Biotechnology of biopreparations and medicinal substances
Before the appearance of genetic and cellular engineering many drugs were possible be to obtained only in small quantities, and their production was very expensive. It was assumed that with the help of new technology it would be possible to obtain full range of these products in sufficient quantities for use in practice. And those expectations were met. To date, hundreds of genes of different proteins, hormones, and enzymes of humans and animals have been cloned. Most of these genes are already expressed in the cells of the hosts, and now their products are tested on the potential use for the treatment of various diseases and improving animal productivity.
Bovine somatotropin (growth hormone in cattle). Even in the the 1930's it was shown that the administration of growth hormone in cows greatly increased their milk yield. Since getting the natural hormone in large quantities was very time-consuming and expensive, it has not found widespread use in the dairy industry. Using recombinant DNA technology gene of somatotropin was cloned into E. coli, and synthetic recombinant hormone was isolated from bacterial cells and purified. As expected, the milk yield of cows, which was introduced by the recombinant product, increased by 25-30%.
Growth hormone found in milk, has been exhaustively tested for safety. In cows that received recombinant hormone its concentration in the milk was not higher than in control animals. Moreover, the drug was inactive in the body, and all toxicity tests did not reveal any adverse effects. Using all available studies, FDA concluded that meat and milk from cows treated with recombinant products were safe for humans. This conclusion was supported by the Office of Technology Assessment in the United States after having been conducted an independent analysis of numerous data on testing of bovine growth hormone. However, recombinant bovine growth hormone was not accepted by society with open arms. One powerful lobby groups acted as a united front for that permission to use the recombinant hormone, issued by FDA, was blocked. The basis of the action was economic considerations relating to the effects of recombinant products for the dairy industry. Members of the group considered that it would lead to the ruin of many small dairy farms, because to get the same amount of milk it will need less cows. In addition, concerns have been raised that the dairy industry will be monopolized by large corporations to the detriment of independent producers. Apparently, these economic arguments are reasonable and, of course, any group of people has the right to protest against the fact that may be a threat to their existence. However, the main reason for the campaign, unfolding against the use of somatotropin, was the consideration that "hormones obtained by recombinant methods," can harm a person and cause formation of tumors. The fact that for the growth hormone was used recombinant DNA technology increased emotional tension. In addition to the economic arguments, opponents of somatotropin express views that its use would increase the frequency of bacterial infection of the mammary glands (mastitis) in cows. This would require the use of l arge amounts of antibiotics, leading to higher concentrations of it in milk and, in turn, such milk can cause allergic reactions in people who use it for consumption. In addition, the increasing the number of antibiotics can lead to the emergence of pathogens resistant to him. However, the Advisory Committee on Veterinary Medicine at FDA, conducting an appropriate analysis, concluded that the incidence of mastitis in cows that received recombinant growth hormone, was not higher than that of cows not treated with this drug.
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Recombinant growth hormone has been licensed in the U.S. for use in the dairy industry in 1994, but in many other countries there is still a temporary prohibition on the sale of milk from cows treated with a hormone. Apparently, the ban is due to socio-economic reasons rather than to concerns of possibile influence of the preparation on human health.
Antibioticsbelong to secondary metabolites which are small molecules that are not required for cell survival and formed at the end of their growth phase, ie, in idiophase, so they are called idiolits. The biological role of antibiotics is to ensure the existence of microbes in competitive environment, by suppressing of microbial life of other groups.
Antibiotic substances are produced by different various groups of soil microorganisms belonging to actinomycetes, bacteria, filamentous fungi. They are saprophytes aerobes, heterotrophs. Antibiotic producers among soil anaerobic bacteria as well as acetone-butyl, disulfate, propionic acid bacteria are rarely found, because the competitive environment is created by the accumulation of anaerobic metabolites of fermentation: butanol, propionic acid, acetic acid, ethanol, and also due to the production of sulfides, hydrogen sulfide, etc. Antibiotic producers are also absent among the acetic acid, thiol, methylotrophic bacteria. These aerobes are out of competition because surrounding microorganisms do not use their specific substrate or may not exist in the highly acidic environment that they create.
Dominant position among the producers of antibiotics are occupied by actinomycetes. More than 4,000 antibiotics produced by this group of microorganisms are known. Only one species of actinomycetes Str. griseus produces about 50 antibiotic compounds. More than 1000 antibiotics of bacterial origin are known, produced by soi and spore-forming bacteria from the genus Bacillus. Filamentous fungi are producers of beta-lactam antibiotics - penicillin and cephalosporin as well as their semi-synthetic derivatives. The main producers are fungi from genera Penicillium, Cephalosporium (Acremonium). Antibiotics, which are synthesized by actinomycetes are classified as: tetracyclines (chlorine, oxytetracycline, and their derivatives (doxycycline methacycline) - Str. Rimosus, Str. Aureofaciens); aminoglycosides (streptomycin - Str. Griseus, neomycin - Str. Fradiae; kanamycin - Str. Kanamyceticus etc.), macrolides (erythromycin - Str. (saccharopolyspora) erythraeus, oleandomycin - Str. antibioticus); aromatic compounds (chloramphenicol - Str. venezuelae); polyene (amfoterritsin B, nystatin - Str. neurasei); rifamycins (rifampin - Str . (Nocardia) mediterranei); anthracyclines and anticancer (bleomycin - Str. verticillius).
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Tetracyclines, aminoglycosides, macrolides, and aromatic compounds inhibit protein translation on the ribosomes of prokaryotes. Polyene antibiotics disturb the permeability of the cytoplasmic membrane of pathogens of leukemia. Rifamycins inhibit RNA – polymerase of bacteria. Anthracyclines violate DNA replication.
Bacteria produce peptide antibiotics: Bacillus brevis-gramicidin C, Bacillus polymyxa - polymyxin B, Bacillus licheniformis - bacitracin. Peptide antibiotics disturb the permeability of the cytoplasmic membrane of prokaryotes.
Filamentous fungi, produce beta-lactam antibiotics and their derivatives which inhibit the synthesis of the peptidoglycan component of the cell walls of gram-negative and especially Gram-positive microorganisms: penicillin (P. chrysogenum, P. notatum); cephalosporin (C. chrysogenum, C.acremonium); cephamycin, etc.
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World annual production of antibiotics is nearly $ 20 billion. The most important antimicrobial and antitumor preparations belong to antibiotics. Consequently, they are widely used in human and veterinary medicine, food and canning industry, agriculture. In medical and veterinary practice, they are used in the treatment of humans and animals from various infections. Nisin (Str. lactis) is using in food and canning industry for the purpose of food preservation. Except conventional amino acids it contains lysine, histidine, proline, methionine, isoleucine. Nisin inhibits the growth of streptococcus, staphylococcus, bacillus. Adding it to canned products allows to reduce temperature and time of sterilization and thus preserve their flavor and nutritional quality. In livestock more than 20 antibiotics (biomitsin, terramycin, Grisinum, flavomicin etc are used as feed additive and growth promoter.
Microbiological synthesis begins with the preparation of the culture medium. Substrate should give a good microbial growth and should be cheap and affordable. The medium is preliminary sterilized in the bioreactor with the help of moist steam under pressure. At the same time inoculum of pure culture is being prepared. Producer strain are sequentially cultivated in flasks, and then in laboratory and pilot fermenters. The next stage is deep aerobic batch fermentation during 7-10 days. In the process of fermentation there is a constant mixing of the culture medium and temperature, pH as well as pO2 are maintained, chemical and mechanical defoaming are used. Then, a biomass processing: filtering, if antibiotics in the culture fluid, and if antibiotics in the cells, the deposition of antibiotics in residue together with the cells is implementtd, and then their release from cells is carried out. In the process of extraction and purification of antibiotics methods of extraction, ion adsorption, precipitation, etc. are used. Antibiotic isolated in a homogeneous state is dried by spray or freeze drying, stabilized with the aim of preserving its biological activity, and dosage form is given.
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Synthesis of new antibiotics. New antibiotics with unique properties and specificity can be obtained by conducting genetic engineering manipulations with genes involved in biosynthesis of known antibiotics.
One of the plasmids of Streptomyces rIJ2303 carrying chromosomal DNA fragment of S. coelicolor with length 32,500 base pairs, contains all genes of the enzymes responsible for the biosynthesis antibiotic aktinorodina out of the acetate. Whole plasmid and various subclones bearing parts of 32 500 pairs of nucleotides fragment (eg rIJ2315) were administered either in Streptomyces sp. (strain AM-7161), synthesizing related antibiotic medermitsin, or S. violaceoruber (strain V1140 or Tu22), synthesizing related antibiotics granaticin and digidrogranaticin. All these antibiotics are acid-base indicators, which give growing culture characteristic color depending on pH. In its turn pH (and color) of the medium depends on what kind of compound is synthesized. Mutants of the parent strain of S. coelicolor, do not capable of synthesizing aktinorodin, they are colorless. The appearance of color after transformation of strain AM-7161 Streptomyces sp. or strains V1140 or Tu22 S. violaceoruber by plasmid carrying all or some of the genes encoding the biosynthetic enzymes of aktinorodina demonstrates the synthesis of a new antibiotic.
Improving the production of antibiotics. With the help of genetic engineering it is possible not only to create new antibiotics, but also to increase the efficiency of synthesis of already known ones. The limiting factor in the industrial production of antibiotics by Streptomyces spp. is the deficiency of oxygen available to the cells. Due to the poor solubility of oxygen in water and high density of Streptomyces’s culture its the amount of oxygen becomes insufficient and cell growth slows, output of antibiotic is reduced. To solve this problem, first, it is necessary to change the design of bioreactors in which the culture of Streptomyces is grown, and secondly, using the techniques of genetic engineering to create strains of Streptomyces which is more efficient in using available oxygen. These two approaches are not mutually exclusive.One strategy used by some aerobic microorganisms for survival in conditions of lack of oxygen, is the synthesis of hemoglobin similar product that can accumulate oxygen and deliver it to the cells. For example, aerobic bacterium Vitreoscilla sp. synthesizes homodimer hem-containing protein, which is functionally similar to eukaryotic hemoglobin. Gene "hemoglobin» Vitreoscilla was isolated, integrated in Streptomyces plasmid vector and introduced into cells of this organism. After his expression proportion of Vitreoscilla hemoglobin was approximately equal to 0.1% of all cellular proteins of S. coelicolor even in the case when the expression was carried out under the control of its own promoter. Transformed cells of S. coelicolor, growing at low levels of dissolved oxygen (about 5% of the saturation concentration), synthesized aktinorodin in 10 times more and had a greater speed of growth than non-transformed ones. This approach can be used to provide oxygen of other microorganisms growing in a lack of oxygen.
Monoclonal antibodies as medicines. The possibility of using radioactive Mab in cancer therapy was first demonstrated in 1979-80 in the treatment of patients with unresectable primary liver cancer (Medical School Johns Hopkins University, state Maryland). Such treatment was more efficient than conventional chemo-and radiotherapy. One of the most effective anticancer drugs of plant origin is ricin - a toxin from the seeds of castor beans. Under the conditions of in vitro this toxin destroys not only cancer, but also normal cells, as they have the ability to bind to the surface of all cells. Therefore, necessary condition in using ricin for the treatment of patients is the selectivity of binding.Ricin in its structure as it is created to perform such a task. It consists of two polypeptide chains: A chain is toxic determinants, and the B-chain contains a section that recognizes galactose, which allows ricin binding to the cell membrane. In SRI Maple Midi in Montpellier (France) A chain of ricin was purified with the aim its conjugation with antitumoral Mab. Ricin without B chain acquired new binding specificity due to antibodies and did not fall into normal cells (Casellas P. and Gros P., 1982). These immunotoxins recognized antigens, peculiar to certain tumors in mice and human neoplasms. It was proven to kill a healthy cell is required 1000-100000 times more poison than to destroy cancer cells. In experiments on animals immunotoxins showed high selectivity and killed only malignant cells in vitro. Mab were used in the selection of biologically active substances (proteins, hormones, toxins) from complex mixtures. One of the first drugs cleared by ICA was interferon. In this case the antibodies were sewn to the carbohydrate granules and used to make immunosorbent column on which crude interferon preparation was purified. After one passage through a column with immobilized Mab preparation was cleared in 5000 times.
Mab also able to neutralize the effect of lymphocytes responsible for rejection of the transplant, and autoantibodies generated in autoimmune diseases (some forms of diabetes, multiple sclerosis, rheumatic diseases). They are used also in enhancing the effects of drugs on target cells reducing the side effects occurring during conventional cancer chemotherapy. For example, Mab were embedded inside liposome bubbles. Last, crossing the cell membrane transport molecules of the drug to the target organ (in accordance with the specificity of the associated Mab), where they leave its contents. Drugs that exhibit high activity during testing in vitro (usually in cell culture), are often less effective in vivo. Decrease their activity due to the fact that they do not reach the body or target cells in the desired concentration.
Increasing the dose of the drug does not solve the problem because it often have side effects. Moreover, in order to avoid such effects, many therapeutic substances are obviously injected in less than optimal dose, which further reduces their effectiveness. To facilitate the delivery of the drug to its site of action several techniques are used: a) enclosing it to special particles - liposomes, lipid envelope which has a high affinity to the desired authorities, b) inserting genes of specific toxins in tumor infiltrating lymphocytes, which release these toxins directly into tumor ) attaching drug molecules to monoclonal antibodies specific for proteins available on the surface of cells, such as tumor, and d) using drugs in an inactive form, and translating them into an active state by enzymes. Such transformation occurs only near the target cells, the enzyme is attached to a monoclonal antibody specific for the surface antigen of the cell.
To implement this approach, it is necessary that: a) Mab attaching to the enzyme that takes the drug to its active form, should be sufficiently cleaned and have the right amount, and b) Mab should be able to bind to cell protein highly specific for the target: a) Mab should be stable under physiological conditions, but at the same time are rapidly excreted from the circulation, d) if necessary, Mab should be able to penetrate into the tumor tissue, providing effect of the drug to all its cells. In this case, the target cells are well-defined that allows to use drug substance in much smaller doses than in the direct injection. The application of mouse monoclonal antibodies in such a system may cause development of immune response, so it is very important to use human antibodies or fragments of antibodies, the most similar to them in structure. We give an example from the practice of medicine. Thromboembolism of brain or heart arteries is the most common cause of death. Thrombus composed of fibrin molecules, binding factor of the blood, forming a network in response to an injury of vascular wall. Normally, the molecules of fibrin in blood clot are split by plasmin, which is formed under the action of plasminogen activator. But often this biological system is not efficient enough, which leads to clogged arteries. In such situations, to increase the level of plasmin in the blood was offered to use plasminogen activator as a therapeutic agent. However, plasmin can destroy fibrinogen which is the fibrin precursor, and if the fibrinogen degree reduces too much as a result of treatment with plasminogen activator, extensive internal bleeding may occur. This has led to the need for thrombolytic drugs that destroy fibrin only in clots. Scientists have proceeded from the fact that if antibodiy specific for fibrin "sew" to plasminogen activator it will be only local increasing of plasmin concentration near thrombus. To test this hypothesis, tissue-specific plasminogen activator has been attached to a monoclonal antibody specific for fibrin. Testing on model systems have shown that the complex joined to blood clots and lysed them without causing significant destruction of fibrinogen. Other types of antibody-plasminogen activator, also leading to the formation of local plasmin destroying blood clots were created. Despite the apparent promise of immunotherapy, this method has a number of limitations associated with the using of mice monoclonal antibodies. The application Mab for treatment of humans and farm animals can result in allergic reactions, so it is important to use homologous immunoglobulin. In the case of multiple injections murine Mab as foreign proteins can cause sensitization of patient. Creating of species specific antibodies is a rather difficult task, since the production of antibodies of human and domestic animals by conventional hybridoma technology faces a number of problems (lack of effective myeloma cell lines, human immunization is not carried out for reasons of ethical nature, etc.). To obtain hybridomas of farm animals it is necessary to have transplantable myeloma cell line derived from a certain species of livestock. Various plasmacytomas of horses, cows, pigs, and dogs, cats and rabbits (P.Pastoret, 1982) were described, but they did not meet the requirements for myeloma cells. For example, transplantable line of farm animals have no stable markers due to genetic mutation of cells. Therefore, some researchers conducting research to obtain heterologous Mab by fusing mouse plasmacytomas with lymphocytes of pets. So, S.Sricumaran et al. (1983) received hybridomas from the mouse myeloma cells and bovine immunoglobulin which producing immunoglobulins more simlar to Ig of cattle. Mouse hybridoma + bull synthesizing Mab against K99 antigen E.coli (DVAnderson et al., 1987) and bovine coronavirus (TJRaybould et al., 1985) were described. DJGroves et al., (1987) obtained mouse + sheep hybridoma producing MAb to testosterone and TJRaybould et al. (1984) described mouse + pig hybridoma.
KHNielsen and MDHenning (1989) obtained Mab-producing cattle hybridoma against lipopolysaccharides of Brucella abortus by fusion of peripheral blood lymphocytes from immunized cows with mouse plasmacytoma. Antibodies produced by interspecies hybridoma weakly agglutinate Brucella cells at neutral pH, but in the acidic environment agglutination ability of Mab significantly increased. These antibodies did not precipitated LPS in RID, but were active in the CFT and indirect ELISA, but could not compete with the homologous murine Mab. However, interspecies hybridoma as expected, were not stable in the production of Mab. Consequently, to obtain antibodies homologous to human and animal Mab it is necessary to develop other approaches.
Antibody production by E. coli. Hybridoma, like most other animal cell cultures, grow relatively slowly, it does not reach the high density and require complex and expensive media. The resulting monoclonal antibodies are very expensive wich does not allow their extensive usage them in the clinic. To solve this problem, there have been attempts to create a sort of "bioreactors" from genetically modified bacteria, plants and animals. Frequently for the effective delivery and functioning of some immunotherapeutic agents one antigen binding region of an antibody (Fab-or Fv-fragment) is enough, ie, the presence of Fc-antibody fragment is optional. The essence of the method for production of functional antibodies using E. coli is the following: using mRNA extracted from the antibody-producing cells (B lymphocytes), cDNA is synthesized. Then separate PCR amplification of cDNAs encoding H-and L-chain is conducted. After that, the amplified cDNA was treated with specific endonucleases, and then inserted into a vector on the basis of bacteriophage l. cDNA of H-and L-chains have different endonuclease sites, which facilitates integration of each specific nucleotide sequence in its vector. At this stage, cloning of many different segments of H-and L-chains is occur. Next, common "combinatorial" vector is inserted into cDNA of one H and one L-chain, so that the bacteriophage synthesized both strands Ig and form a "full» Fv-fragment. Synthesis of H-and L-chains occurs during the lytic cycle of bacteriophage l, therefore it is possible to screen libraries of combinatorial phage clones to determine their antigen-binding activity. At the connection of cDNA H-and L-chains in a single vector a wide spectrum of different antibody genes is formed. Some of them encode unique binding sites and obtain them with conventional hybridoma technology would be impossible. Pool of mammals antibodies includes 106-108 different antibodies. Phage library contains about the same amount of clones, so we may expect that one combinatorial library will generate the same amount of different antibodies (Fv-molecules), just like any mammal. In addition, once created initial combinatorial library allow us to combine L-and H-chains and get Fv-fragments, which recognize unusual epitopes. Even greater diversity can be achieved using non-specific mutagenesis. Identification of Fv-fragments with the desired specificity takes from 7 to 14 days. For comparison, screening hundreds of hybridoma cell lines usually takes months. Vectors based on bacteriophage 1 is not very suitable for the production of large amounts of protein molecules. To solve this problem, such a vector was constructed in which DNA of H-and L-chains were integrated into site, flanked by plasmid DNA. Such plasmid containing the DNA of H-and L-chain can be cut out of the vector and transformed to E. coli. As part of plasmid DNA of Fv-fragments will be repeatedly replicated in E. coli with the formation of a large number of products which can be used for diagnostic and therapeutic purposes. Screening of combinatorial libraries of antibody fragments can be carried out by ELISA. The essence of the method is the following: samples (aliquots) from the library placed in wells of plates containing the target antigen. The wells is washed to remove unbound phage particles. Conjugate composed of an antibody that binds to the protein shell of the phage and enzyme is added in each well. The wells is washed to remove unbound conjugate and chromogenic substrate splitting by the enzyme associated with the phage is added to each of them. In this case those wells are stained which contain phage particles carrying antibodies to the target antigen. Selecting phage that synthesizes desired antibody it is possible to extract encoding DNA fragment and subclone it into the expression vector. Thus, the tools of biotechnology offer new promising perspectives in the creation of unique medicines and biological products for the needs of medicine and veterinary.
Test questions: 1. What are the reasons that constrain the use of growth hormone in animal breeding? 2.What antibiotics synthesized by actinomycetes have been widely used in veterinary medicine and animal husbandry? 3. Tell us about the modern approach in the technology of antibiotics production; 4. Give examples of using monoclonal antibodies for therapeutic purposes, 5. Production of monoclonal antibodies by Escherichia coli.
Lecture №6
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