Division of Biosciences, Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, Middlesex, UK.
Department of Chemical Engineering, College of Engineering, Design & Physical Sciences, Brunel University London, Uxbridge, Middlesex, UB8 3PH, UK.
J Virol Methods. 2022 Jan;299:114305. doi: 10.1016/j.jviromet.2021.114305. Epub 2021 Oct 6.
Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH)SO (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at -20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.
腺病毒（AdVs）被用作基因治疗载体来治疗人类疾病，也被用作针对 COVID-19 的疫苗。AdVs 通过用 AdV 质粒载体转染人胚肾 239（HEK293）或 PER.C6 病毒生产细胞，或用含有复制缺陷型 AdV 的细胞裂解物感染这些细胞来产生。细胞裂解物可以通过氯化铯或色谱方案进一步纯化，以研究病毒种子储备（RVSS）进行高纯度主病毒种子储备（MVSS）和工作病毒种子储备（WVSS）的特性，然后再进行纯高滴度 AdV 的下游生产。裂解物的传染性差，会堵塞过滤柱，并且存储能力有限。双水相系统（ATPS）是 AdV 纯化的替代方法，可快速生成更清洁的 RVSS 进行 MVSS 的特性研究。在测试了多种 ATPS 配方后，发现 20%PEG600 和 20%（NH4）2SO4（w/w）的水相混合物最有利于 AdV 的分配，可产生高达 97%+3%的高滴度病毒，既有效在体外和体内均无可见的细胞毒性。重要的是，储存在-20°C 或 4°C 的 AdV 制剂的滴度损失可忽略不计，并且适合下游加工到临床级别，以满足 AdV 疫苗的需求。
