Adenovirus Purification by Q XL

1. Grow 1 × 10⁹ 293EF cells  in a Nunc cell factory , or 18 of 5 layer (875 cm²   ) flasks

6 well plate==10 cm²==>6.25 x 10⁵  293 cells. 1X 10⁹ : need 18 of 5-layer multi-flasks (875 cm² per flask).

For 1 X 10^12 particles:

3 of 5-layer multi-flasks are required. Total area: 2625  cm²

Or 15 of T175  flasks

Or 20 of 15 cm dish (135 cm²)

Medium: ~500 ml.

2. Infect cells with 1 × 10¹² particles of Adenovirus. (1:1000).

For making 1 X 10¹² particles:

5 X 10¹⁰ particles of Adenovirus are required

3. After 60 hours of infection--> maximum cytopathic effect can be seen.

4. Collect cells by centrifugation for 20 minutes at 1 000 × g.

5. Resuspend in 10 or 5 ml of 50 mM Tris-HCl (pH 8.0) in 5% glycerol.

6. Freeze and thaw cells three times.

7. Spin to remove cell debris.

8. Load adenovirus in supernantant to a 20 ml Q Sepharose XL column. Flow rate: flow rate: 30 cm/h
9. Elute with 0 to 1.0 M NaCl gradient in 50 mM Tris/HCl, pH 8.0, in 5% glycerol, for 20 column volumes (CV). 5 ml per fraction. Flow rate: 150 cm/h

Identify virus peak by  A260/280 ratio.Virus should be eluted by three 5 ml fractions.

For 16/10 Q XL column, virus should be eluted between 60 ~ 70 min, or 65 min. Fraction is 17, 18 and 19. (vol: 85~95 ml).

The ratio of 260:280 readings should be close to 1.3 for purified adenovirus.
calculate the particle number:
OD260 reading x dilution factor x 1.1 x 1012 particles = number of
particles per mL of sample.
Example: OD260 reading of 0.15
0.15 x 4 x 1.1 x 1012 = 6.6 x 1011 particles per mL of your sample

Regenerate the column  with four CV of 0.1 M NaOH between each run.
10. Further purification using a Sartobind IDA membrane unit charged with Zn(2+) ions as affinity ligands. or Gel filtration.

11. Concentrate adenovirus using a 100 KDa spin column.

Yield:

6.7 x 10^12 particles from 10⁹ cells.

1 X 10^12 particles from 15 of T175 flasks, or 20 of 15 cm dish (135 cm²)

Particle/PFU: 40