CHO细胞表达的抗炭疽保护性抗原人源化抗体的纯化及质量控制分析

Purification and Quality Control Analysis of the CHO-Derived Humanized Anti Anthrax Protective Antigen Monoclonal Antibody

目的:建立CHO细胞表达的抗炭疽保护性抗原人源化单抗纯化工艺和质量控制方法。方法:收获50 L生物反应器中无血清悬浮培养的CHO工程细胞培养液,通过高速离心去除细胞及碎片后超滤浓缩上清液,经亲和层析、SPFF阳离子交换层析后,将所得目的蛋白质经G25凝胶柱更换缓冲液以完成纯化;对纯化的产品进行单抗鉴别(Western印迹)、相对分子质量(SDS-PAGE和MOLDI-TOF)、纯度(SEC-HPLC)、生物学活性(毒素中和试验)、产品相关杂质(SEC-HPLC检测聚集体、降解产物)、工艺相关杂质(ELISA分析残余宿主蛋白、残余蛋白A)、安全性(凝胶法检测内毒素、薄膜过滤法考察无菌)分析。结果:样品回收率达61.7%;单抗鉴别实验阳性;MOLDI-TOF测定完整分子的相对分子质量为147 995,与预期相符;单体比例为99.25%,二聚体比例为0.75%;EC50值为0.1516μg/m L。残余宿主蛋白、残余蛋白A、内毒素、无菌检查结果符合药典要求。结论:初步建立了纯化工艺和质量控制方法,为抗炭疽人源化单抗药物的研制奠定了基础。

Objective： To develop a purification process and quality control analysis method of humanized anti？body against anthrax protective antigen（PA） expressed by CHO cells. Methods： Serum-free medium cultured engi？neering CHO cells were harvested from Current AP50L bioreactor, and then were centrifuged at 12 000 r/min for 20 min and ultrafiltrated to obtain concerntrated supernatant, from which the humanized antibody was purified by affinity chromatography and SPFF cation exchange chromatography, followed by G25 sieve chromatography to change buffer. Every purification step was monitored by protein concentration measurement（UV detection）. The pu？rified product was subjected to specificity identification against PA（Western blotting）, molecular weight analysis （SDS-PAGE and MOLDI-TOF）, and biological activity determination（TNA）, purity determination（SEC-HPLC） and residual host cell proteins and protein A detection（Cygnus ELISA kits）, endotoxin and aseptic examination（meth？ods in current Chinese Pharmacopoeia）. Results： The total recovery of product reached 61.7% finally. Western blotting showed that the antibody was specifically bound to anthrax PA. The EC50 of the antibody determined by TNA test was 0.1516 μg/mL. The molecular weight of the antibody was 147 995 as respected. The proportion of monomer in the final products was 99.25% and the dimer was 0.75%. Other results of quality control items met the standards of the current Chinese Pharmacopoeia. Conclusion： The established purification process and quality control analysis method will help for pharmaceutical research of humanized monoclonal antibody against anthrax.