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首页> 外文期刊>BMC Biotechnology >Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture
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Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

机译：灌注HEK293细胞培养中流感产生的代谢和动力学分析。

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Background Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 106 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products. Results To further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 106 cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 1011 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 1015 IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles. Conclusions These results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.
机译：背景技术基于细胞培养的流感疫苗的生产仍然是基于蛋的生产的有吸引力的替代方法。响应时间短和高产量是在大流行和季节性流感疫苗的工业生产中广泛采用细胞培养技术的关键成功因素。最近，HEK293SF细胞已成功用于生产流感病毒，在迄今报道的最高范围内实现了血凝素（HA）和传染性病毒颗粒（IVP）效价。在同一项研究中，有人提出，超过4×10 6 细胞/ mL时，由于缺乏营养或有毒产品的积累，病毒的生产受到了限制。结果为了进一步提高高细胞密度时的病毒滴度，评估了灌注培养模式。在感染细胞密度为6×10 6 细胞/ mL的情况下，比较了灌注和分批培养模式的生产率。广泛记录了病毒感染前后两种培养方式的代谢，包括糖酵解，谷氨酰胺分解和氨基酸利用，以及诸如活力和凋亡等生理指标，以确定潜在的代谢限制。一个3 L的生物反应器，灌注率为0.5 vol / day，使我们达到的最大滴度为3.3×10 11 IVP / mL和4.0 logHA单位/ mL，感染后3天后的总产量为1.0×10 15 IVP和7.8 logHA单位。总体而言，灌注模式滴定度比分批培养模式高出近一个数量级。葡萄糖和谷氨酰胺消耗率分别提高了1.5倍和4倍，这种改善与细胞代谢的激活有关。还观察到病毒生产动力学的变化，导致具有更高比生产的更多活细胞的积累，并导致感染性流感颗粒的总体积生产增加。结论这些结果证实，HEK293SF细胞是高产量生产流感病毒的优良底物。此外，通过更好地控制细胞培养环境和病毒生产动力学，在进一步提高产量方面具有巨大潜力。一旦完成，该细胞系就可以作为工业平台推广，以具有成本效益的方式生产流感季节性疫苗，以及在大流行期间达到高峰需求时期。

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