Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate

Chem. J. Chinese Universities ›› 2010, Vol. 31 ›› Issue (11): 2239.

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Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate

HAO Su-Juan1, WANG Yin-Jue2,3, KANG Ai-Jun4, LIU Yong-Dong2*, LI Xiu-Nan2, SHI Hong2, MA Run-Yu1, MA Guang-Hui2, SU Zhi-Guo2

1. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China;
2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
3. Graduate School of Chinese Academy of Sciences, Beijing 100049, China;
4. Department of Laboratory Animal Science, Peking University Health Science Center, Beijing 100191, China

Received:2010-01-21 Online:2010-11-10 Published:2010-11-10
Contact: LIU Yong-Dong. E-mail: ydliu@home.ipe.ac.cn
Supported by:
国家自然科学基金(批准号: 20976178, 20636010, 20820102036)和国家“八六三”计划项目(批准号: 2007AA021604)资助.

Abstract

Abstract: 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.

Key words: Erythropoietin, Non-glycosylated, PEGylation, mPEG-ALD

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HAO Su-Juan, WANG Yin-Jue, KANG Ai-Jun, LIU Yong-Dong*, LI Xiu-Nan, SHI Hong, MA Run-Yu, MA Guang-Hui, SU Zhi-Guo. Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate[J]. Chem. J. Chinese Universities, 2010, 31(11): 2239.

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Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate

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