生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
6期
817-820
,共4页
张金龙%李冰%任军%宋晓红%张章%侯利华%于长明%付玲%杨秀旭%李建民
張金龍%李冰%任軍%宋曉紅%張章%侯利華%于長明%付玲%楊秀旭%李建民
장금룡%리빙%임군%송효홍%장장%후리화%우장명%부령%양수욱%리건민
炭疽%保护性抗原%人源化抗体%纯化%质量控制
炭疽%保護性抗原%人源化抗體%純化%質量控製
탄저%보호성항원%인원화항체%순화%질량공제
anthrax%protective antigen%humanized monoclonal antibody%purification%quality control
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147995，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/mL。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。
目的：建立CHO細胞錶達的抗炭疽保護性抗原人源化單抗純化工藝和質量控製方法。方法：收穫50 L生物反應器中無血清懸浮培養的CHO工程細胞培養液，通過高速離心去除細胞及碎片後超濾濃縮上清液，經親和層析、SPFF暘離子交換層析後，將所得目的蛋白質經G25凝膠柱更換緩遲液以完成純化；對純化的產品進行單抗鑒彆（Western印跡）、相對分子質量（SDS-PAGE和MOLDI-TOF）、純度（SEC-HPLC）、生物學活性（毒素中和試驗）、產品相關雜質（SEC-HPLC檢測聚集體、降解產物）、工藝相關雜質（ELISA分析殘餘宿主蛋白、殘餘蛋白A）、安全性（凝膠法檢測內毒素、薄膜過濾法攷察無菌）分析。結果：樣品迴收率達61.7%；單抗鑒彆實驗暘性；MOLDI-TOF測定完整分子的相對分子質量為147995，與預期相符；單體比例為99.25%，二聚體比例為0.75%；EC50值為0.1516μg/mL。殘餘宿主蛋白、殘餘蛋白A、內毒素、無菌檢查結果符閤藥典要求。結論：初步建立瞭純化工藝和質量控製方法，為抗炭疽人源化單抗藥物的研製奠定瞭基礎。
목적：건립CHO세포표체적항탄저보호성항원인원화단항순화공예화질량공제방법。방법：수획50 L생물반응기중무혈청현부배양적CHO공정세포배양액，통과고속리심거제세포급쇄편후초려농축상청액，경친화층석、SPFF양리자교환층석후，장소득목적단백질경G25응효주경환완충액이완성순화；대순화적산품진행단항감별（Western인적）、상대분자질량（SDS-PAGE화MOLDI-TOF）、순도（SEC-HPLC）、생물학활성（독소중화시험）、산품상관잡질（SEC-HPLC검측취집체、강해산물）、공예상관잡질（ELISA분석잔여숙주단백、잔여단백A）、안전성（응효법검측내독소、박막과려법고찰무균）분석。결과：양품회수솔체61.7%；단항감별실험양성；MOLDI-TOF측정완정분자적상대분자질량위147995，여예기상부；단체비례위99.25%，이취체비례위0.75%；EC50치위0.1516μg/mL。잔여숙주단백、잔여단백A、내독소、무균검사결과부합약전요구。결론：초보건립료순화공예화질량공제방법，위항탄저인원화단항약물적연제전정료기출。
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.
