Purification of Adeno-Associated Virus Particles by Polyethylene Glycol Precipitation and Ultracentrifugation

Take a filtered suspension of adeno-associated virus that contains full viral particles with genomes, empty viral particles without genomes, and host-derived contaminants.

Add a salt solution containing polyethylene glycol (PEG), then incubate under agitation.

PEG causes molecular crowding that brings the viruses closer together, while the salt neutralizes the viral surface charge and reduces electrostatic repulsion, allowing the viruses to aggregate.

Spin down the virus, discard the PEG-rich supernatant containing contaminants, and resuspend in a buffer.

Overlay it onto a discontinuous density gradient with layers of increasing iodixanol concentration.

Ultracentrifuge to separate components by density.

Full viral particles migrate to denser layers, while empty particles remain above.

Extract the layer with full viral particles and load it into a dialysis cassette.

Dialyze to remove the iodixanol and excess salts. Concentrate the dialyzed virus using a centrifugal filter unit.

Collect the purified virus for downstream analysis.

Now add 40% PEG sodium chloride solution to the AAV solution, making the final concentration up to 8% PEG. Place the mixture on a low speed orbital rotator overnight