Modern technologies for production of antibiotics

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 laboratoryand 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. 

Improvingthe 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.

The use of monoclonal antibodies for therapeutic purposes

Monoclonal antibodiesas 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.    


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