Generation of a high-titer viral stock from a recombinant adenovirus. Plaque purification prior to amplification is highly recommened to insure the purity and stability of the recombinant adenovirus. Amplified adenoviruses are purified by CsCl banding (ultracentrifugation)

Scale: 1 L AdExpress-293A culture

Time Required: 2-6 weeks

Includes:

• Amplification of a recombinant adenovirus in AdExpress-293A cells at the desired volume.
• Harvest adenovirus. Three freeze/thaw cycles are performed and cell debris is removed by centrifugation.
• Purification on a CsCl step gradient followed by a continuous CsCl gradient using an ultracentrifuge.
• Desalting and elute virus using a storage buffer containing glycerol
• Make viral stock aliquots.

Deliverable: Purified adenovirus. The final titer is variable and depends on the nature of the recombinant adenovirus to be amplified. On average 2.5x10e¹³ particles are obtainined. For an accurate infectious titer, a TCID₅₀ or plaque assay can be performed on the final product.