AAV Particle Purification

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.





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Why OptiPrepTM

Not destructive 
At the densities used to purify viruses, sucrose and CsCI are highly hyperosmotic. However, OptiPrep is iso-osmotic at all densities and consequently it does not change the biological features of the purified virus.

Saves time 
Unlike sucrose and CsCl, if further analysis of the purified particles is required, OptiPrep does not need to be removed by pelleting. CsCl is also known to lead to poor recovery and low infectivity of rAAV isolates. Furthermore, the low viscosity of OptiPrep allows rapid sedimentation of the AAV particles.

Greater recovery rate 
Compared to CsCl gradients, the virus has at least a ten times greater recovery rate with OptiPrep, as well as a significantly higher infectivity titer (up to 100x).

Higher infectivity titer
Measurements and analyses following purification, e.g. infectivity assays can be performed without the need to remove the medium e.g. by dialysis, as OptiPrep does not cause any toxic effects. Thus avoiding the risk of decreasing infectivity titers due to further propagation of the purified material.

Key Features

Density Gradient Medium of Choice

In AAV gene therapy, OptiPrep is widely regarded as the gradient medium of choice for rAAV purification. Although originally OptiPrep was developed as an X-ray contrast medium. Its use in a medical context ensured thorough and comprehensive clinical testing.

Today, it is more and more often used as a convenient density gradient medium due to its non-ionic, non-toxic and metabolically inert features. OptiPrep allows the generation of solutions of all densities with low viscosity and osmolality.

OptiPrep Advantages

  • OptiPrep = 60% iodixanol in water (density: 1.320 g/ml)
  • Iso-osmotic at all densities
  • Non-ionic and inert nature
  • Ready-made, sterile and endotoxin-tested solution
  • Manufactured, packed and released in compliance with GMP and ISO13485.

OptiPrepTM Applications

Uses

Beside its use for AAV particle purification, OptiPrep is commonly used to separate cell types from blood and other tissue e.g. monocytes, lymphocytes, spermatozoa or plant protoplasts.

Due to its chemical features and the high quality grade production it is also suitable for fractionating cell organelles, membranes and ribonucleoproteins, to separate viable from non-viable cells or to purify proteins, plasma lipoproteins, plasmid DNA.

Techniques

OptiPrep is used for different density gradient designs including discontinuous or continuous gradients due to its high density. It is most commonly used for the preparation of discontinuous gradients.

These kind of gradients can be generated in two specific ways:

  1. The most common technique is the overlaying technique. Starting with the densest solution and continuing to layer solutions of successively lower densities on top. To avoid mixing the two adjacent layers of different densities, it is important to place the tip of the pipette or syringe against the wall of the tube and apply the solution in a steady, gentle flow.

  2. The second technique is the underlaying technique. Starting with the lowest density solution and underlaying successively denser solutions beneath the lighter solutions. This technique is supposed to be easier than the overlaying technique. However, it is very important to avoid the generation of air bubbles since they disturb the layers and can hardly be removed.

(Ultra)centifugation

Whatever technique is chosen to prepare the gradient; the actual purification is performed by (ultra)centrifugation following the application of the sample on top of the gradient. The specifications of the centrifugation strongly depend on the sample applied. However, always finish with the fractionation of the different components of the sample according to their density.

For many applications such as electrophoresis, infection of cultured cells, in vivo animal experiments, removal of the iodixanol is not a requirement. If a removal or at least reduction of the iodixanol concentration is required for any reason some form of ultrafiltration is widely regarded as the most effective method.

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