羟基磷灰石分离纯化抗TNF-α单克隆抗体

Purification of monoclonal antibody against TNF-α with macro-prep ceramic hydroxyapatite

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 the monoclonal antibody（m Ab）against tumor necrosis factor（TNF）-α with macro-prep ceramic hydroxyapatite（MPC）chromatography. Methods Crude m Ab agasint TNF-α was purified by protein A affinity chromatography followed by a MPC chromatography step. The elution velocity, p H value and salt concentration of MPC were optimized by the Design of Experiment（Do E）. The m Ab was purified under the optimized condition, and determined for concentration, polymer content and purity by HPLC and capillary electrophoresis. Results The recovery rate and purity of m Ab against TNF-α after purification were 96. 1% and 96. 0% respectively, while the polymer content decreased to 1. 9%. The optimal range of flow rate, sodium chloride concentration and p H value obtained by Do E were 1 ~ 3 ml / min,0. 25 ~ 0. 30 mol / L and 7. 0 ~ 7. 3 respectively, based on which the elution condition was optimized as follows：flow rate 1 ml / min, sodium chloride concentration 0. 25 mol / L, and p H 7. 0. The recovery rate, purity and polymer content of m Ab under the optimal condition were 93. 1%, 97. 3% and 0 respectively, which were superior to those of reference.Conclusion The anti-TNF-α m Ab meeting the requirements for quality of reference was obtained by moderate purification by MPC.