非糖基化促红细胞生成素的聚乙二醇定点修饰及修饰产物性质

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

临床用重组人促红细胞生成素(rhEpo)是中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO)表达的糖蛋白,糖基对稳定蛋白的结构和生物活性非常重要,但CHO表达体系生产成本高、产量低.以大肠杆菌表达的促红细胞生成素为非糖基化蛋白(rh-ngEpo),对其进行聚乙二醇(PEG)修饰可以提高蛋白稳定性和体内循环半衰期.本文采用分子量为20000的N-末端专一性的单甲氧基聚乙二醇-丙醛(mPEG-ALD)修饰rh-ngEpo,对影响修饰反应的因素进行了考察.结果表明,在最佳反应条件下,单修饰率可达55%.修饰混合物经离子交换层析分离,获得了纯度大于95%的单修饰产物,其二、三级结构证明与原蛋白相似.肽图分析结果表明,PEG绝大部分修饰在蛋白N-末端的氨基酸残基上.ELISA分析表明,单修饰产物的体外活性虽然比修饰前减少30%,但热稳定性得到显著增强,在SD大鼠体内的药代动力学性质得到显著提高.研究结果表明,PEG可以在一定程度上替代糖基的作用,PEG修饰的非糖基化Epo有望成为一种新型的促红细胞生成蛋白药物.

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.How-ever,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 monoPEGylat-ed rh-ngEpo was higher than 95% as indicated by HPSEC and RP-HPLC.The secondary and tertiary struc-tures 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 erythro-poiesis-stimulating drugs.