重组人干扰素α-2b反相层析分离纯化工艺的研究  

Separation and Purification Process of Recombinant Human Interferonα-2b by Reversed Phase Chromatography

作　　者：王文博 李秀男[2] 赵大伟[2] 张松平[2] 张万忠 陈超[2] WANG Wen-bo;LI Xiu-nan;ZHAO Da-wei;ZHANG Song-ping;ZHANG Wan-zhong;CHEN Chao(College of Pharmaceutical and Biological Engineering,Shenyang University of Chemical Technology,Shenyang 110142,China;State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]沈阳化工大学制药与生物工程学院,沈阳110142 [2]中国科学院过程工程研究所生化工程国家重点实验室,北京100190

出　　处：《中国生物工程杂志》2023年第9期9-18,共10页China Biotechnology

基　　金：国家自然科学基金(21821005)。

摘　　要：2020版《中国药典》对重组人干扰素α-2b提出了新的质量标准,增加了相关蛋白的分析检测并明确了含量上限。而相关蛋白的结构与重组人干扰素α-2b的结构仅有微小差别,现有的常规分离方法难以去除。研究建立了重组人干扰素α-2b的反相层析纯化工艺,实现了重组人干扰素α-2b与其相关蛋白的分离。筛选和优化了反相层析填料、洗脱液乙腈浓度及上样量,确定了反相层析分离纯化工艺,包括层析填料:HPLCONE-10C18C3(300?,10μm);洗脱条件:0.1%TFA-48%乙腈水溶液等度洗脱;上样量:不超过柱体积的1.6倍;检测波长210 nm。中试放大结果表明制备得到的重组人干扰素α-2b原液符合2020版《中国药典》的质量标准,原液活性高,纯度大于99%。该工艺分离效果好、成本低、工艺过程简单、易于大规模生产,为干扰素和其他生物大分子药物的生产企业提供了有价值的参考。The 2020 edition of the Chinese Pharmacopoeia proposed new quality control standards for recombinant human interferonα-2b,increased the analysis and detection of related proteins and clarified the upper limit of related proteins.However,the related proteins are only slightly different from recombinant human interferonα-2b in structure,and it is difficult to remove them by conventional separation methods.A reversed phase chromatographic purification process of recombinant human interferonα-2b was established in this paper to separate recombinant human interferonα-2b from related proteins.First,the reversed-phase chromatographic packing and the concentration of acetonitrile in the eluent and the loading amount were screened and optimized.Next,the reversed-phase chromatographic separation and purification process of recombinant human interferonα-2b was determined,which include chromatographic packing:HPLCONE-10C18C3(300?,10μm);elution conditions:0.1%TFA-48%acetonitrile aqueous solution equivalent elution;loading amount:no more than 1.6 times of column volume;detection wavelength:210 nm.Second,the scale up test was carried out.The results showed that the prepared recombinant human interferonα-2b stock solution,with high activity and purity greater than 99%,met the quality control standards of the 2020 edition of the Chinese Pharmacopoeia.The process has the advantages of good separation results,low cost,simple preparation technology and easy commercial production,which provides a valuable reference for the production enterprises of interferons and other biological macromolecular drugs.

关 键 词：重组人干扰素Α-2B 纯化 反相层析 中试 

分 类 号：Q511[生物学—生物化学]