论文部分内容阅读
目的用填料为羟基磷灰石的层析柱纯化抗TNF-α单克隆抗体。方法经protein A为填料的层析柱纯化抗TNF-α单克隆抗体后,通过Do E实验设计,对填料为羟基磷灰石的层析柱的洗脱流速、p H值及盐浓度进行优化,确定最佳洗脱条件后,进行抗TNF-α单克隆抗体的纯化,纯化产物经HPLC及毛细管电泳法检测其浓度、多聚体含量及纯度。结果抗TNF-α单克隆抗体经protein A层析纯化后,回收率达96.1%,纯度为96.0%,多聚体含量下降至1.9%。通过Do E优化,筛选出最佳条件范围为:流速1~3 ml/min,0.25~0.30 mol/L Na Cl,p H 7.0~7.3,选定1 ml/min流速、0.25 mol/L Na Cl、p H 7.0、作为洗脱条件进行纯化,回收率为93.1%,纯度为97.3%,多聚体含量为0,优于参比制剂。结论将羟基磷灰石用于抗TNF-α单克隆抗体的中度纯化,获得符合参比制剂质量的产品。
OBJECTIVE To purify anti-TNF-α monoclonal antibody with column packed with hydroxyapatite. Methods After the purification of anti-TNF-α monoclonal antibody by chromatography on protein A, the elution flow rate, p H value and salt concentration of the column packed with hydroxyapatite were optimized by Do E experimental design , Determine the best elution conditions, the anti-TNF-α monoclonal antibody purification, purification of the product by HPLC and capillary electrophoresis to detect the concentration, polymer content and purity. Results After purification by protein A chromatography, the recovery of anti-TNF-α monoclonal antibody reached 96.1% with a purity of 96.0% and the polymer content decreased to 1.9%. The optimal conditions were as follows: flow rate 1 ~ 3 ml / min, 0.25 ~ 0.30 mol / L NaCl, pH 7.0 ~ 7.3, flow rate 1 ml / min, 0.25 mol / L NaCl , P H 7.0, purified as elution conditions with a recovery of 93.1%, a purity of 97.3% and a polymer content of 0, superior to the reference formulation. Conclusion Hydroxyapatite was used for the moderate purification of anti-TNF-α monoclonal antibody, and the products with the quality of reference preparation were obtained.