高质量重组人干扰素α2b的分离纯化研究  

Isolation and Purification of Recombinant Human Interferon α2b of High Quality

作　　者：胡敏进 颜法宝 严伟 王强 华子春 HU Min-jin;YAN Fa-bao;YAN Wei;WANG Qiang;HUA Zi-chun(Jiangsu Target Pharma Laboratories Inc.,Changzhou 213164,China;Nanjing Genrecom Laboratories,Ltd.,Nanjing 210043,China;Institute of Pharmaceutical Biotechnology of Jiangsu Industrial Technology Research Institute(Changzhou High-Tech Research Instituteof Nanjing University),Changzhou 213164,China)

机构地区：[1]江苏靶标生物医药研究所有限公司,江苏常州213164 [2]南京吉芮康生物技术研究院有限公司,江苏南京210043 [3]江苏省产业技术研究院医药生物技术研究所(常州南京大学高新技术研究院),江苏常州213164

出　　处：《药物生物技术》2022年第4期353-357,共5页Pharmaceutical Biotechnology

基　　金：常州市科技局(No.CZ20210010,No.CJ20210024);上海华新生物高技术有限公司;江苏苏青生物技术有限公司资助。

摘　　要：重组人干扰素2b作为一种上市药物,被广泛用于治疗慢性丙型肝炎、慢性乙型肝炎、恶性黑色素瘤等多种疾病的治疗,产生了良好的临床疗效和经济效益。目前国内厂家生产的重组人干扰素以大肠杆菌基因工程表达为主。大肠杆菌表达的人干扰素2b以包涵体的形式表达,包涵体产物需通过变性复性以及其后的纯化才能获得生物活性。干扰素复性得率低,纯化时需要经过四步色谱处理,工艺繁琐,成本高,而且无法去除未完全复性的中间产物,从而影响产品质量和疗效。建立可溶性重组人干扰素α2b的高密度发酵工艺,并在此基础上,建立其高效、快速和稳定的纯化工艺。通过发酵工艺优化获得可溶性重组人干扰素α2b的高密度发酵,收集菌体后,通过高压破菌、硫酸铵分级沉淀、phenyl Sepharose Fast Flow疏水层析、Q Sepharose Fast Flow阴离子交换层析、SP Sepharose Fast Flow阳离子交换层析、Octyl Sepharose Fast Flow疏水层析进行分离纯化,并对纯化产物进行生物活性分析。重组人干扰素α2b工程菌的发酵密度A_(600)达到38.5,可溶性重组人干扰素α2b占菌体总蛋白的14.0%;100克湿菌可平均纯化获得240 mg纯度接近100%的重组人IFNα2b,比活为1.81×10^(8) IU/mg。建立的重组人干扰素α2b原液生产工艺具有良好的效率、回收率和活性,产品质量远高于国家药典规定的质量标准。因此,本文所建立生产工艺可用于重组人干扰素α2b工业化生产。Human interferon α2b is widely used for the therapy of various diseases such as chronic hepatitis C,chronic hepatitis B,malignant melanoma and other diseases,it has displayed good efficacy and achieved great commercial success.The present domestically produced human interferon α2b is in the form of inclusion body and needs the complicate denaturation and refolding and four steps of chromatography processes.The yield of refolding is low and purification process is complicated and costly.To develop the high density fermentation process of recombinant human interferonα2 b engineering bacteria,and develop efficient,rapid and stable purification process of soluble recombinant human interferon α2b,the engineered bacteria expressing soluble recombinant human interferon α2b was fermented at high density.After the bacteria were collected,the protein was purified by high-pressure bacteria breaking,ammonium sulfate fractional precipitation,phenyl Sepharose fast flow hydrophobic interaction chromatography,Q Sepharose fast flow ion exchange chromatography,SP Sepharose fast flow ion exchange chromatography and octyl Sepharose fast flow hydrophobic interaction chromatography,and the biological activity of the purified products was analyzed.The fermentation density at OD_(600) of engineering strain reached 38.5,and the soluble recombinant human interferon α2b accounted for 14.0% of the total bacterial proteins.240 mg of IFNα2b with the purity close to 100% can be purified from 100 g of wet bacteria pellets,with the specific activity of IFNα2 b of 1.81×10^(8) IU/mg.The established production process of soluble recombinant human interferon α2b has good efficiency,recovery and activity,and its product quality is much higher than the quality standard stipulated in the national pharmacopoeia.Thus the process can be used in industrial production of recombinant human interferonα2 b.

关 键 词：重组人干扰素Α2B 高密度发酵 纯化 离子交换层析 疏水层析 国产填料 

分 类 号：Q503[生物学—生物化学]