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Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate
Alternative TitleChem. J. Chin. Univ.-Chin.
Hao Su-Juan2; Wang Yin-Jue1,3; Kang Ai-Jun4; Liu Yong-Dong1; Li Xiu-Nan1; Shi Hong1; Ma Run-Yu2; Ma Guang-Hui1; Su Zhi-Guo1
2010-11-10
Source PublicationCHEMICAL JOURNAL OF CHINESE UNIVERSITIES-CHINESE
ISSN0251-0790
Volume31Issue:11Pages:2239-2245
AbstractRecombinant 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.
KeywordErythropoietin Non-glycosylated Pegylation Mpeg-ald
SubtypeArticle
WOS HeadingsScience & Technology ; Physical Sciences
URL查看原文
Indexed BySCI
Language英语
WOS KeywordHAMSTER OVARY CELLS ; POLYETHYLENE-GLYCOL ; GROWTH-FACTOR ; IN-VITRO ; CARBOHYDRATE ; STABILITY ; HIRUDIN ; ANEMIA
WOS Research AreaChemistry
WOS SubjectChemistry, Multidisciplinary
WOS IDWOS:000285538100024
Citation statistics
Cited Times:1[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/6481
Collection研究所(批量导入)
Affiliation1.Chinese Acad Sci, State Key Lab Biochem Engn, Inst Proc Engn, Beijing 100190, Peoples R China
2.Beijing Univ Chem Technol, Coll Life Sci & Technol, Beijing 100029, Peoples R China
3.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China
4.Peking Univ, Dept Lab Anim Sci, Hlth Sci Ctr, Beijing 100191, Peoples R China
Recommended Citation
GB/T 7714
Hao Su-Juan,Wang Yin-Jue,Kang Ai-Jun,et al. Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate[J]. CHEMICAL JOURNAL OF CHINESE UNIVERSITIES-CHINESE,2010,31(11):2239-2245.
APA Hao Su-Juan.,Wang Yin-Jue.,Kang Ai-Jun.,Liu Yong-Dong.,Li Xiu-Nan.,...&Su Zhi-Guo.(2010).Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate.CHEMICAL JOURNAL OF CHINESE UNIVERSITIES-CHINESE,31(11),2239-2245.
MLA Hao Su-Juan,et al."Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate".CHEMICAL JOURNAL OF CHINESE UNIVERSITIES-CHINESE 31.11(2010):2239-2245.
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