我们使用源于小鼠白血病病毒（MLV）的逆转录病毒载体进行体细胞基因治疗应用（如对造血干细胞的遗传修饰）。最近，我们报道了建立了一种悬浮型病毒包装细胞系（VPC）用于生产MLV载体。我们利用跳跃子载体技术对人胚胎肾细胞293-F（HEK293-F）进行了基因修饰，以实现这一目的。在这里，我们展示了使用该细胞系建立连续高细胞密度（HCD）工艺。首先，我们在小尺度上比较了不同培养基在最大可达到的活细胞浓度（VCC）方面的差异。接下来，我们将这个工艺转移到搅拌槽生物反应器，并应用了增产策略。具体而言，我们建立了一种使用交替切向流过滤系统的灌流工艺。在这里，我们实现了高达27.4E + 06细胞/mL的VCC和高达8.6E + 06传导单位/mL的MLV载体滴度。最后，我们使用管式膜进行细胞滞留和连续病毒载体收获，建立了连续HCD工艺。这里，空间-时间产量比相比相应的批次培养提高了18倍。总体而言，我们的结果明确证明了高密度培养对病毒载体的高产量生产是可行的，特别是与连续病毒载体收获相结合时。关键点： • 建立了用于MLV载体生产的连续高细胞密度工艺 • 管式细胞滞留膜实现了连续载体收获 • 建立的工艺的空间-时间产量比相比批次培养提高了18倍。© 2023. 作者。

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.© 2023. The Author(s).