Biotechnology Development, Schering-Plough Research Institute, 1011 Morris Avenue, 07083, Union, USA.
Cytotechnology. 2001 Nov;37(3):189-98. doi: 10.1023/A:1020555310558.
Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells.
人胚肾 293（HEK293）细胞通过逐步血清驯化步骤适应无血清悬浮培养基，用于生产腺病毒（AdV）基因治疗载体。培养基配方中存在肝素钠可显著减少悬浮培养中的细胞聚集。适应的细胞可以在含血清的培养基中作为附着培养物生长，也可以在无血清的悬浮培养基中生长。在摇瓶中开发了可扩展的生产工艺，然后在搅拌罐生物反应器中进行了评估。该工艺包括分批式生长阶段，随后是涉及培养基灌注和补充的生产阶段。病毒接种后添加氯化钙可使病毒产量至少增加一倍。感染后添加刺激剂，如丁酸钠、N-乙酰-L-半胱氨酸（NAC）、二甲基亚砜（DMSO）或乙醇，可进一步以剂量依赖的方式提高病毒产量。此处描述的无血清悬浮工艺应适用于其他 E1 缺失的 AdV 载体的制造，并可能可用于通过 HEK293 细胞生产重组蛋白。
