AAV vectors for gene therapy clearly benefit from isolation methods which are both effective and cause little or no damage to the viral particles. Density gradient centrifugation has always played an important part in the concentration and purification of virus particles.
The most prominently used gradient media, sucrose and CsCl, are highly hyperosmotic at the densities used to band viruses and generally have to be removed either by pelleting the virus or by dialysis, prior to further processing or analysis. CsCl also leads to poor recoveries and low infectivity of rAAV isolates.
In contrast to sucrose and CsCl, OptiPrep™ (iodixanol) is widely regarded as the gradient medium of choice for rAAV purification. Advantages of OptiPrep™compared to CsCl gradients:
♦ Recovery of virus from the gradient is at least ten times greater
♦ Particle:infectivity titer is up to 100x lower
♦ Infectivity measurements and many add-on techniques can be carried out without the need to dialyze the medium.