Cesium Chloride-Based Purification of Genome-Containing Adenoviral Vectors

Begin with mammalian cells infected with an adenovirus.

Perform repeated freeze-thaw cycles to lyse the cells and release the virions.

Centrifuge the lysate to pellet the debris. Collect the supernatant containing viral particles.

Layer the viral supernatant onto a stepwise cesium chloride gradient for density-based separation.

Ultracentrifuge to allow the viral particles to migrate according to their buoyant densities.

Intact virions containing the full adenoviral genome are denser due to the packaged DNA and settle into a lower band, while lighter, empty capsids and debris remain in the upper layers.

Remove the upper layers. Collect the lower band of genome-containing virions.

Resuspend the virions in uniform-density cesium chloride and ultracentrifuge again to refine the separation and concentrate the full particles.

Discard the upper layers and recover the lower band.

Dialyze the sample to remove the cesium chloride.

The high-purity, genome-containing adenoviral vectors are now ready for downstream applications.

To prepare the viral lysate for cesium chloride gradient purification, freeze/thaw the cell virus suspension four times. Then, centrifuge the viral lysate at 500 times g for eight