阻断剂单克隆抗体制备及表达工艺优化

Preparation and technology optimization of monoclonal antibody of blocker

[目的]体外制备阻断剂单克隆抗体,并对表达工艺进行优化。[方法]通过RLM-RACE技术钓取杂交瘤细胞抗体基因序列,经分子克隆构建至真核表达载体pCHO1.0,在CHO-S细胞中构建稳定表达细胞株;运用DOE实验设计方法,筛选最佳培养基、培养温度及补料方式;在5 L生物反应器进行工艺验证,蛋白A亲和纯化后用化学发光法进行阻断性能验证。[结果]目的基因序列钓取成功,稳定表达单克隆细胞株11H7表达量为2.90 g/L;使用CHO Max A培养基、梯度补料策略、32℃培养可将表达量提升至5.28 g/L,5 L生物反应器表达量为5.47 g/L,纯度均>95%,且在磁微粒G17和ProGRP项目异常样本中检测满足阻断性能需求。[结论]阻断剂单克隆抗体构建成功,通过表达工艺优化,表达量由2.90 g/L提升至5.28 g/L,且在生物反应器中得到初步验证。

[Objective]To prepare and optimize the technology of monoclonal antibody of blocker.[Method]The monoclonal antibody of blocker was prepared in vitro,and the expression process was optimized to catch hybridoma cells by RLM-RACE technique.The antibody gene sequence of hybridoma cells was obtained by RLM-RACE technique,and the eukaryotic expression vector pCHO1.0 was constructed by molecular cloning,and stable expression cell lines were constructed in CHO-S cells.The optimal medium,culture temperature and feeding method were selected by DOE experimental design method.The process was verified in a 5-L bioreactor,and the blocking performance was verified by chemiluminescence after protein A affinity purification.[Result]The target gene sequence was successfully harvested,and the stable expression of 11H7 cell line was 2.90 g/L.The expression level of CHO Max A culture medium,gradient feeding strategy and 32℃culture could be increased to 5.28 g/L,and the expression was increased to 5.47 g/L in a 5-L bioreactor,both of which were more than 95%purity.The blocking performance requirements were met in the abnormal samples of magnetic particle G17 and ProGRP projects.[Conclusion]The monoclonal antibody of blocker was successfully prepared,and the expression level was increased from 2.90 g/L to 5.28 g/L through the optimization of the expression process,which was preliminarily verified in the bioreactor.