重组人血清白蛋白-干扰素β融合蛋白的分离纯化及质控方法的建立  

Isolation,purification and development of method for quality control of recombinant human serum albumin-interferon β fusion protein

作　　者：李芝芹[1] 邱爽[1] 雷楗勇[1] 陈蕴[1] 金坚[1] 

机构地区：[1]江南大学医药学院药物设计与分子药理学实验室,江苏无锡214122

出　　处：《中国生物制品学杂志》2012年第10期1325-1328,共4页Chinese Journal of Biologicals

基　　金：国家自然基金资助项目(30970029)

摘　　要：目的建立中试制备重组人血清白蛋白-干扰素β(Recombinant human serum albumin-interferonβ,rHSA-IFNβ)融合蛋白的纯化工艺及质控方法。方法采用50 L发酵罐诱导表达rHSA-IFNβ融合蛋白,发酵液经超滤浓缩及脱盐后,再经BlueSepharose FF亲和层析、G25凝胶过滤层析和SP Sepharose FF阳离子交换层析纯化。SDS-PAGE(银染法)和HPLC测定融合蛋白纯度,Western blot分析反应原性,质谱法测定相对分子质量,Edman降解法测定N-末端氨基酸序列,等电聚焦电泳法测定等电点,细胞病变抑制法测定rHSA-IFNβ融合蛋白的生物学活性,其余检测项目均按《中国药典》三部(2010版)要求进行。结果纯化的3批融合蛋白纯度可达96%以上,具有人血清白蛋白和人干扰素β的双重反应原性,相对分子质量分别为89 070、89 035和88 669,N-末端氨基酸序列为:NH2-D-A-H-K-S,等电点为6.34,比活性分别为1.9×106、1.5×106和1.1×106IU/mg,其余各项指标均符合要求。结论已成功建立了中试制备rHSA-IFNβ融合蛋白的纯化工艺及质控方法。Objective To develop a procedure for purification and a method for quality control of recombinant human serum albumin-interferon β （rHSA-IFNβ） fusion protein produced in a pilot scale. Methods The expression of rHSA-IFNβ fusion protein was induced in 50 L fermenter. The fermentation broth was concentrated by ultrafiltration and purified by Blue Sepharose FF affinity chromatography, G25 gel filtration chromatography and SP Sepharose FF cation exchange chromatography, then determined for purity of fusion protein by SDS-PAGE after silver staining and HPLC, for reactogenicity by Western blot, for relative molecular mass by mass spectrometry, for biological activity by CPE inhibition test, and for other quality indexes according to the requirements in Chinese Pharmacopoeia （Volume 11I, 2010 edition）. Results Three batches of the expressed fusion protein, with relative molecular masses of about 89 070, 89 035 and 88 669 respectively, reached purities of more than 96% after purification and showed both reactogenicitics of rHSA and IFN[3, of which the amino acid sequences at N-terminus were NH2-D-A-H-K-S, while the isoelectric points were 6. 34, and the activities were 1.9 x 10^6 , 1.5 x 10^6 and 1. 1 x 10^6 IU/mg respectively. All the other indexes met the requirements in Chinese Pharmacopoeia （Volume Ill , 2010 edition）. Conclusion A procedure for purification and a method for quality control of recom- binant human serum albumin-interferon β（rHSA-IFNβ） fusion protein produced in a pilot scale were successfully developed.

关 键 词：干扰素Β 血清白蛋白 毕赤酵母 融合蛋白 质量控制 

分 类 号：Q511[生物学—生物化学] R392-33[医药卫生—免疫学]