基于细胞周期调控的CHO细胞强化流加补料工艺提高重组抗体表达效率

Development of an intensified fed-batch process to enhance recombinant antibody productivity of CHO cells based on cell cycle regulation

摘要:

中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞是生物药品生产的最主要宿主，但其较低的表达效率阻碍了产业化的发展。为提高CHO细胞的抗体表达效率，本研究以表达抗EGFRv III抗体的Cell A和Cell B细胞株为对象，开展了基于细胞周期调控的强化流加工艺研究。首先通过流式细胞术分析并建立细胞周期与抗体比生产速率(specific antibody production rate, Qp)的关系，结果表明随着培养时间的延长，G0/G1期细胞比例显著增加，在第12天分别达到50%和64%；同时Qp较初期分别提高了1.5倍(Cell A细胞株)和1.0倍(Cell B细胞株)。然而，较高的G0/G1期细胞比例虽显著提升了抗体Qp，但抑制了细胞生长与积累，导致抗体表达总产量下降。为此，本研究在N-1种子阶段(表示生产阶段的上一个步骤)进行补料并延长时间培养，收获时Cell A细胞株和Cell B细胞株的细胞密度分别达到26.3×10⁶ cells/mL和17.7×10⁶ cells/mL，G0/G1期细胞比例分别提升至45%和38%。将该高密度种子接种到5 L生物反应器中进行重组抗体表达培养14 d后，Cell A和Cell B细胞株的累积活细胞密度(integrated viable cell density, IVCD)均超过常规培养工艺，Qp分别提高了11.0%和9.1%，最终抗体浓度分别达到9.5 g/L和5.0 g/L，且对抗体关键质量属性无显著影响。综上，本研究建立的基于细胞周期调控的强化CHO细胞流加补料工艺，可同时提升细胞积累与抗体比生产速率，最终显著提高CHO细胞的抗体表达水平。强化的流加工艺为中国仓鼠卵巢细胞工业规模生产重组抗体蛋白药物提供了一种新的策略。

Abstract:

Chinese hamster ovary (CHO) cells are the most widely used hosts for biopharmaceutical production, yet their low recombinant antibody expression efficiency remains one of key bottlenecks in industrial applications. To enhance anti-EGFRv III antibody expression in CHO cells, an intensified fed-batch process was successfully developed for Cell A and Cell B cell strains based on cell cycle regulation. First, flow cytometry was used to establish the relationship between cell cycle distribution and specific antibody production rate (Qp). The results showed that the proportions of cells in the G0/G1 phase increased significantly over time, reaching 50% and 64% on day 12 for Cell A and Cell B, respectively. Meanwhile, the single-cell Qp increased by 1.5 folds (Cell A) and 1.0 fold (Cell B) compared with initial levels. Higher proportions of cells in the G0 and G1 phases could significantly boost Qp, however, impair cell growth and accumulation, leading to a reduction in total antibody titer. To address this, we implemented feeding during the N-1 seed stage and extended the culture time. At harvest, the densities of Cell A and Cell B reached 26.3×10⁶ cells/mL and 17.7×10⁶ cells/mL, with the proportions of cells in G0/G1 phase increasing to 45% and 38%, respectively. After inoculation of high-density seed cultures into 5 L bioreactors and continuing culture for 14 days, the integrated viable cell density (IVCD) of both Cell A and Cell B exceeded that of the conventional process. In addition, the treatment increased the Qp by 11.0% and 9.1% and achieved final antibody titers of 9.5 g/L and 5.0 g/L for Cell A and Cell B, respectively, with no significant impact on critical quality attributes of recombinant antibody. In summary, the intensified fed-batch process established in this study for CHO cells, based on cell cycle regulation, simultaneously enhances cell accumulation and specific productivity, ultimately significantly improving the recombinant antibody expression levels in CHO cells. Therefore, this intensified fed-batch process can be adopted for recombinant antibody production by CHO cells cultivated at an industrial scale.