响应面法优化人凝血因子Ⅸ肝素亲和层析条件研究

Parameters optimization in Heparin affinity chromatography purification of human coagulation factor Ⅸ

目的利用响应面法对肝素亲和层析在人凝血因子Ⅸ纯化中的纯化条件进行优化,确立最佳层析工艺参数。方法通过单因素试验分析上样温度对纯化效果的影响,三因素三水平响应面法优化层析洗脱条件,福林酚法和一期凝固法分别测定总蛋白含量和人凝血因子Ⅸ效价,以比活性指标以及工艺回收率评价层析纯化效果。结果优化的最佳层析条件为5℃上样,洗涤4 CV,洗脱液配方枸橼酸钠20 mmol/L、盐酸精氨酸18.7 mmol/L、NaCl 611.6 mmol/L、pH7.5;在此最佳层析参数条件下工艺回收率为(46.6±2.9)%,人凝血因子Ⅸ效价达到(68.4±4.7) IU/mL,比活性(62.8±3.3) IU/mg。结论经本研究优化的肝素亲和层析工艺参数可对人凝血因子Ⅸ的纯化起到很好的指引作用。

Objective To optimize the purification conditions of heparin affinity chromatography in the purification of human coagulation factor Ⅸ by response surface method and establish the optimal chromatography process parameters.Methods The effect of sample loading temperature on purification efficiency was analyzed through single factor test. Three-factor three-level response surface method was used to optimize the chromatographic elution conditions. The Folin phenol method and the automatic hemagglutination analyzer were used to determine the total protein content and human coagulation factor Ⅸ titer, respectively. The purification effect was evaluated by activity index and process recovery rate.Results The optimized optimal chromatographic conditions were loading at 5 ℃, washing 4 CV, eluent formulation of sodium citrate 20 mmol/L, arginine hydrochloride 18.7 mmol/L, NaCl 611.6 mmol/L and pH 7.5;under this optimal setting, the recovery rate of the chromatographic process was(46.6±2.9) %, titer of factor Ⅸ rated to(68.4±4.7) IU/mL and specific activity was(62.8±3.3) IU/mg. Conclusion The optimized parameters of heparin affinity chromatography process by response surface method can produce better purification effect on human coagulation factor Ⅸ.