Affinity Chromatography-Based Purification of Adeno-Associated Virus Vectors

Begin with a filtered cell lysate containing adeno-associated virus (AAV) vectors and host-derived contaminants.

Pass the lysate through a heparin-based affinity column.

The viral capsid proteins bind electrostatically to the negatively charged heparin groups.

Rinse the column with a buffer to remove unbound contaminants.

Apply a detergent-containing buffer to solubilize membranous debris.

Rinse again with the detergent-free buffer.

Introduce a buffer supplemented with cations to create an ionic environment, then wash with a moderate-salt buffer to release weakly bound impurities.

Add a high-salt buffer to disrupt capsid-heparin interactions and elute the viral particles.

Collect the elution fractions enriched in intact viral particles and transfer them to centrifugal concentrator tubes fitted with filter membranes.

Centrifuge the tubes to remove excess buffer, then collect the concentrated viral fractions in a tube.

Rinse the membrane with the buffer to recover residual viral particles and maximize yield.

Thaw the crude lysate at four degrees Celsius overnight.

Once thawed, filter it using a 0.22 micron filter. Immediately before the purification steps, passivate the centrifugal concentrator