Chem. J. Chin. Univ.-Chin.

Recombinant human erythropoietin (rhEpo) is a glycoprotein expressed in Chinese hamster ovary (CHO) cell. Carbohydrates play an important role in maintaining the protein's stability and bioactivity. However, mammalian expressing system has low yields and high costs of production. In this article, a strategy of PEGylating E. coli expressed recombinant human non-glycosylated Epo (rh-ngEpo) by a 20000 site-specific monomethoxy polyethylene glycol propionaldehyde(mPEG-ALD) was investigated. The modification reaction was optimized and 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 by peptide mapping analysis. The in vitro bioactivity of the monoPEGylated rh-ngEpo decreased 30% compared with its unmodified counterpart while the thermal stability was greatly enhanced. The in vivo pharmacokinetic parameters were greatly enhanced. These results show 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.Recombinant human erythropoietin (rhEpo) is a glycoprotein expressed in Chinese hamster ovary (CHO) cell. Carbohydrates play an important role in maintaining the protein's stability and bioactivity. However, mammalian expressing system has low yields and high costs of production. In this article, a strategy of PEGylating E. coli expressed recombinant human non-glycosylated Epo (rh-ngEpo) by a 20000 site-specific monomethoxy polyethylene glycol propionaldehyde(mPEG-ALD) was investigated. The modification reaction was optimized and 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 by peptide mapping analysis. The in vitro bioactivity of the monoPEGylated rh-ngEpo decreased 30% compared with its unmodified counterpart while the thermal stability was greatly enhanced. The in vivo pharmacokinetic parameters were greatly enhanced. These results show 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