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

תקציר

Source: Zhang, T., et al. Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform. J. Vis. Exp. (2024).

This video demonstrates the purification of adeno-associated virus (AAV) from an infected mammalian cell culture. It shows how polyethylene glycol and salt are used to aggregate the viral particles, followed by centrifugation and density gradient separation to isolate full AAV capsids. Finally, the process includes dialysis and concentration steps to obtain purified virus for downstream analysis.

פרוטוקול

1. AAV purification by iodixanol gradient ultracentrifugation

1. Prepare the iodixanol gradients freshly before loading the AAV according to the number of ultracentrifugation tubes used (Table 1).
   NOTE: Phenol red was used to observe the layers.
2. Overlay each solution into a round-top polypropylene ultracentrifuge quick-seal tube slowly using a 10 mL syringe attached with a 16 G punted long needle. Avoid bubbles.
3. Carefully add up to 17 mL of AAV solution on top of the gradient with a 22 G syringe. Use AAV dialysis buffer to top off the tube.
   NOTE: Add the AAV solution by loading drops against the wall in the ultracentrifuge tube. Don't disturb the gradient layers.
4. Seal the ultracentrifuge tubes with an electric sealer.
   NOTE: Balance the tubes before sending them to ultracentrifuge with ±0.2 g difference.
5. Centrifuge at 350,000 x g for 2 h in a Ti70 rotor at 4 °C.
6. Carefully remove the tubes from the rotor and place them in the ultracentrifuge tubes rack.
   NOTE: Ensure that the gradients are not disturbed.
7. Collect AAV fractions following the steps below:
   1. Stabilize the tube on a stand holder.
   2. Puncture the ultracentrifuge tubes slightly below the 40%-60% interface with a 19 G needle attached to a 10 mL syringe.NOTE: The opening of the needle should be up, facing the 40% gradient.
   3. Punch a hole in the top of the tube with a 16 G needle.
   4. Collect up to 5 mL per tube. Avoid collecting the protein contamination at the 25%-40% interface.
   5. Repeat for each ultracentrifuge tube.
      NOTE: Transfer the AAV fractions to a 50 mL conical tube.

2. Second round iodixanol gradients ultracentrifugation

NOTE: This step is optional. This step is to reduce the empty AAV ratio for a higher quality full AAV capsid.

1. Dilute AAV >1:1 with AAV dialysis buffer.NOTE: AAV collected from the first round of ultracentrifugation was at 40% iodixanol layer. It should be diluted by at least 50% to be able to be loaded on top of the 30% iodixanol layer.
2. Overlay each solution (Table 2) into an ultracentrifugation tube using a 16 G punted needle and slowly attach a 10 mL syringe. Avoid bubbles.
3. Carefully add 20 mL of diluted AAV solution on top of the gradient with a 22 G syringe. Use AAV dialysis buffer to top off the tube.
4. Repeat the steps 6.4 to 6.7.

3. AAV dialysis and concentration

1. Transfer the AAV virus into the dialysis cassette using a new 18 G needle and 10 mL syringe. Slowly inject the virus into the cassette and remove air from it.
2. Add a stir bar to the beaker containing AAV dialysis buffer and place it on a stir plate.
3. Put the dialysis cassette into the dialysis buffer with a float buoy. Stir at 4 °C. Change 2-3 times the AAV dialysis buffer.
   NOTE: Use enough buffer to allow the cassette to float and perform the buffer exchange. Cover the beaker with aluminum foil.
4. Using a 10 mL syringe attached with an 18 G needle, collect the virus from the cassette into a centrifugal filter unit.
5. Centrifuge at 4,000 x g for 15-30 min at 4 °C.
   NOTE: Rince the AAV with AAV dialysis buffer 2-3 times. Concentrate the AAV until ~200 µL of remaining is on top of the filter.
6. Collect the concentrated AAV from the filter unit. Rinse the filter with another 300 µL of AAV dialysis buffer and transfer it to the concentrated AAV.
7. Filter the virus through a 0.22 µm syringe filter using a 1 mL tuberculin luer slip syringe. To ensure the least amount of volume loss, use the 10 mL syringe to push air through the filter 2-3 times.
8. Store the purified AAV virus at 4 °C temporally for titration.
   NOTE: Titrate the virus, aliquot, and store at -80 °C within 1 week.

Table 1. Preparation of iodixanol gradient.
Volume of each layer (mL)	Layer		Number of gradients
	%	Solutions (mL)	1	2	4	6	8
6	15	DPBS + high salt	5	9.9	19.8	29.7	39.6
		60% iodixanol	1.7	3.3	6.6	9.9	13.2
6	25	DPBS + low salt	3.9	7.7	15.4	23.1	30.8
		60% iodixanol	2.8	5.5	11	16.5	22
		Phenol red	0.1	0.1	0.2	0.3	0.4
5	40	DPBS + low salt	1.8	3.7	7.3	11	14.7
		60% iodixanol	3.7	7.3	14.7	22	29.3
5	60	DPBS + low salt	5.5	11	22	33	44
		Phenol red	0.1	0.1	0.2	0.3	0.4

Table 2. Preparation of second round iodixanol gradient.
Volume of each layer (mL)	Layer		Number of gradients
	%	Solutions (mL)	1	2	4	6	8
8	30	DPBS + low salt	4	8	16	24	32
		60% iodixanol	4.1	8.2	16.4	24.6	32.8
		Phenol red	0.1	0.2	0.4	0.6	0.8
5	40	DPBS + low salt	1.8	3.7	7.3	11	14.7
		60% iodixanol	3.7	7.3	14.7	22	29.3
5	60	DPBS + low salt	5.5	11	22	33	44
		Phenol red	0.1	0.1	0.2	0.3	0.4

חומרים

רשימת החומרים שנעשה בהם שימוש במאמר זה

שם	חברה	מספר קטלוג	הערות
40% PEG solution	Sigma	P1458-50ML
AAV6 reference full capsids	Charles River Laboratories	RS-AAV6-FL
Centrifugal Filter Unit	MilliporeSigma	UFC905024
Corning PES Syringe Filters	Fisher Scientific	09-754-29
Dialysis Cassettes, 10 K MWCO	Fisher Scientific	PI66810
Disposable PES Filter Units 1 L 0.2 µm	Fisher Scientific	FB12566506
Disposable PES Filter Units 1 L 0.45 µm	Fisher Scientific	FB12566507
Disposable PES Filter Units 500 mL 0.2 µm	Fisher Scientific	FB12566504
pAAV-CMV-GFP	Addgene	105530
pAAV-DJ	Cell BioLab	VPK-420-DJ
pAAV-RC6	Cell BioLab	VPK-426
pAdDeltaF6	Addgene	112867
PEG 8000	Promega	V3011
Type 70 Ti Fixed-Angle Titanium Rotor	Beckman Coulter	337922