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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (8): 50-58    DOI: 10.13523/j.cb.20180807
技术与方法     
基于PAT的PCV2 VLPs生产过程优化与控制研究
化磊召,易小萍,储炬,庄英萍(),张嗣良
华东理工大学生物反应器工程国家重点实验室 上海 200237
Process Control and Optimization of PCV2 Production of VLPs Based on PAT
Lei-zhao HUA,Xiao-ping YI,Ju CHU,Ying-ping ZHUANG(),Si-liang ZHANG
East China University of Science and Technology,State Key Laboratory of Bioreactor Engineering,Shanghai 200237,China
 全文: PDF(1145 KB)   HTML
摘要：

昆虫细胞-杆状病毒载体表达系统(baculovirus expression vector system,BEVS)是病毒样颗粒亚单位疫苗(virus like particles,VLPs)的理想生产平台。动物细胞培养过程分析技术(process analytical technology,PAT)研究的重点在于更多地获取与细胞生理状态相关的过程参数,以及实现过程敏感参数的在线表征和过程控制关键时间节点的在线判定,从而指导过程优化和控制。通过昆虫Sf9细胞的分批和补料悬浮培养,发现细胞代谢活性与在线特征频率(fc)之间存在相关性。以fc作为细胞代谢活性的在线指征,在细胞代谢活性下降之前补料,使得Sf9细胞在代谢活性明显增强的基础上,最高活细胞密度提高1.75倍。通过分批培养与补料分批培养过程细胞生理特性参数的相关性分析,发现比电容增长速率(με)与培养体系S-期细胞比例之间存在相关性,με作为细胞增殖活性状态的在线指征参数,可以作为最佳接毒时间的判定依据。此外,研究还发现在线检测参数电容(ε)与最高疫苗产量之间存在相关性,可以作为疫苗最佳收获时间的在线表征参数。以在线fc值作为补料时间指征,以在线με值作为病毒感染时间指征,以在线ε值作为病毒收获时间指征,实现了猪圆环病毒2型(PCV2)VLPs的高效生产。相比于批培养,基于PAT的生产工艺疫苗单位体积产量提高76%,生产周期缩短了24h,为PCV2病毒样颗粒亚单位疫苗大规模生产提供了一种新的高效生产模式。
关键词： VLPs;  昆虫细胞-杆状病毒载体表达系统;  PAT    
Abstract:

Baculovirus expression vector system (BEVS) is the ideal platform for Virus-Like Particles (VLPs) production. The key points of the Process Analytical Technology (PAT) in animal cell culture include obtaining cell physiological characteristic parameters, monitoring process sensitive parameters, and controlling critical time points. These parameters in turn can promote process optimization and process control. A close correlation between cell metabolic status and on-line parameter critical frequency (fc) was found in Sf 9 cell batch culture. Using the fc as on-line indictor of cell metabolic status and feeding the culture, a 1.7 time increase of Peak Viable Cell Density (PVCD) was obtained. Besides, the metabolic status was also improved. A close correlation was found between the proportion of S-phase cells and specific capacitance growth rate (με). It turned out that με could indicate the optimized infection time point. Additionally, a high correlation was found between ε and the maximum VLPs productivity. The parameter of ε can act as an on-line indicator. Overall, an effective product mode was established by using fc as on-line feeding indicator, με as on-line infecting indicator, ε as on-line harvesting indicator. Compared with traditional process, the volume productivity was increased by 76%, and the culture duration was reduced by 24h. A new VLPs production mode has been established successfully.
Key words: VLPs    BVES    PAT
收稿日期: 2018-04-15 出版日期: 2018-09-11
ZTFLH:  Q813  
通讯作者: 庄英萍     E-mail: ypzhuang@ecust.edu.cn
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引用本文:

化磊召,易小萍,储炬,庄英萍,张嗣良. 基于PAT的PCV2 VLPs生产过程优化与控制研究[J]. 中国生物工程杂志, 2018, 38(8): 50-58.

Lei-zhao HUA,Xiao-ping YI,Ju CHU,Ying-ping ZHUANG,Si-liang ZHANG. Process Control and Optimization of PCV2 Production of VLPs Based on PAT. China Biotechnology, 2018, 38(8): 50-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180807    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I8/50

图1  分批培养中细胞生长动力学研究
图2  分批培养中ε与离线活细胞密度的线性拟合
图3  分批培养中Sf9细胞活性变化分析
图4  分批培养中Sf9细胞的代谢活性状态分析
图5  昆虫Sf9细胞培养最佳补料时间的确定(箭头代表补料操作)
图6  分批和补料培养体系的细胞生长对比 (箭头代表补料操作)
图7  分批和补料培养体系的细胞比生长速率对比
图8  分批和补料培养体系细胞S-期细胞比例分布对比(箭头代表补料操作)
图9  分批和补料培养体系细胞比葡萄糖消耗速率对比(箭头代表补料操作)
图10  分批和补料培养体系细胞比耗氧速率对比(箭头代表补料操作)
图11  分批培养中细胞增殖活性变化
图12  分批补料培养中细胞增殖活性变化(左箭头代表补料,右箭头代表感染)
图13  分批培养中VLPs产量与ε变化情况
图14  补料培养体系VLPs产量与在线电容ε变化情况
图15  分批培养与补料分批培养中最高活细胞密度对比
图16  基于PAT的优化工艺与传统工艺产量对比
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