正交试验设计筛选scFv包涵体变性和复性工艺

Screening of Denaturation and Renaturation Process for scFv Inclusion Body through Orthogonal Experimental Design

单链抗体(single-chain variable fragment,scFv)包涵体的变性和复性受p H值、温度、盐浓度、氧化还原电对、添加剂等多种因素影响,快速找到合适的变、复性条件是蛋白质纯化的重点和难点。本试验以scFv-Z2为研究对象,通过正交试验设计筛选scFv-Z2包涵体的变性和复性条件,找出关键的影响因素,得到最适变性条件:6 mol/L盐酸胍变性,变性时间24 h;最适复性条件:以4 mol/L尿素、1 mol/L精氨酸(Arg)、1 mmol/L还原型谷胱甘肽(GSH)和0.02 mmol/L氧化型谷胱甘肽(GSGG)的溶液为复性液,样品与复性液比例为1∶30,复性时间7 d。通过SP阳离子柱分离后得到高纯度(99.2%)scFv-Z2单体,经工艺放大重复验证,最终开发出稳定可靠的纯化工艺路线。本研究建立了正交试验设计在scFv-Z2包涵体纯化工艺开发中的新应用。

Denaturation and renaturation of single-chain variable fragment （scFv） inclusion body are influenced by pH value, temperature, salt concentration, redox couples, additives and other factors. How to quickly find the right operating conditions is the important and difficult point in proteins＇ purification. This study takes the scFv-Z2 as the study example, the experimental conditions of denaturation and renaturation of scFv inclusion body were screened by the orthogonal experimental design. It helped us to analyze the key influencing factors and to find out the optimum conditions. The optimized denature conditions were determined as follows： 6 mol/L guanidine hydrochloride as the denature solution, with the denaturation period of 24 h. The optimum renaturation dilution was composed by 4 mol/L urea, 1 mol/L arginine （Arg）, 1 mmol/L GSH and 0.02 mmol/L GSGG. The ratio of sample and renaturaiton dilution was 1 ： 30, with the renaturaiton time of 7 days. As a result, scFv-Z2 with monomer purity of 99.2 % was separated by SP cation column chromatography. The conditions obtained through the orthogonal experiments were verified by scale-up test and proved to be a repeatable and scaled purification process. The content above demonstrated a new application of orthogonal experimental design in protein purification process development.