Expressing The Epo-fc Fusion Protein Engineered Cell Line Identification And The Nature Of The Fusion Protein And The Quality Control Standards

Erythropoietin (EPO) is a hormone produced by the kidney. It is a glycoprotein that promotes the proliferation and differentiation of erythrocyte (red blood cell) precursors in the bone marrow and regulates hemoglobin production. Recombinant human erythropoietin(rHuEPO), a 165-amino-acid glycoprotein, is used to treat chronic renal disease, anemia of cancer and anemia associated with chemotherapy. The rHuEPO was obtained fromn Chinese hamster ovary(CHO)cells by genetic engineering. Since 1989, rHuEPO year sale room had been $10 billion. It was one of the greatest biotechnological product all over the world. Alought rHuEPO allows for the optimal correction of renal anemia in dialysis patients, thus reducing blood transfusion and improving their physical fitness and general wellbeing, the serum half-life of rHuEPO is short and the therapeutic protocol used in the treatment of patient requires frequent injections of rHuEPO, which increases the suffering and unconvenient, and severely reduces the clinical compliableness. Many research institutions had been prolonging half-life of rHuEPO though many kinds of technique. Now, a few products have been approved to sale by FDA. The products were named long-lasting EPO which prolong half-life.Immunoglobulin G (IgG), a class of antibodies which is predominant in serum, has a half-life of up to 21 days. It was a good idea to combine EPO molecule with the Fc fragment of human IgG to constructed EPO-Fc fusion protein molecule which had a prolonged in vivo half-life. EPO-Fc fusion protein not only had the biological effects of EPO but also had the effects of long half-life of Fc fragment. Now, we have obtained recombinant CHO cell expressed EPO-Fc. This paper focus on three aspects, one aspect was the safty and stability of recombinant CHO cell expressed EPO-Fc. one aspect was the physico-chemical properties, bioactivity, immunogenicity and long-lasting properties of EPO-Fc fusion protein. Another aspect was the requirements and methods for quality control and bioassay of EPO-Fc fusion protein.The research showed that the safty and stability of recombinant CHO cell expressed EPO-Fc had been all analysed according to the Chinese pharmacopoeia( 2005.volumeⅢ) and guidelines for quality control of recombinant DNA products. The results of bioassay were reliable and duplicable. The physico-chemical and biological properties were revealed. Immunogenicity study of EPO-Fc fusion protein indicated that most rhesus monkeys emerged antibody and neutralization antibody after subcutaneous injections once weekly for 16 weeks. Some antibody titer in some monkeys descended significantly, and other antibody titer even descended to normal level at the end of recovery phase. EPO-Fc fusion protein has certain immunogenicity to rhesus monkeys because it is a extrinsic protein, but it should have little immunogenicity to human as a endogenesis recombinant protein. We ensure that EPO-Fc has a bright prospect of clinical application.The research revealed The physico-chemical and biological properties of EPO-Fc fusion protein for the first time. Recombinant CHO cell expressed EPO-Fc conformed to the standard of recombinant biological products. The complete system of quality control and bioassay of EPO-Fc fusion protein had been established first. The reference standard material had been established. EPO-Fc fusion protein has immunogenicity to rhesus monkeys,but it shoud be observed in clinic trial.