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 by adding four milliliters of filter pretreatment buffer for each heparin-Sepharose column being used. Place the tubing in a peristaltic pump.

Sequentially run the solutions and media. Prepare for chromatography by attaching a five milliliter heparin-Sepharose column to the tubing, and running 25 milliliters of Bazell DMEM through the column. Then load 0.2 micromolar of the filtered crude lysate onto the column at a flow rate of one to two drops per second.

Wash the column sequentially to elute five milliliters five times with 300 millimolar of sodium chloride HBSS, with magnesium and calcium, and label the five elutions as E1 to E5.When ready, spin the centrifugal concentrator containing the pretreatment buffer at 900 times g for two minutes. Discard the flow-through. Wash the centrifugal concentrator filter with four milliliters of HBSS with magnesium and calcium by spinning at 1000 times g for two minutes.

Then add the elution E2 to the centrifugal concentrator, and spin at 1000 times g for five minutes. Discard the flow-through. Similarly spin the elution E3 into the centrifugal concentrator until the concentrated virus is approximately one milliliter.

Remove the concentrated virus from the centrifugal concentrator using a P200 filtered tip, and collect it into a sterile 1.5 milliliter centrifuge tube. After collection of the virus, rinse the centrifugal concentrator with 200 microliters of HBSS with magnesium and calcium. Pipette up and down vigorously for 30 seconds to dislodge any virus adhered to the membrane, and collect the concentrated virus in the same 1.5 milliliter centrifuge tube.

Mix the tube well.