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Title: Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology
Authors: Suleman, S
Schrubaji, K
Filippou, C
Ignatova, S
Hewitson, P
Huddleston, J
Karda, R
Waddington, SN
Themis, M
Keywords: adenovirus type 5;aqueous two-phase partitioning system;caesium chloride;vaccine;viral vector purification
Issue Date: 6-Oct-2021
Publisher: Elsevier
Citation: Suleman, S., Schrubaji, K., Filippou, C., Ignatova, S., Hewitson, P., Huddleston, J., Karda, R., Waddington, S.N. and Themis, M. (2022) 'Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology', Journal of Virological Methods, 299, 114305, pp. 1-9. doi: 10.1016/j.jviromet.2021.114305.
Abstract: Copyright © 2021 The Author(s). 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 % (NH4)2SO4 (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.
ISSN: 0166-0934
Other Identifiers: 114305
Appears in Collections:Dept of Life Sciences Research Papers

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