Fast and inexpensive purification of adenovirus vectors using optimized two-phase aqueous technology
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J Virol methods. October 6, 2021: 114305. doi: 10.1016 / j.jviromet.2021.114305. Online ahead of print.
Adenoviruses (AdV) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney virus 239 (HEK293) or PER.C6 producing cells with AdV plasmid vectors or by infecting these cells with cell lysates containing replication defective AdVs. Cell lysates can be further purified by cesium chloride or chromatographic protocols to screen for virus seed stocks (RVSS) for characterization into high quality master virus seed stocks (MVSS) and virus seed stocks. (WVSS) before downstream production of high titer pure AdV. The lysates are not very infectious, block the filtration columns and have a limited storage capacity. Two-phase aqueous systems (ATPS) are an alternative method for the purification of Adv that rapidly generates cleaner RVSS for characterization in MVSS. After testing several ATPS formulations, an aqueous mixture of 20% PEG 600 and 20% (NH4)2SO4 (w / w) was found to be most efficient for AdV partition, producing up to 97 + 3% yield of high titer virus that lacked aggregates both effective in vitro and in vivo with no observable cytotoxicity . It is important to note that AdV preparations stored at -20 ° C or 4 ° C exhibit negligible titer loss and are suitable for downstream processing to clinical grade to meet the need for AdV vaccines.
PMID: 34626684 | DOI: 10.1016 / j.jviromet.2021.114305