Currently, the mammalian biomanufacturing industry explores process intensification (PI) to meet upcoming demands of biotherapeutics while keeping production flexible but, more importantly, as economic as possible. However, intensified processes often require more development time compared with conventional fed-batches (FBs) preventing their implementation. Hence, rapid and efficient, yet straightforward strategies for PI are needed. In this study we demonstrate such a strategy for the intensification of an N-stage FB by implementing N-1 perfusion cell culture and high inoculum cell densities resulting in a robust intensified FB (iFB). Furthermore, we show successful combination of such an iFB with the addition of productivity enhancers, which has not been reported so far. The conventional CHO cell FB process was step-wise improved and intensified rapidly in multi-parallel small-scale bioreactors using N-1 perfusion. The iFBs were performed in 15 and 250 ml bioreactors and allowed to evaluate the impact on key process indicators (KPI): the space–time yield (STY) was successfully doubled from 0.28 to 0.55 g/L d, while product quality was maintained. This gain was generated by initially increasing the inoculation density, thus shrinking process time, and second supplementation with butyric acid (BA), which reduced cell growth and enhanced cell-specific productivity from ~25 to 37 pg/(cell d). Potential impacts of PI on cell metabolism were evaluated using flux balance analysis. Initial metabolic differences between the standard and intensified process were observed but disappeared quickly. This shows that PI can be achieved rapidly for new as well as existing processes without introducing sustained changes in cellular and metabolic behavior.

中文翻译：

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快速强化已建立的 CHO 细胞分批补料工艺

目前，哺乳动物生物制造行业正在探索工艺强化 (PI)，以满足即将到来的生物治疗需求，同时保持生产灵活性，更重要的是，尽可能经济。然而，与传统的补料批次 (FB) 相比，强化工艺通常需要更多的开发时间，从而阻碍了其实施。因此，需要快速有效但直接的 PI 策略。在这项研究中，我们通过实施 N-1 灌注细胞培养和高接种细胞密度来展示这种强化 N 阶段 FB 的策略，从而产生强大的强化 FB (iFB)。此外，我们展示了这种 iFB 与添加生产力增强剂的成功组合，目前尚未报道。传统的 CHO 细胞 FB 工艺在使用 N-1 灌注的多平行小型生物反应器中得到逐步改进和快速强化。iFB 在 15 和 250 ml 生物反应器中进行，并允许评估对关键工艺指标 (KPI) 的影响：时空产率 (STY) 从 0.28 g/L d 成功翻倍至 0.55 g/L d，同时保持产品质量。这种增益是通过最初增加接种密度而产生的，从而缩短了处理时间，以及第二次补充丁酸 (BA)，这减少了细胞生长并将细胞比生产力从 ~25 提高到 37 pg/(细胞 d)。使用通量平衡分析评估 PI 对细胞代谢的潜在影响。观察到标准过程和强化过程之间的初始代谢差异，但很快就消失了。