Adenovirus Purification by Chromatography

1. 48 hours after infection, spin down cells.
2. Resuspend cell pellet in 10 ml of resuspension buffer (5 mM Tris–HCl, 75 mM NaCl, 1 mM MgCl2 , 5% sucrose, 1% Polysorbate-80 (PS-80), pH 8.0)

3. Transfer into two 50-ml conical tubes (∼25 ml each).
4. Freeze the tubes at –70 oC for at least 1 h. The frozen harvested cells can be held indefinitely at this point.
5. Thaw the tubes in a 37 °C water bath.
6. Repeat steps 3 and 4 two times.

7. Centrifuge cell lysate for 10 min at 9000 × g.
8. Collect the supernatant and transfer to another bottle.
9. Add Benzonase to a final concentration of 150 U/ml, mixing thoroughly
10. Incubate 1 h at room temperature with gentle mixing on a stir plate, (or overnight at 4 oC).
11. Concentrate virus by Diafiltration/ultrafiltration using 50 cm2 300 kDa regenerated cellulose tangential flow ultrafiltration cartridge and Diafiltration buffer: 50 mM HEPES, 2 mM MgCl2 , 1 M NaCl, pH 7.5.

12. Pack a column with 8 ml of Source 15Q resin.
13. Sanitize the column using 0.1 N NaOH though the column at 1.2 ml/min until the pH is elevated in the effluent. Disconnect the column and hold in NaOH for 30 min.
14. Wash with buffer B.
15. Wash with buffer A.
16. Equilibrate column with 5 CV (40 ml) of buffer A at 1.2 ml/min.
17. Load samples to column at 1.2 ml/min.
18. Wash column with 5 CV (40 ml) of buffer A
19. Elute adenovirus using a linear gradient to 100% buffer B over 4 column volumes. The virus peak will have an A260/A280 ratio of approximately 1.25, and the adenovirus should elute in approximately 1–1.5-column volumes
20. Pre-equilibrate 1 PD-10 desalting column (GE Healthcare PD-10) for each 2.5 ml of batch volume, using 25 ml of stabilizing buffer (5 mM Tris–HCl, 75 mM NaCl, 1 mM MgCl2 , 5% sucrose, 0.005% PS-80, pH 8.0).
21. Allow column to drain completely. If virus will not be applied to the column within about 5 min, recap so that the column will not dry out.
22. Within a biosafety cabinet, pipette 2.5 ml of virus fraction on each column, and allow to drain.
23. Add 3.5 ml of stabilizing buffer and collect this fraction as product.
24. Pool the product fractions.
25. Inside a biosafety cabinet, carefully draw the product into a luer-lock syringe.
26. Attach a sterile 0.22 μm PVDF Millex GV-25 syringe filter.
27. Slowly push the product through the filter. If resistance increases, replace the filter and continue

Adjustment buffer: 50 mM HEPES, 2 mM MgCl2 , 0.1% Polysorbate-80, pH 7.5.
Chromatography buffer A: 50 mM HEPES, 2 mM MgCl2 , 0.39 M NaCl, 0.1% Polysorbate-80, pH 7.5 (see Note 6).
Chromatography buffer B: 50 mM HEPES, 2 mM MgCl2 , 0.47 M NaCl, 0.1% Polysorbate-80, pH 7.5
Diafiltration buffer: 50 mM HEPES, 2 mM MgCl2 , 1 M NaCl, pH 7.5.
Resuspension buffer: 5 mM Tris–HCl, 75 mM NaCl, 1 mM MgCl2 , 5% sucrose, 1% Polysorbate-80 (PS-80), pH 8.0.

Note: The capacity of 8 ml 15Q column: the column can be used to process 10^13 vp.