Investigation On The Site-specific PEGylation Of Recombinant Human Non-glycosylated Erythropoietin

Erythropoietin (Epo) is a cytokine which stimulates division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. Recombinant human Epo (rhEpo) has been used in the treatment of anemia resulting from chronic renal failure (CRF), AIDs and cancer therapies. Carbohydrates play an important role in maintaining stability and in vivo hematopoietic activity of the protein. At present, clinical used rhEpo was mostly expressed in CHO cells. However, mammalian expressing system has low yields and high costs of production. In contrast, Escherichia coli (E.coli) expression system allows simple culture conditions, economic and fast production of large amounts of homogeneous protein. Although E.coli expressed recombinant human non-glycosylated Epo (rh-ngEpo) had almost no in vivo bioactivity because deglycosylation greatly inceased its clearance rate in body, this deficiency may be overcome by covalent PEG conjugation. PEGylation can improve protein drugs'pharmacokinetics and in vivo activity through reducing renal clearance, immunogenicity, and degradation by metabolic enzymes. In this article, a strategy of PEGylating rh-ngEpo by 20 kDa and 30 kDa monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD) was investigated. It was found that the optimized reaction conditions for the highest yield of the monoPEGylated rh-ngEpo was as follows:20 mmol/L PB,0.5 mol/L mannitol, pH 6.0, concentration of rh-ngEpo 1 mg/mL, molar ratio of PEG/rh-ngEpo 10:1, molar ratio of NaBH3CN/PEG 10:1, and reaction time 24 h at 4℃. Under the optimized reaction condition, a high mono-modification yield of 55% was achieved. Ion exchange chromatography was then used to separate the monoPEGylated rh-ngEpo from the reaction mixture. The purity of the monoPEGylated rh-ngEpo was higher than 95% as indicated by HPSEC and RP-HPLC.The secondary and tertiary structures of rh-ngEpo were not changed by PEGylation. rh-ngEpo was PEGylated mostly at the N-terminus studied by peptide mapping analysis. The in vitro immunoactivity of the 20k-PEG-ALD-rh-ngEpo and 30k-PEG-ALD-rh-ngEpo decreased 30% and 45.6% respectively compared with its unmodified counterpart while the thermal stability was greatly enhanced. Moreover, the in vivo pharmacokinetic parameters were greatly enhanced.In summary, we have optimized the reaction conditions of rh-ngEpo PEGylation, separated and analyzed the structure, physicochemical properties, pharmacokinetics of the monoPEGylated rh-ngEpos. These results showed that PEG could replace carbohydrates in enhancing the in vivo stability of nonglycosylated Epo. This research provides a direction for the development of new erythropoiesis-stimulating drugs.