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Abstract：
Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 × 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 × 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimize inhibitory metabolite accumulation and vector exposure at 37°C. Similar specific productivities of infectious LV and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimize downstream processing volumes. The optimized process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titers. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titers, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of 7.7% ± 2.9% and 11.9% ± 3.0% for infectious and physical LV titers, respectively. The optimized flask process was successfully transferred to the iCELLis Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 × 108 TU.
摘要：
在8天的过程中，使用连续稳定的生产细胞系，采用准灌注培养来加强慢病毒载体（LV）的制造。初步研究旨在确定可扩展的播种密度，具有3、4和5×104个细胞cm-2，可提供相似的感染性LV比生产率。选择3×104个细胞cm-2的种子，并且调节准灌注以使抑制代谢物积累和在37°C下的载体暴露最小化。在每天1、2和3个血管体积（VVD）时，感染性LV和物理LV的特定生产率相似。选择1个VVD以最小化下游处理量。优化后的工艺放大了50倍，达到1,264cm2烧瓶，达到相似的LV滴度。然而,扩大到6320cm2多层烧瓶，降低了滴度，可能来自次优的气体交换。在25cm2至6,320cm2烧瓶中的三个独立过程中，重现性高，感染和物理LV滴度的变异系数为7.7％±2.9％和11.9％±3.0％，分别。优化的烧瓶工艺成功转移到iCELlisNano（Cytiva）固定床生物反应器中，在1VVD下准灌注产生1.62×108TU。
