High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf-9-BEV system
High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf-9-BEV system
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DOI：
10.1002/bit.21224
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发表时间：
2007-06-01
影响因子：
3.8
通讯作者：
Elias, Cynthia
中科院分区：
文献类型：
--
作者：
Jardin, Barbara Ann;Montes, Johnny;Elias, Cynthia
以稳定连续方式表达重组蛋白的昆虫细胞的开发是用于重组蛋白生产的BEV系统的有吸引力的替代方案。可以设计高细胞密度补料分批和连续灌注工艺以最大化稳定转化细胞的生产率。通过脂质体介导法转染Sf-9昆虫细胞，经筛选获得稳定表达SEAP报告蛋白的细胞株Sf-9 SEAP。将Sf-9 SEAP细胞的表达与BEVS系统进行比较。观察到在BEVS中获得的产率与批次Sf-9 SEAP相似，分别为8和7 IU/mL。该外源基因产物与稳定细胞的生产率通过采用分批补料和灌注培养模式的生物过程强化来提高，以增加培养物中的细胞密度。补料分批工艺产生的最大细胞密度为28 × 10（6）个细胞/mL，SEAP为12 IU/mL。使用灌注方法可以进一步提高生产率，这证明了长时间的稳定生产率。该过程维持43天，稳态细胞密度为17-20 x 10（6）个细胞/mL，SEAP为7 IU/mL。灌注工艺中获得的总产率（394 IU）分别约为分批工艺（17.6 IU）和补料分批工艺（46.1 IU）的22倍和8倍。
The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.