Site-specific PEGylation of Recombinant Human Non-glycosylated Erythropoietin and Characterization of the Mono-PEGylated Conjugate
文献类型：期刊论文
| 作者 | 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 |
| 刊名 | CHEMICAL JOURNAL OF CHINESE UNIVERSITIES-CHINESE
 |
| 出版日期 | 2010-11-10 |
| 卷号 | 31期号:11页码:2239-2245 |
| 关键词 | Erythropoietin Non-glycosylated PEGylation mPEG-ALD |
| ISSN号 | 0251-0790 |
| 其他题名 | Chem. J. Chin. Univ.-Chin. |
| 中文摘要 | 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. |
| 英文摘要 | 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. |
| WOS标题词 | Science & Technology ; Physical Sciences |
| 类目[WOS] | Chemistry, Multidisciplinary |
| 研究领域[WOS] | Chemistry |
| 关键词[WOS] | HAMSTER OVARY CELLS ; POLYETHYLENE-GLYCOL ; GROWTH-FACTOR ; IN-VITRO ; CARBOHYDRATE ; STABILITY ; HIRUDIN ; ANEMIA |
| 收录类别 | SCI |
| 原文出处 | |
| 语种 | 英语 |
| WOS记录号 | WOS:000285538100024 |
| 公开日期 | 2013-11-28 |
| 版本 | 出版稿 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/6481]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 作者单位 | 1.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 |
| 推荐引用方式 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. |
入库方式： OAI收割
来源：过程工程研究所
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