病毒载体
细胞培养
HEK 293细胞
逆转录病毒
生物
生物反应器
连续生产
胚胎干细胞
转染
小鼠白血病病毒
遗传增强
造血
干细胞
细胞生物学
化学
材料科学
基因
生物化学
遗传学
重组DNA
植物
复合材料
作者
Marc D. Hein,Daniel Kazenmaier,Yasemin van Heuvel,Tanya Dogra,Maurizio Cattaneo,Sascha Young Kupke,Jörn Stitz,Yvonne Genzel,Udo Reichl
标识
DOI:10.1007/s00253-023-12689-9
摘要
Abstract Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting. Key points • A continuous high cell density process for MLV vector production was established • The tubular cell retention membrane allowed for continuous vector harvesting • The established process had a 18-fold higher space time yield compared to a batch
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