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

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.

Keywords: Adenovirus type 5; Aqueous two-phase partitioning system; Caesium chloride; Vaccine; Viral vector purification.

Graphical abstract

Fig. 1

Infection of HepG2 cells by AdVRSVβgal. (a). HepG2 infection by CsCl prepared vector after partitioning by 3 ATPS formulations. Positive staining for β-galactosidase activity was identified in each ATPS upper phase, with highest infection by PEG 600/(NH₄)₂SO₄. Uninfected HepG2 cells appear negative for enzyme activity. (b). Quantification of cell infection: PEG1000/K₂HPO₄; 55.15 ± 2.74 %, PEG8000/K₂HPO₄; 59.85 ± 9.84 %, PEG600/(NH₄)₂SO₄; 97.00 ± 3.0 %, (n = 5).

Fig. 2

Analysis of IMRS2 cell permissiveness to AdVRSVβgal infection. (a). Infected IMRS2 cells at various dilutions of AdV particles (vg/mL). Infection was detected by analysis of β- galactosidase activity demonstrating IMRS2 cells are highly permissive to infection. (b). Quantification of positive cells after exposure to serial dilutions of CsCl prepared AdRSVβgal. IMRS2 infection was: 10 ¹¹:100 ± 0%, 10 ¹⁰: 80 ± 3.84 %, 10 ⁹: 60 ± 1.15 %, 10 ⁸: 46 ± 2.19 %, 10 ⁷: 15 ± 1.76 % and 10 ⁶: 1 ± 0.88 % (n = 3).

Fig. 3

Analysis of AdV recovery direct from 293 T cell lysates (a). Infection of HepG2 and IMRS2 cells using AdVRSVβgal purified directly from 293 T cell lysate using 3 ATPS formulations. Infection of AdV was characterised by staining for β- galactosidase activity. (b). Quantification of HepG2 and IMRS2 cell infection by AdV purified directly from 293 T cell lysate. The upper phase presented the highest recovery of AdVRSVβgal particles and was used for infection. For HepG2 and IMRS2 cells, respectively, 93.3 ± 0.9 % and 85.3 ± 1.8 % cells were infected using PEG 1000/K₂HPO₄. 91.7 ± 2.4 % and 30.0 ± 1.2 % cells stained positive for AdV infection using PEG 8000/K₂HPO₄. 91.3 ± 1.5 % and 93.7 ± 2.2 % cells were infected using PEG 600/(NH₄)₂SO₄ (n = 3).

Fig. 4

Analysis of cell survival after AdV/ATPS treatments. Percentage cell survival after treatment with upper phase ATPS +/- AdVRSVβgal. HepG2 cells + ATPS only (dark grey), + CsCl AdV (light grey) and + 293 T AdV (hash). PEG 1000/K₂HPO₄ treatment showed viability of 77.33 ± 5.06 % (+ ATPS), 90.502.88) (+ CsCl AdV ATPS) and 93.25 ± 0.63 % (+293 T AdV). Cells treated with PEG 8000/K₂HPO₄ upper phase showed a viability of 87.25 ± 4.40 % (+ATPS), 92.20 ± 2.99 % (+CsCl AdV) and 91.00 ± 1.83 % (+293 T AdV). PEG 600/(NH₄)₂SO₄ upper phase treatment showed a viability of 83.75 ± 4.12 % (+ATPS), 93.50 ± 0.99 % (+CsCl AdV) and 90.25 ± 1.49 % (+293 T AdV). No significant difference in viability was observed (P < 0.05). This demonstrates the low toxicity of ATPS subsequent to AdV purification and recovery (n = ≥4).

Fig. 5

Application of PEG 600/ (NH₄)2SO₄ ATPS purified AdVLuc in vivo. AdVLuc virus was purified using PEG 600/(NH₄)₂SO₄ ATPS or CsCl ultracentrifugation. 1 × 10 ⁹ vg/mL AdVluc particles recovered from the ATPS upper phase or CsCl were injected into CD-1 mice via their tail vein mice to determine in vivo gene transfer. Imaging for luciferase activity showed gene expression was comparable between the ATPS and CsCl purified vector thus demonstrating ATPS AdV can be used successfully in vivo without obvious side effects (a single representative mice of n = 3 shown only).