基于实验设计法针对抗VEGFR2-MICA融合蛋白进行发酵工艺优化（英文）  

Fermentation Optimization of an Anti-VEGFR2-MICA Fusion Protein Adopting Design of Experiments(DOE)

作　　者：柳芳[1] 王佑富 唐铭英 杜晓典 张航[1] 王旻[1] 张娟[1] 

机构地区：[1]中国药科大学生命科学与技术学院抗体工程实验室,江苏南京210009

出　　处：《药物生物技术》2017年第3期211-217,共7页Pharmaceutical Biotechnology

基　　金：supported by the National Natural Science Foundation of China(No.NSFC81273425 and NSFC81473125);Natural Science Foundation of Jiangsu Province(No.BK20161459);Jiangsu Province Qinglan Project(2014);Specialized Research Fund for the Doctoral Program of Higher Education(No.20130096110007);The Graduate Innovation Program of Jiangsu Province(No.KYLX15_0670);A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions

摘　　要：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1×106cells/mL接种密度将细胞接种于商业培养基CDM4PerM Ab,每2 d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3 L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 thera- peutic antibodies. The cultivation of CHO cells and expression of antibody are extremely related to culturing environ- ment. 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 Minitabl6.0, 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 sus- tain 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 CDMdPerMAb, supplemented with Boost2 ＋ BoostS, 12% （V/V） volume, 1 x 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 func- tion on MDA-MB-231 cells and K562 cells,the MTT 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

关 键 词：中国仓鼠卵巢细胞 实验设计法 补料分批培养 过程开发 细胞密度 发酵条件 产品滴度 

分 类 号：TQ460.72[医药卫生—药物分析学]