表达人源化抗血管内皮生长因子单克隆抗体的重组CHO细胞传代稳定性评价

目的评价表达人源化抗血管内皮生长因子(vascular endothelial growth factor,VEGF)单克隆抗体(简称抗VEGF单抗)的重组CHO细胞(K11细胞)传代稳定性。方法细胞培养瓶中进行K11细胞连续传代,于培养1、4和7个月时测定K11细胞的倍增时间、单细胞抗体表达量和基因拷贝数。在传代培养过程中,分别于传代培养2、3、4、5和7个月时将K11细胞接种至全自动生物反应器中进行发酵培养,测定发酵培养产物的单抗表达量,测序单抗轻、重链基因;采用三步层析(亲和、阴/阳离子交换层析)纯化人源化抗VEGF单抗,分析人源化抗VEGF单抗的纯度、电荷异质性、一级结构和生物学活性。结果 K11细胞在细胞培养瓶中连续传代培养1、4和7个月时,对数生长期K11细胞的倍增时间分别为25、23和34 h,单细胞抗体表达量分别为15. 6、15. 0和11. 5 pg/(个·d),单细胞基因拷贝数分别为200、219和204 copies/个。生物反应器发酵培养5批单抗,纯化后单抗表达量为1. 18~2. 06 g/L,K11细胞单抗轻、重链基因测序结果均与理论序列一致,人源化抗VEGF单抗SEC-HPLC单体纯度为96. 23%~98. 21%,电荷异质性和一级结构相似,生物学活性为(0. 859~0. 901)×10^4U/mg。结论 K11细胞在工业化生产规模的培养周期内保持稳定,可满足人源化抗VEGF单抗的商品化的生产需要。

Stability during subculture of recombinant CHO cells expressing humanized monoclonal antibody against vascular endothelial growth factor

Objective To evaluate the stability of recombinant CHO cells(K11 cells)expressing humanized monoclonal antibody(McAb)against vascular endothelial growth factor(VEGF)during subculture. Methods K11 cells were subcultured in flask for 1,4 and 7 months,and determined for the doubling time(DT),antibody expression level in a single cell and gene copy number. In the process of cell subculture,the K11 cells were inoculated into automated bioreactor 2,3,4,5 and 7 months after subculture,and determined for McAb expression level in fermentation culture. The light and heavy chains of the McAb were sequenced. The humanized McAb against VEGF was purified by affinity,anion and cation exchange chromatography, and analyzed for purity, charge heterogeneity, primary structure and biologic activity.Results After subculture in flask for 1,4 and 7 months,the DTs of K11 cells in logarithmic phase were 25,23 and 34 h,while the antibody expression levels were 15. 6,15. 0 and 11. 5 pg/(cell·d),and the gene copy numbers were 200,219 204 copies/cell,respectively. Five batches of McAbs were cultured by fermentation in bioreactor,of which the expression level was 1. 18 ~ 2. 06 g/L,and the sequences of light and heavy chains were consistent with those in theory.The SEC-HPLC purity of the McAb was 96. 23% ~ 98. 21%. The charge heterogeneities and primary structures of the five batches were in agreement,while the biological activities were(0. 859 ~ 0. 901)× 10^4 U/mg. Conclusion K11 cells were stable in the culture cycle for industrial production scale,which met the requirements for commercial production of humanized McAb against VEGF.