疏水层析法和凝胶过滤法分离HI47抗CD20F(ab’)_2之比较

Comparison of hydrophobic interaction chromatography and gel filtration chromatography for purification of HI47 anti-CD20 F(ab')_2

目的 比较疏水层析和凝胶过滤两种方法分离纯化HI4 7抗CD2 0F(ab’) 2 的优缺点。方法 采用亲和层析柱protein G对发酵菌体裂解液进行粗纯化,分别采用疏水层析法和凝胶过滤法进行抗CD2 0F(ab’) 2 的分离纯化:疏水层析采用梯度洗脱,80 %B液洗脱2 0min ,10 0 %B液洗脱30min ;凝胶过滤以5 0mmol LPB缓冲液(pH 7.0 ) ,0 .8mL min洗脱4h。结果 粗纯化产物主要含F(ab’) 2 、Fab’,含量分别为19.6 %、5 9.7%。采用疏水层析法1h即可完成分离,F(ab’) 2 回收率为6 7% ,纯度为89.3% ;凝胶过滤4h完成分离,F(ab’) 2 回收率为6 5 % ,纯度为90 .5 %。FACS结果表明,两者结合Raji细胞的阳性率分别为99.93%和99.97%。结论 疏水层析分离纯化抗CD2 0F(ab’) 2 简单、快速、回收率高、纯度高,适用于大规模的工业化生产。

Objective To compare the efficiency of hydrophobic interaction chromatography and gel filtration chromatography for purification of HI47 anti-CD20 F(ab') 2. Methods The fermentation paste was prepurified using protein G column, then the samples were purified by hydrophobic interaction chromatography and gel filtration chromatography, respectively. The HI47-CD anti-CD20 F(ab') 2 purified by hydrophobic interaction chromatography was washed with 80% B buffer for 20 min, and then with 100% B buffer for 30 min. The HI47-CD anti-CD20 F(ab') 2 purified by gel filtration chromatography was washed with PB buffer (PH 7.0) for 4 h. Results The product of prepurification mainly contained F(ab') 2 and Fab' with purities of 19.6% and 59.7%, respectively. The hydrophobic interaction chromatography was finished in 1 h, and the yield of F(ab') 2 was 67%, and the purity was 89.3%. The gel filtration chromatography was finished in 4 hour, and the yield and purity were 65% and 90.5%, respectively. The fluorescence-activated cell sorting (FACS) results showed that the fragments purified by the two methods could bind to Raji cells with positive rates of 99.3% and 99.7%, respectively. Conclusion Hydrophobic interaction chromatography is a simple and timesaving method to obtain high yield and purity of anti-CD20 F(ab') 2 fragment.