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 minutes at room temperature.

Then, collect the supernatant containing the high-capacity adenovirus. Carefully and slowly pipette cesium chloride solutions into the tubes in the following order, 0.5 milliliters of 1.5 grams per cubic centimeter solution, three milliliters of 1.35 grams per cubic centimeter solution and 3.5 milliliters of 1.25 grams per cubic centimeter solution. Overlay approximately 4.5 milliliters of cleared vector supernatant on top of the 1.25 grams per cubic centimeter layer.

Centrifuge the gradients in an ultracentrifuge using a swing out rotor at 12 degrees Celsius for at least two hours at 226,000 times g to separate the high-capacity adenovirus genome containing viral particles from empty particles and cell debris. Following the centrifugation, a diffuse band of cell debris forms on top of the tubes. Below this, two white bands can be observed.

The upper band contains empty particles, whereas the lower band is the high-capacity adenovirus. Carefully remove the layers of cell debris and empty particles. Then, collect one milliliter of the lower bands from each tube.

Transfer the virus into a sterile 50 milliliter tube. Add up to 24 milliliters of 1.35 grams per cubic centimeter cesium chloride solution to the collected virus particles and mix carefully. Fill the centrifuge tubes to the top with 1.35 grams per cubic centimeter cesium chloride virus solution.

Then centrifuge overnight at 226,000 times g at 12 degrees Celsius in an ultracentrifuge using a swing out rotor with slow acceleration and deceleration. Remove the upper layers from the top using a pipette. Then, collect the high-capacity adenovirus present in the prominent lower band using a fresh pipette tip.

Finally, dialyse the collected virus particles for buffer exchange. And then, titer the final preparation according to the detailed instructions in the written protocol.