Suleman, S;
Schrubaji, K;
Filippou, C;
Ignatova, S;
Hewitson, P;
Huddleston, J;
Karda, R;
... Themis, M; + view all
(2022)
Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology.
Journal of Virological Methods
, 299
, Article 114305. 10.1016/j.jviromet.2021.114305.
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Abstract
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.
| Type: | Article |
|---|---|
| Title: | Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology. |
| Location: | Netherlands |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.jviromet.2021.114305 |
| Publisher version: | https://doi.org/10.1016/j.jviromet.2021.114305 |
| Language: | English |
| Additional information: | Copyright © 2021 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license |
| Keywords: | Adenovirus type 5, Aqueous two-phase partitioning system, Caesium chloride, Vaccine, Viral vector purification |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL EGA Institute for Womens Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL EGA Institute for Womens Health > Maternal and Fetal Medicine |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10137091 |
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