Glaxo Institute for Molecular Biology, 14 Chemin des Aulx, CH-1228, Plan-les-Ouates, Switzerland.
Cytotechnology. 1995 Jan;18(3):183-92. doi: 10.1007/BF00767766.
We demonstrate here that transient expression with COS cells can be performed at the one litre scale for a period of more than 10 days. Cells grown in T225 flasks were transfected by electroporation, transferred into spinners, and then grown either in suspension or on microcarriers. A daily medium change significantly extented culture life and production time, compared with standard protocols.Concentrations of the product, the secreted fusion protein CD40-Fc, were comparable in microcarrier and suspension culture. Cultures were started in fetal calf serum containing medium and the subsequent production process was performed in a low protein serum free medium which allowed easy downstream processing. 10 litres of supernatant, collected from one transfected batch of cells, yielded 30 mg of purified and biologically active protein.In addition to developing a simplified protocol for generation of cells we also reduced the material (DNA, cuvettes) required for electroporation. Our results show that scale up of transient expression to the litre scale can be successfully acieved. This provides a new tool to generate milligram quantities of protein within weeks of gene cloning.
我们在此证明，COS 细胞的瞬时表达可以在 1 升规模下进行超过 10 天的时间。在 T225 瓶中生长的细胞通过电穿孔转染，转移到旋转器中，然后在悬浮液或微载体上生长。与标准方案相比，每天更换培养基可显著延长培养寿命和生产时间。产物（分泌融合蛋白 CD40-Fc）的浓度在微载体和悬浮培养中相当。培养物在含有胎牛血清的培养基中起始，随后在低蛋白无血清培养基中进行生产，这有利于下游处理。从转染的一批细胞中收集了 10 升上清液，得到了 30 毫克纯化的、有生物活性的蛋白质。除了开发简化的细胞生成方案外，我们还减少了电穿孔所需的材料（DNA、小瓶）。我们的结果表明，可以成功地将瞬时表达扩大到 1 升规模。这为在基因克隆后的数周内生成毫克级蛋白质提供了一种新工具。
