Development bioactive of human recombinant GM-CSF produced in Pichia Pastoris yeast

In the clinic, preparations of recombinant GM-CSF are used for recovery after cancer chemotherapy and bone marrow transplantation. Currently, only two preparations of recombinant GM-CSF are available for clinical use - Sargramostim (produced by S. cerevisiae) and Molgramostim (produced by E. coli). The methylotrophic yeast Pichia pastoris is an alternative host cell system due to its non-immunogenic glycosylation pattern, high yield of secreted protein, and ease of culture conditions. In the present work, a strain of P. pastoris provided efficient expression and synthesis of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was obtained. The yield of GM-CSF during shake-flask fermeпtation was 95-100 mg/L. The recombinant GM-CSF was purified by three-stage ion-exchange chromatography to about 95% purity. The bioactivity of the GM-CSF preparation was assessed both by the ability to stimulate the proliferation of the TF1 erythroid line cells аs well as by its ability to induce the maturation of dendritic cells from human mononuclear cells. It was found that yeast-derived GM-CSF exhibited a 2-fold proliferative effect on BFU-E at a concentration 1.7 times less than GM-CSF produced by biosynthesis in E. coli. Dendritic cells prepared by incubation with yeast GM-CSF exhibited a similar effect to commercial bacterial GM-CSF in terms of their ability to induce lymphocyte proliferation.