强化病毒载体悬浮细胞培养物的澄清深度过滤。

Depth filtration for clarification of intensified lentiviral vector suspension cell culture.

机构信息

Genomic Medicine Unit CMC, Global CMC Development, Waltham, Massachusetts, USA.

Process and Analytical Development (PAD), Gene Therapy Franchise, National Resilience Inc., Waltham, Massachusetts, USA.

出版信息

Biotechnol Prog. 2024 Mar-Apr;40(2):e3409. doi: 10.1002/btpr.3409. Epub 2023 Nov 20.

Abstract

Depth filtration significantly impacts efficiency of lentiviral (LV) vector purification process. However, it is often deprioritized in the overall scope of viral vector manufacturing process optimization. The demand for LV vectors has increased with the rise in disease indications, making it crucial to improve current manufacturing processes. Upstream bioreactor process intensification has enabled cell densities of over 10 viable cells/mL, creating challenges for harvest unit operations. The larger size of LV vectors and their physiochemical similarity to host cell-DNA (HC-DNA) and poor clarification performance causes significant challenges for the subsequent chromatography-based purifications. As a result, a robust and scalable harvest of LV process is needed, especially for LV in vivo therapeutic quality needs. In this study, we systematically evaluated the overlooked yet important issue of depth filtration systems to improve enveloped LV functional vector recovery. We found that an established depth filtration system in process A that provided 94% (n = 6) LV functional recovery could not be translated to intensified Process B cell culture. Hence, the depth filtration process became a bottleneck for the purification performance in an intensified process. We demonstrated an improvement in LV functional vector recovery from 34% to 82% via filter train optimization for an intensified suspension cell culture system (>10 cells/mL with higher titer), while still maintaining a loading throughput of ≥82 L/m and turbidity ≤20 NTU. It was demonstrated that the two or three-stage depth filtration scheme is scalable and more suitable for high cell density culture for large scale for LV manufacturing process.

摘要

深度过滤对慢病毒 (LV) 载体纯化工艺的效率有显著影响。然而,在病毒载体制造工艺优化的整体范围内,深度过滤往往被置于次要地位。随着疾病适应症的增加,对 LV 载体的需求也在增加,因此改进当前的制造工艺至关重要。上游生物反应器工艺强化使细胞密度超过 10 个活细胞/mL,这给收获单元操作带来了挑战。LV 载体的尺寸较大,其理化性质与宿主细胞 DNA(HC-DNA)相似,且澄清性能不佳,这给随后基于色谱的纯化带来了重大挑战。因此,需要一种强大且可扩展的 LV 收获工艺,特别是对于 LV 体内治疗质量的需求。在这项研究中,我们系统地评估了深度过滤系统这一被忽视但却很重要的问题,以提高包膜 LV 功能性载体的回收率。我们发现,在工艺 A 中建立的深度过滤系统可以提供 94%(n=6)的 LV 功能性回收,但无法转化为强化的工艺 B 细胞培养。因此,深度过滤工艺成为强化工艺中纯化性能的瓶颈。我们通过优化过滤组件,使强化悬浮细胞培养系统(细胞密度>10 个/mL,滴度更高)中的 LV 功能性载体回收率从 34%提高到 82%,同时仍保持≥82 L/m 的装载通量和≤20 NTU 的浊度。结果表明,两阶段或三阶段深度过滤方案可扩展,更适合用于大规模 LV 制造工艺的高密度细胞培养。

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