Motronenko V. Biotechnology of the substance of recombinant human interleukin-7

Human's interleukin-7 (IL-7) is a lymphopoietic growth factor that holds a special position among other cytokines because of its unique function in hematopoiesis, which is not duplicated by other factors. Lack of functional IL-7 may be one of the causes of severe combined immunodeficiency. The result of functional’s lack IL-7 in the body is lymphopenia and, as a consequence, severe immunodeficiency. The level of B- and T-lymphocytes in the blood increases with the increase in the number of IL-7, IL-7 is involved in the lymph nodes’ formation. The major producers of IL-7 are bone marrow and thymus stromal cells, as well as some other cells in the human body. Scientific novelty of the obtained results. The composition of the nutrient medium for rIL-7 biosynthesis was optimized by E. coli bacteria (yield 0.95mg/ml) with high in vitro and in vivo biological activity, which increased the rIL-7 yield 1.3 times compared to the basic technology. The stimulating effect extract of Cliviaminiata and Zephyranthes grandiflora (0.5-1.0%) and liposomal form of vitamin K1 (15-25mg/ml) as stimulating additives to the nutrient medium is shown (leading to an increase in the yield of the target product 1.69 times). The biologically active substances of the investigated phytoextracts interact with the pores of the outer membrane of E. coli, which is likely to cause an increase in the biosynthetic activity of the recombinant producer. It has been shown that administration of rIL-7 (5mg rIL-7 daily intraperitoneally, daily before wound healing) accelerates the rate of wound healing in experimental animals and the elimination of Pseudomonas aeruginosa from them. The results of the work are in close connection with the current scientific and methodological approaches for the creation of recombinant proteins for therapeutic purposes. The practical significance of the results obtained. The technological and instrumental scheme of obtaining substance rIL-7 was developed and the critical points of the production process were analysed. The rational quantitative composition of organic components and minerals of the nutrient medium for the cultivation of recombinant producer E. coli was experimentally substantiated, which allowed to increase the yield of RIL-7 1.3 times. The stimulating effect of clover extracts of cinnabar and zephyrantes large-flowered as additives to the nutrient medium in the cultivation of recombinant E. coli bacteria in the range of 0.5-1.0%, which provided an increase in the yield of the target product 1.69-1.82 times. The stimulating effect of the liposomal form of vitamin K1 at a concentration of 15-25mg/ml on the level of rIL-7 biosynthesis (an increase of 1.74-1.81 times) is shown. The mechanisms of biological activity of phytoextracts for the synthesis of rIL-7 by bacterial producers have been shown to include interaction with the surface structures of a bacterial cell. An advanced scheme for purification of rIL-7 was developed by introducing a highly specific immunoaffinity chromatography step, which allowed the substance to be obtained at 98%. For the tetraethoxysilane-synthesized immunoaffinity column, rational technological parameters of chromatographic purification have been determined, which allow to obtain a highly purified product over 15 cycles of continuous operation and over a wide range of product concentrations (0.005-20μg/ml). The efficacy of the use of rIL-7 in the treatment of P. aeruginosa wound infections has been experimentally proven. It was shown that in mice treated with rIL-7 (5μg per day intraperitoneally), wound healing and pathogen elimination occurred 5 days earlier than in mice without rIL-7 treatment. The technological and equipment-technological scheme of obtaining the substance of human rIL-7 based on the obtained experimental data was developed. The risks of the process of obtaining the substance rIL-7 were evaluated and the most critical stages were identified, which allowed the development of a production control system to prevent and minimize risks. The feasibility study of the developed technology of obtaining substance rIL-7 has been carried out: the cost of the final product and the production capacity have been determined in view of the safety of patients of Ukraine and the prevalence of relevant pathologies. The estimated production capacity was 240g of substance rIL-7 per year, and its cost was 43 478UAH. The estimated raw material cost of the ready-to-use injection form of the drug rIL-7 (3-60µg per dose) is 38-1512UAH, which determines its social accessibility for the population of Ukraine.