Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology

Saqlain Suleman,Kuteiba Schrubaji,Chrysovalanto Filippou,Svetlana Ignatova,Peter Hewitson,Jonathan Huddleston,Rajvinder Karda,Simon N Waddington,Michael Themis

doi:10.1016/j.jviromet.2021.114305

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.

Highlights

Adenoviruses (AdVs) are highly suited to effective gene transfer and commonly used in vaccine production to target both dividing and nondividing cells and their biology is well understood (Cortin, Thibault et al 2004; Ura, Okuda et al 2014) The vectors replicate and are assembled within the nucleus of infected cells before undergoing lysis for the progeny virus to infect adjacent cells
First generation vectors have early genes E1 and/or E3 regions removed allowing ~8 kb of transgenic sequences to be carried. These early genes that are necessary for virus replication have been inserted into the producer cells to Abbreviations: AdV, Adenoviruses; ATPS, Aqueous two-phase systems; HEK293, human embryonic kidney 293. * Corresponding author at: Institute of Environment, Health & Societies, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London, Kingston Lane, Uxbridge, Middlesex, UB8 3PH, UK
Because ATPS technology is amenable to scale-up, we propose this technology ideal for the rapid manufacture of large amounts of research virus seed stocks (RVSS) for characterisation to master virus seed stocks (MVSS) under good manufacture practice-certified (GMP) conditions to support clinical grade therapeutic AdV and AdV-based vaccines

Summary

Introduction

Adenoviruses (AdVs) are highly suited to effective gene transfer and commonly used in vaccine production to target both dividing and nondividing cells and their biology is well understood (Cortin, Thibault et al 2004; Ura, Okuda et al 2014) The vectors replicate and are assembled within the nucleus of infected cells before undergoing lysis for the progeny virus to infect adjacent cells. AdV vectors are designed to carry a large transgene payload and particles are usually produced at high titre from human embryonic kidney 293 (HEK293) or PER.C6 producer cells (Xie, Pilbrough et al 2002). First generation vectors have early genes E1 and/or E3 regions removed allowing ~8 kb of transgenic sequences to be carried. These early genes that are necessary for virus replication have been inserted into the producer cells to Abbreviations: AdV, Adenoviruses; ATPS, Aqueous two-phase systems; HEK293, human embryonic kidney 293.

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Discussion

Conclusion