超声截留灌流培养重组CHO细胞工艺优化  

Process optimization of ultrasonic intercept perfusion culture recombinant CHO cells

作　　者：庄宗兰 程杰 彭杰 周云 祁碧玉 杨文静 杨世龙 谭小东 ZHUANG Zong-lan;CHENG Jie;PENG Jie;ZHOU Yun;QI Bi-yu;YANG Wen-jing;YANG Shi-long;TAN Xiao-dong(The Research Office 3 of R&D Center,Anhui Zhifei Longcom Biopharmaceutical Co.,Ltd..,Hefei 230088,Anhui Province,China)

机构地区：[1]安徽智飞龙科马生物制药有限公司研发中心研究三室,安徽合肥230088

出　　处：《微生物学免疫学进展》2022年第3期43-49,共7页Progress In Microbiology and Immunology

摘　　要：目的采用超声截留控制器超声使得细胞聚集、沉降的方法截留悬浮细胞,优化重组CHO细胞灌流培养工艺。方法基于无血清化学限定(CD)培养基,选择表达水痘-带状疱疹病毒(varicella-zoster virus,VZV)糖蛋白E(VZV gE)的重组CHO细胞在5 L生物反应器中灌流培养。对初步确定的培养条件进行响应面分析,优化培养温度、培养基pH、灌流速度;并采用上述优化条件验证超声截留控制器灌流培养重组CHO细胞工艺。结果本实验条件下,优化后的灌流培养条件为:温度33.2℃、pH 7.0、灌流速度4.1 mL/min。3批验证结果显示,相较批培养,灌流培养时间延长5~7 d,收获体积增加5~7倍。其中,葡萄糖和乳酸含量、细胞密度和活性批间变化趋势一致;细胞截留效率均高于90%,批间差异无统计学意义(P>0.05);目的蛋白比活性约为0.5,批间差异无统计学意义(P>0.05);Western blot结果显示,相对分子质量和糖基化修饰与批培养一致。结论实现了5 L生物反应器灌流培养重组CHO细胞表达gE蛋白工艺,为后续大规模生产提供了依据。Objective To optimize the perfusion culture process of recombinant CHO cells,suspending cell was trapped by ultrasound retention controller(URC),which induced cell aggregation and sedimentation under ultrasonic waves.Methods Recombinant CHO cells expressing varicella-zoster virus(VZV)glycoprotein E(gE)were chosen to perform URC perfusion culture in 5 L bioreactor based on the serum-free Chemical defined(CD)medium.Preliminarily determined culture conditions were analyzed by Response Surface Methodology(RSM),and culture temperature,medium pH and irrigation speed were optimized,then these optimized conditions were utilized to verify the process of URC perfusion culture.Results Under the experimental conditions,optimized perfusion culture conditions were determined as follows:temperature 33.2℃,pH 7.0,and perfusion speed 4.1 mL/min.Verification results of three batches showed that,compared with batch culture,perfusion culture maintain time prolonged 5-7 d,and harvest volume increased 5-7 times.Variation of glucose and lactic acid contents,cell density and activity were consistent among batches;cell retention efficiencies of over 95%have been observed,and there was no statistical difference between batches(P>0.05);specific activity of target protein detected was approximately 0.5,and there was no statistical difference between batches(P>0.05);results of Western blot showed that the relative molecular mass and glycosylation were consistent with batch culture.Conclusion The process of recombinant CHO cells expressing gE protein was established in 5 L bioreactor perfusion culture,which provide basis for subsequent large-scale production.

关 键 词：灌流培养 重组CHO细胞 生物反应器 水痘-带状疱疹病毒糖蛋白E 

分 类 号：Q813.11[生物学—生物工程]