稳定表达抗HER2单克隆抗体的FUT8基因敲除细胞株的建立

本研究在实验室前期构建的敲除α-1,6-岩藻糖基转移酶(FUT8)基因的中国仓鼠卵巢(CHO)细胞株(FUT8-/--CHO-S)基础上,建立了稳定表达IgG1型抗HER2单克隆抗体的稳转细胞株。以曲妥珠单抗为模式蛋白,构建了带有嘌呤霉素抗性基因的表达载体pIRES-HC和pIRES-LC,并用电穿孔法共转染入FUT8-/--CHO-S细胞,通过嘌呤霉素加压培养、Dot Blot和WesternBlot等检测方法,经过两轮有限稀释筛选出了高表达的稳转细胞株Tru-FUT8-/--CHO-S。该细胞株在批次培养(Batch)中最高生长密度达每毫升6.05×106个,抗体产量为168 mg/L。在补料培养(Fed-Batch)中,通过补料CHO Feed4的添加,最高生长密度提高到每毫升17.1×106个,抗体产量达到437 mg/L,试验结果表明改造的FUT8-/--CHO-S细胞株具有开发成工业细胞株的潜力。

Establishment of FUT8 Knockout CHO-S Cell Line with Stable Expression of Anti-HER2 Monoclonal Antibody

In order to evaluate potential capability of the α-1,6-fucosyltransferase(FUT8) gene knockout Chinese hamster ovary(CHO) cell line(FUT8-/--CHO-S) in antibody expression, a stable cell line expressing the IgG1 monoclonal antibody was established. We chose trastuzumab as model protein. An anti-puromycin gene was cloned into vector pIRES, which followed by insertion of antibody heavy chain(HC) or light chain(LC) encoding regions respectively. Expression vectors pIRES-HC and pIRES-LC were co-transfected into FUT8-/--CHO-S cells by electroporation(1 800 V, 20 ms and 1 pulse). Transfected cells were selected with 16 μg/ml puromycin for 21 days. Then cell clones were isolated by limiting dilution and the antibody expression were evaluated by Dot Blot and Western Blot. Finally, a stable cell line(Tru-FUT8-/--CHO-S) with reasonable productivity and optimal growth was obtained. In batch culture, the highest cell density of Tru-FUT8-/--CHO-S cells was 6.05×106 cells/ml and antibody titer was 168 mg/L. Then we increased antibody production sequentially by Fed-Batch culture, the highest cell density reached 17.1×106 cells/ml and antibody titer was 437 mg/L with the addition of a mixture of nutrients. The results demonstrated that the engineered cell line FUT8-/--CHO-S was potential to be developed as a biopharmaceutical industrial cell line for antibody production.