高渗胁迫和灌流培养策略提高HEK 293细胞生产重组腺病毒载体产量

Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells

摘要:

由于各种疾病在全球范围内的肆虐，国际市场对重组腺病毒载体(adenoviral vector,Adv)疫苗的需求量急剧增加，而工艺研究是解决这一问题的有效手段之一。在细胞接毒前施加高渗胁迫可以提高分批培养模式下的Adv产量，新兴的灌流培养也可以显著提高Adv的产量。将高渗胁迫工艺与灌流培养相结合，有望进一步提升高细胞密度生产过程中的Adv产量。本研究利用摇瓶结合拟灌流培养作为生物反应器灌流培养的缩小模型，使用渗透压为300–405 mOsm的培养基研究了高渗胁迫对细胞生长和Adv生产的影响。结果显示，在细胞生长阶段使用370 mOsm的高渗透压培养基，在病毒生产阶段使用300 mOsm的等渗透压培养基的灌流培养工艺有效地提高了Adv的产量。进一步研究发现这可能归因于病毒复制后期HSP70蛋白的表达量增加。将这种工艺放大至生物反应器中，Adv的产量达到3.2×10¹⁰ IFU/mL，是传统灌流培养工艺的3倍。本研究首次将高渗胁迫工艺与灌流培养相结合的策略应用于HEK 293细胞生产Adv，同时揭示了高渗胁迫工艺增产Adv的可能原因，为HEK 293细胞生产其他类型Adv的工艺优化提供了借鉴。

Abstract:

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×10¹⁰ IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.