The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Gower St, London WC1E 6BT, UK.
Division of Advanced Therapies, National Institute for Biological Standards and Control, South Mimms EN6 3QG, UK.
Viruses. 2021 Feb 9;13(2):268. doi: 10.3390/v13020268.
Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
慢病毒载体(LVs)是将目的基因递送至哺乳动物细胞的有效工具,目前在细胞和基因治疗领域得到广泛应用,用于治疗单基因疾病和过继性治疗,如嵌合抗原 T 细胞(CAR-T)疗法。本文全面综述了 LV 生产中使用的各个生物工艺操作。我们强调了包膜蛋白在载体设计中的作用及其对慢病毒载体生物加工的影响。概述这些操作的现状为生物工艺发现和改进提供了机会,重点强调了在开发和临床生产过程中优化和可扩展处理 LV 的注意事项。描述了用于 LV 生成的上游培养,并比较了不同的转染方法和各种用于悬浮和贴壁生产细胞培养的生物反应器。考察了 LV 的纯化,评估了小规模和大规模生产要求的下游工艺操作的不同序列。对于可扩展操作,重点是开发色谱纯化,此外还深入研究了切向流过滤的应用。还介绍了载体定量和表征分析的总结。最后,讨论了整个 LV 生产的生物工艺评估,以从对不同工艺选择的潜在相互作用的更广泛理解中受益。本文综述旨在帮助实现高质量、高浓度的慢病毒载体,来自稳健和可扩展的工艺。