基于MDCK悬浮细胞灌注培养的流感病毒生产过程开发

doi:10.13560/j.cnki.biotech.bull.1985.2019-1228

摘要/Abstract

摘要：

为了提升用MDCK悬浮细胞培养生产流感疫苗的效率,克服目前存在的产能低下问题,以期满足日益增长的流感疫苗市场需求和提升应对大流行爆发的能力。通过病毒驯化、稀释流加条件下的细胞生长和产毒能力分析、Semi-perfusion中的细胞高密度培养可行性分析以及ATF灌注反应器过程验证完成过程开发。驯化的病毒加速了其在细胞内的感染扩增,且在稀释流加培养中获得（3.57±0.17）log₁₀（HAU/100 μL）的病毒滴度。Semi-perfusion可实现细胞高密度培养至40×10⁶ cells/mL以上,病毒产量达4 log₁₀（HAU/100 μL）以上,同时发现MOI和胰酶浓度对病毒的复制过程存在显著影响。在反应器中的ATF灌注培养过程得到了与Semi-perfusion相近的细胞密度及更高的病毒产量4.37 log₁₀（HAU/100 μL）,并且通过降温措施维持了高密度下的单细胞病毒产量,克服了“细胞密度效应”。建立了基于MDCK悬浮细胞ATF灌注培养的流感病毒生产平台,通过同时提高细胞密度和单细胞病毒产率提升了流感病毒的生产效率,为基于细胞培养的流感疫苗产业化生产提供了新选择。

关键词: MDCK细胞, 流感病毒, 灌注培养, semi-perfusion

Abstract:

This work aims to increase the production efficiency of influenza vaccine,i.e.,overcome the current drawback of low production,and to meet the growing market demand for influenza vaccines and be full preparedness to pandemic outbreaks. Process development was carried out using MDCK suspension cells by virus adaptation,extended batch cultivation to evaluate cell growth and virus production,semi-perfusion to verify the feasibility of MDCK high cell density cultivation,and ATF perfusion process verification in the bioreactor. Faster infection with adapted seed virus contributed to high virus titer of（3.57±0.17）log₁₀（HAU/100 μL）in extended batch cultivation. The high cell concentration over 40×10⁶ cells/mL and high virus titer over 4 log₁₀（HAU/100 μL）in the semi-perfusion model proved the feasibility of high cell density cultivation using MDCK suspension cells. MOI and trypsin concentration presented significant impacts on the cell growth and virus propagation during the infection phase. Similar cell growth profile and higher virus titer of 4.37log₁₀（HAU/100 μL）in the ATF-based perfusion process were obtained in the bioreactor;and comparable cell-specific virus yield（CSVY）to the batch cultivation was maintained by implementing the temperature reduction strategy during infection phase,which overcame the “cell density effect”. In conclusion,the platform of producing influenza virus based on the MDCK suspension cell ATF perfusion process is established,and it enhanced the influenza virus productivity by increasing the cell density and CSVY,which provides a new option for the cell-based industrialization of influenza vaccine.

Key words: MDCK cells, influenza virus, perfusion process, semi-perfusion

吴熠潇, Thomas Bissinger, Yvonne Genzel, 刘旭平, Udo Reichl, 谭文松. 基于MDCK悬浮细胞灌注培养的流感病毒生产过程开发[J]. 生物技术通报, 2020, 36(10): 247-255.

WU Yi-xiao, Thomas Bissinger, Yvonne Genzel, LIU Xu-ping, Udo Reichl, TAN Wen-Song. Perfusion Process Development of MDCK Suspension Cells for Influenza Virus Production[J]. Biotechnology Bulletin, 2020, 36(10): 247-255.

图/表 8

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技术开发流程	技术成果	瓶颈与展望
病毒驯化	加快病毒复制,减小工作种毒体积,增加种毒感染性。	/
初步建立批培养流感病毒生产平台	悬浮生长,细胞密度至千万级别,病毒产量位于前列	/
高密度培养可行性分析	细胞密度可至四千万,病毒产量突破4 log₁₀（HAU/100 μL）,建立scale-down模型	由于体系限制最高支持五千万的细胞密度,可进一步设计和优化;灌注过程为半连续,与反应器连续过程仍存差异
灌注培养生物反应器验证	病毒产量突破4 log₁₀（HAU/100 μL）,目前最高,且提高了单细胞病毒产率。	ATF存在病毒透过情况;需手动更改灌注速率,无实现自动化控制;副产物累积对病毒产量的潜在影响
最终：建立了高效的MDCK细胞高密度培养的流感病毒生产平台

技术开发流程	技术成果	瓶颈与展望
病毒驯化	加快病毒复制,减小工作种毒体积,增加种毒感染性。	/
初步建立批培养流感病毒生产平台	悬浮生长,细胞密度至千万级别,病毒产量位于前列	/
高密度培养可行性分析	细胞密度可至四千万,病毒产量突破4 log₁₀（HAU/100 μL）,建立scale-down模型	由于体系限制最高支持五千万的细胞密度,可进一步设计和优化;灌注过程为半连续,与反应器连续过程仍存差异
灌注培养生物反应器验证	病毒产量突破4 log₁₀（HAU/100 μL）,目前最高,且提高了单细胞病毒产率。	ATF存在病毒透过情况;需手动更改灌注速率,无实现自动化控制;副产物累积对病毒产量的潜在影响
最终：建立了高效的MDCK细胞高密度培养的流感病毒生产平台