Produção do fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) em biorreator

Abstract

The granulocyte colony-stimulating factor (G-CSF) is a major hematopoietic cytokine involved in the immune defense against infectious agents, stimulating and regulating the proliferation, survival and differentiation of neutrophils precursor cells in the bone marrow. The G-CSF molecule has a 174 amino acids sequence, with a molecular weight of approximately 18.8 kDa. As a strategy for neutropenia prevention, G-CSF (or filgrastim) has been successfully used in cancer patients whose treatment requires high doses of chemotherapy, in both adults and children. Moreover, this biopharmaceutical may be used to strengthen the immune system in patients with HIV, pneumonia, infections resulting from diabetes, leukemia and febrile neutropenia. Currently, filgrastim is not produced in Brazil, which obligates the government to import this medicine. Due to its wide clinical application, the large scale production of G-CSF is required to supply the demand of national market. In this work, a protocol to obtain this protein was developed using overexpression and cultivation in a bioreactor, solubilization and purification of recombinant G-CSF. The protein was expressed in Escherichia coli host cells C41(DE3) cultures grown in bioreactor using a fed-batch strategy. The expression of the recombinant protein was induced by IPTG. A linear ascending feeding strategy permitted high plasmid stability, low accumulation of acetate in the culture medium, a biomass of approximately 31 g/ L and high expression levels of the protein in the form of inclusion bodies which were solubilized using 2 M urea and alkaline pH. The recombinant protein was purified and yielded approximately 1.22 mg of homogeneous recombinant protein per gram of wet cells, corresponding to a volumetric yield of 151.5 mg of rhG-CSF per liter of culture medium. A mass spectrometry analysis was performed, confirming the identity of the recombinant protein. Our work shows a simple and effective strategy to obtain recombinant hG-CSF, stimulating and encouraging a future production of a national biosimilar.