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中国仓鼠卵巢细胞(CHO)是目前生产治疗性抗体的主要表达系统,该细胞的培养和抗体表达与培养条件密切相关.本实验室构建出了一种靶向VEGFR2单抗融合MICA的融合蛋白,具有良好的抗肿瘤活性,但是抗体产量较低,限制了该抗体的进一步研发和应用.为了探究适合该抗体的最佳发酵体系,本研究通过Minitab16.0正交法设计多种流加培养方式,探索了4种培养基、流加物以及培养条件对发酵细胞密度和抗体产量的影响,并优化出最佳的发酵方式,即以1×106 cells/mL接种密度将细胞接种于商业培养基CDM4PerMAb,每2d加入12％ (V/V)的流加物Boost2+ Boost5.为保持抗体的稳定性和质量一致性,将此发酵方式扩大至3L发酵罐以提高抗体的批次产量.最终抗体产量可达54.45 mg/L,并且Western blot实验结果验证了融合抗体装配的正确性.MTT实验结果表明JZB01能有效抑制HUVEC、MDA-MB-231和K562的细胞增殖,具有显著的抗血管生成作用以及比母体单抗更有效的抗肿瘤活性.综上,本研究开发出了一种有利于抗体生产的发酵工艺,能够满足实验室抗体研发需求.本实验室将进一步扩大该工艺适用的发酵规模,以满足抗体的临床前研究以及后续工业化生产.“,”Chinese hamster ovary (CHO) cells have become the mainstream in expression systems for producing therapeutic antibodies.The cultivation of CHO cells and expression of antibody are extremely related to culturing environment.We previously generated a fusion antibody named JZB01 targeting vascular endothelial growth factor receptor 2 (VEGFR2) and natural killer cells with significant anti-tumor activity but limited production.In this study,we aim to develop a robust method for fed-batch fermentation using design of experiments (DOE) with Minitabl 6.0,and evaluate the influence of commercial media,supplements and culture conditions on cell viability and antibody production.To improve the yield of fusion protein and establish a strategy for further study,the optimization of the production was undertook with orthogonal experimental analysis for four factors and the level was assessed by the response.Furthermore,in order to sustain the stability and uniformity of fusion protein for further research and diagnosis,the bioprocess was scaled up to a 3-L bio-reactor under the conditions of commercial medium CDM4PerMAb,supplemented with Boost2 + Boost5,12％ (V/V) volume,1 × 106 cells/mL of initial density and 2 days of feeding.The final yield was 54.45 mg/L and Western blot was adopted to identificated the affinity of fusion protein preliminarily,which was characterized as a homodimer with binding ability to each receptor.By the investigation for anti-angiogenesis function of JZB01 on HUVEC cells and anti-tumor function on MDA-MB-231 cells and K562 cells,the MrTT assay confirmed that the fusion protein had anti-angiogenic effect and enhanced anti-tumor activities.Thus,by establishing a platform to obtain fusion protein with high molecular weight in CHO cells system along with moderate costs,this study provides a robust methodology for producing therapeutic antibodies at laboratory level.And more trials will be taken to enlarge the application to industrial scale which could be applied in vitro and vivo preclinical tests.