Lysis, extraction and purification of adeno-associated virus and adenovirus from host cells
View Patent ↗The present disclosure provides compositions and methods for extracting viral particles of a recombinant adeno-associated virus (AAV) or an adenovirus (AdV) from a sample comprising cells enclosing the viral particles. The method can include lysing the cells with the addition of an alkaline lysis solution to the sample such that the sample comprises compound(s) buffering pH value about 9.0 to about 11.5 and about 0.1% to about 1% of a detergent precipitating host cell proteins by adding a salting solution to the sample such that the sample comprises about 0.5 M to about 2.0 M of a salt and has a pH of about 4.0 to about 7.0.
1. A method of extracting viral particles of a recombinant adeno associated virus (AAV) or an adenovirus (AdV) from a sample comprising cells enclosing the viral particles, comprising:
lysing the cells by the addition of an alkaline lysis solution to the sample such that the sample comprises about 0.1% to about 1% of a detergent comprising a salt of deoxycholic acid and has a pH of about 9.5 to about 11 under suitable conditions to lyse the cells; then
precipitating proteins released from the lysed cells by adding a salting solution to the sample such that the sample comprises about 0.5 M to about 2.0 M of a salt selected from the group consisting of sodium acetate, potassium acetate and combinations thereof and has a pH of about 4.0 to about 7.0; then
removing the precipitated proteins to obtain a supernatant comprising the viral particles; and then
precipitating the viral particles by adding a precipitation solution to the supernatant such that the supernatant comprises about 5% to about 12% of a polyethylene glycol (PEG),
wherein the method does not include use of ultracentrifugation or freeze-thaw cycles.
2. The method of claim 1 , wherein the viral particles are of any one of AAV serotypes 1-11 or any one of AdV species A-G.
3. The method of claim 1 , wherein the detergent further comprises sodium dodecyl sulfate (SDS).
4. The method of claim 1 , wherein the alkaline lysis solution has a pH of about 9.5 to about 11 and comprises, in addition to the detergent, one or more reagents selected from the group consisting of N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and combinations thereof.
5. The method of claim 1 , wherein the salting solution does not include an ammonium salt.
6. The method of claim 1 , wherein the PEG has an average molecular weight of about 3000 to about 10000.
7. The method of claim 1 , further comprising subjecting the precipitated viral particles to chromatography.
8. The method of claim 7 , wherein the chromatography is silica-based chromatography, resin chromatography or ion exchange chromatography columns.
9. The method of claim 1 , wherein the method does not include the use of an organic solvent.
10. The method of claim 1 , wherein the method does not include the use of affinity chromatography.