The isolated protein must be rigorously purified to eliminate contaminants from the final product. In many cases, this separation and purification process is more expensive than the original fermentation. or revert to an earlier genetic state during fermentation, making the products of the fermentation useless. The separation and purification of products from dilute aqueous solutions presents another set of problems. In many pharmaceutical applications, this phase of production costs more than the fermentation itself. In addition to such standard techniques as distillation, drying, and precipitation, bioprocess engineers are experimenting with the use of ultrafiltration, high-performance liquid chromatography, electro- phoresis, and antibody technology to recover products. There is a pressing need in biotechnology for microorganisms better suited to fermentation technologies. For instance, the bacterium Esch- erichia coli, which has been widely used in genetic engineering, manufactures its products intracellularly and also produces highly toxic substances called endotoxins that must be rigorously eliminated

24 BIOTECHNOLOGY from the final product. Researchers are investigating other kinds of bacteria and higher microorganisms like yeast that can be induced to secrete their products into the surrounding medium and that do not produce toxic compounds. Ways must also be developed to grow other kinds of cells, including plant, animal, and human cells, in cultures for industrial purposes. These cells will ultimately be the most useful producers of many valuable substances in biotechnology. However, their nutritional re- quirements are poorly defined, and they are much more fragile and complex, and hence more difficult to grow, than are one-celled micro- organisms like bacteria and yeast. Microorganisms for Use in the Environment In addition to their uses in fermentation processes, genetically engineered microorganisms will find direct application in the environ- ment. The agricultural uses of genetically engineered organisms are discussed in Chapter 3. But that leaves a variety of other industrial processes to which biotechnology could contribute. Liquid and solid wastes are broken down in waste treatment plants largely through the action of microbes. Biotechnology could produce enzymes or other substances that hasten or further this process. For example, biologically derived flocculants would be very useful for separating and thickening solids during treatment. Cellulases, proteases, amylases, and polysaccharide hydrolases could help release the water retained in sludge before it is disposed of. It may even be possible to genetically engineer properties into microorganisms that would enhance their ability to break down certain waste substances— not only sludge but slime, grease, and scum as well. Genetically engineered microorganisms or their products may also be able to remove heavy metals or organic pollutants, including suspected carcinogens, from drinking water and industrial wastewater. Proteins known as metallothioneins can bind various kinds of heavy metals, and other proteins can polymerize aromatic compounds so that they can be removed by flocculation. Microbiologists have either found or produced through conventional genetic techniques organisms that can break down a variety of toxic substances, including 2,4-D and 2,4,5-T. Once the genes controlling these processes are isolated and characterized in an organism, they could be transferred to other organisms via recombinant DNA. The ability of enzymes to recognize and bind metals is important in another possible environmental application of biotechnology: microbial

MOLECULAR AND MICROB1AL PRODUCTS 25 mining. Microorganisms are already used for leaching low-grade ores and concentrating metals; in fact, more than 10 percent of the copper produced in the United States is leached from ores by microbes. Genetic engineering could improve these organisms in any number of ways: by increasing their tolerance to saline or acidic conditions, by decreasing their toxicity to certain metals, or by increasing their ability to withstand high temperatures in underground mines. Another use for microorganisms or their products might be to enhance the extraction of oil from wells. Only about half the world's supply of subterranean oil reserves can be recovered using conven- tional techniques. Biologically derived surfactants and viscosity decreasers could be injected into wells to enable some of this additional oil to be pumped out. Furthermore, if organisms were found or genetically engineered that could live under the harsh conditions of oil wells and give off the proper products, they could be directly introduced into wells to repressurize or condition the oil for removal. However, as with many of the other environmental applications of biotechnol- ogy, considerable additional research is necessary before this will be possible. Monoclonal Antibodies Recombinant DNA is just one of the techniques that have led to the development of biotechnology over the past decade. A panoply of other procedures, from protein sequencing to tissue culturing, have also contributed to the growth of the field. One of the most prominent of these procedures is cell fusion. In this process, the constituents of two different cells are combined to form a single hybrid cell. Cell fusion has given rise to a variety of exotic organisms, such as the hybrid plants mentioned in Chapter 3. But the most important outgrowth of cell fusion, accounting at this point for more commercial products than recombinant DNA has generated, is the production of monoclonal antibodies. Antibodies are complex proteins that are produced and secreted by B lymphocytes, a type of white blood cell that forms an important component of the body's immune system. Antibodies have the ability to recognize and attach themselves to foreign substances in the body— known collectively as antigens—setting in motion a process that will eliminate the antigen from the body. Each lymphocyte produces only a single kind of antibody, but there are a virtually unlimited number of different lymphocytes, and each proliferates rapidly when it detects its corresponding antigen. In this way the immune system offers protec-

26 BIOTECHNOLOGY CELL FUSION Mouse Spleen Cells Mouse Myeloma Cells Hybridoma Cells Grow Hybridomas Separately and Test Antibodies Select Cell Line and Grow in Volume Monoclonal Antibodies To produce monoclonal antibodies, antibody-producing spleen cells from a mouse that has been immunized against an antigen are mixed with mouse myeloma cells. Under the proper conditions, pairs of the cells fuse to form antibody-producing hybrid-myeloma ("hybridoma") cells, which can live indefinitely in culture. Indi- vidual hybridomas are grown in separate wells, and the antibodies they produce are tested against the antigen. When an effective cell line is identified, it is grown either in culture or in the body cavities of mice to produce large quantities of chemically identical, monoclonal antibodies. tion against a wide range of infectious agents and other foreign substances. The traditional means of producing antibodies for research and medical purposes has been to inject an animal with an antigen and collect the antibodies that result. This method has several drawbacks, however. The injection of an antigen generates many different anti- bodies that react with the antigen; the supply of antibodies produced in this way is limited; and an injected antigen usually contains other materials, leading to the production of antibodies against a variety of antigens. In 1975 Cesar Milstein and Georges Kohler of the British Medical

MOLECULAR AND MICROB1AL PRODUCTS 27 A mouse spleen cell and tumor cell fuse to form a hybridoma. As the hybridoma divides, it gives rise to a "clone" of identical cells, giving the name "monoclonal" to the antibodies those cells produce. Research Council Laboratory of Molecular Biology in Cambridge happened on a way around these problems. They fused myeloma tumor cells from a mouse, which have the capacity to grow indefinitely in culture, with cells derived from mouse B lymphocytes, which have a limited lifetime. The resulting hybrid-myeloma or hybridoma cells combined just the right qualities of each parent. They prospered in cell culture and at the same time produced virtually unlimited quantities of chemically identical or monoclonal antibodies (so named because they are produced by the cloned copies of a single hybridoma). Monoclonal antibodies have already begun to find many valuable applications in research, according to William Paul of the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. Because of their great specificity, they are ideal tools for separating and purifying proteins and other cellular components, tasks that were often difficult with conventional techniques. "In certain circumstances, one might even consider using monoclonal antibodies for large-scale [industrial] purification," says Paul, and in fact it is already being used for such purposes. However, this is only possible "where it is economically feasible to do so, which probably limits its use

28 BIOTECHNOLOGY to molecules that are active in exquisitely low concentrations and that would be difficult to purify by more conventional means." Monoclonal antibodies also have "enormous potential" in medicine, according to Paul. "It is in principle possible to develop a measurement technique for essentially any molecule that is immunogenic or could be made immunogenic through chemical manipulation," he points out. This has already led to in vitro diagnostic tests for detecting and monitoring pregnancy, venereal diseases like chlamydia and herpes, viral infections leading to hepatitis and AIDS, bacterial infections causing meningitis, and some forms of cancer. Research is also being conducted on the use of monoclonal antibodies in vivo, although the more rigorous safety testing demanded of such products has so far limited their use. Most obviously, monoclonal antibodies can be used to confer short-term passive immunity, as opposed to the long-term active immunity conveyed by lymphocytes. Researchers are also investigating the use in the body of monoclonal antibodies tagged with radioisotopes or other chemicals. By binding to blood clots or tumors in the body, such constructs could reveal the location of these pathologies to scanning devices. Monoclonal antibod- ies injected into the body may be able to halt the spread of certain tumors, and research is being conducted into the possibility of attach- ing toxic agents to the antibodies that would be delivered directly to a tumor. "The great difficulty is the preparation of antibodies that truly distinguish tumor cells from normal cells," says Paul. "At the current time the numbers of situations in which really good results may be obtained are very limited." Hybridoma technology faces other limitations that have hampered its full effectiveness. For one thing, it remains hard to create mono- clonal antibodies against antigens that generate only a weak im- mune response. Also, it has proved very difficult to create hybridomas from human cells. "The monoclonal antibodies that have been produced thus far of great value have been derived ultimately from either mice or rats," says Paul. "Successes with cells from more distantly related animals have been rare indeed, and those results are very disappointing." In the future it may become possible, using recombinant DNA techniques, to genetically engineer cells to produce unlimited quanti- ties of specific antibodies. But for the immediate future, researchers are working on forming hybridoma cell lines from human cells rather than from mouse cells. Such cell lines would have a number of advantages. The body would be less likely to generate an immune response against human antibodies than against mouse antibodies. Also, human mono-

MOLECULAR AND MICROBIAL PRODUCTS 29 clonal antibodies might have even greater specificities than those now available. Pairs of human tumor cells and human lymphocytes have been induced to fuse in laboratories, but the resulting hybridomas are difficult to grow in culture and tend to be genetically unstable. "Although some successes have been achieved, and it would be wrong to discount those successes, this technology has not yet reached a state in which one can reliably produce human monoclonal antibodies," says Paul. "I have very little doubt that, with the very large number of individuals who have great interest in these areas, progress will be made. But we should not overemphasize how far we have come along what is an exciting but still very difficult pathway." Additional Readings Cesar Milstein. 1980. "Monoclonal Antibodies." Scientific American 243(Octo- ber):66-74. Office of Technology Assessment. 1981. Impacts of Applied Genetics: Micro- Organisms, Plants, and Animals. Washington, D.C.: U.S. Government Printing Office. Office of Technology Assessment. 1984. Commercial Biotechnology: An Interna- tional Analysis. Washington, D.C.: U.S. Government Printing Office. Scientific American. 1981. Industrial Microbiology and the Advent of Genetic Engineering. San Francisco: W. H. Freeman. James D. Watson, John Tooze, and David T. Kurtz. 1983. Recombinant DNA: A Short Course. New York: W. H. Freeman.

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