Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture
Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture
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DOI：
10.1002/bit.21603
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发表时间：
2008-02-15
影响因子：
3.8
通讯作者：
Schmidt, Stefan R.
中科院分区：
文献类型：
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作者：
Rozkov, Aleksei;Larsson, Bert;Schmidt, Stefan R.
以100-L规模在哺乳动物细胞培养物中瞬时表达重组蛋白需要大量的质粒，这是用摇瓶培养物和市售质粒纯化试剂盒实现的非常劳动密集型的。在本文中，我们描述了一种工艺质粒生产在100毫克规模。发酵在4 L补料分批培养中用基本培养基进行。批次结束的检测和触发指数（0.1 h（-1））进料曲线是无人值守的，并由多发酵罐控制系统控制。在补料分批培养中的限制性比生长速率增加了比质粒产量相比，分批培养与最低和丰富的媒体。这与高生物质浓度（68-107 gL（-1）湿重）一起实现了质粒的高体积产率（95-277 mgL（-1），取决于构建体）。纯化工艺包括碱裂解、裂解液澄清和超滤、Triton X-114两相萃取去除内毒素、阴离子交换色谱作为精制步骤、超滤和无菌过滤。发酵和纯化过程均未进行优化，用于生产四种质粒，产生39至163 mg质粒，内毒素含量为2.5 EU mg（-1）或更低。
Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L, fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 gL(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mgL(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.