Lysis, extraction and purification of adeno-associated virus and adenovirus from host cells

Abstract

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 and has a pH of about 9.0 to about 11.5; and   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.   
     
     
         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 is a mild detergent. 
     
     
         4 . The method of  claim 3 , wherein the detergent is selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside, alkyl Sulfates, Polysorbate 20 (Tween-20), Tergitol-type NP-40 and the combinations thereof. 
     
     
         5 . The method of  claim 3 , wherein the detergent is deoxycholate. 
     
     
         6 . The method of  claim 3 , wherein the detergent is deoxycholate and sodium dodecyl sulfate (SDS). 
     
     
         7 . The method of  claim 1 , wherein the alkaline lysis solution has a pH of about 9.0 to about 11.5 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. 
     
     
         8 . The method of  claim 1 , wherein the sample, following addition of the alkaline lysis solution, comprises about 0.1% to about 1.0% of the detergent. 
     
     
         9 . The method of  claim 1 , wherein the salt is selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate and combinations thereof. 
     
     
         10 . The method of  claim 1 , wherein the salting solution does not include an ammonium salt. 
     
     
         11 . The method of  claim 1 , further comprising precipitating the viral particles by adding a precipitation solution to the sample such that the sample comprises about 5% to about 12% of a polyethylene glycol (PEG). 
     
     
         12 . The method of  claim 11 , wherein the PEG has an average molecular weight of about 3000 to about 10000. 
     
     
         13 . The method of  claim 11 , further comprising subjecting the precipitated viral particles to chromatography. 
     
     
         14 . The method of  claim 13 , wherein the chromatography is silica-based chromatography, resin chromatography or ion exchange chromatography columns. 
     
     
         15 . The method of  claim 1 , wherein the method does not include the use of an organic solvent. 
     
     
         16 . The method of  claim 1 , wherein the method does not include the use of ultracentrifugation. 
     
     
         17 . The method of  claim 1 , wherein the method does not include extraction by freeze-thaw cycles. 
     
     
         18 . The method of  claim 1 , wherein the method does not include the use of affinity chromatography. 
     
     
         19 . A preparation of viral particles of a recombinant adeno-associated virus (rAAV) or an recombinant adenovirus (rAdV), extracted and purified from a sample of cells enclosing the viral particles, comprising at least 10 13  of the particles per milliliter (ml), wherein the preparation:
 contains at least 10 10  transducing units (TU) per 10 13  of the particles;   contains less than 1/10,000 host cell proteins relative to the particles (w/w); and   does not contain detectable cesium chloride (CsCl) or iodixanol.   
     
     
         20 . The preparation of  claim 19 , wherein no more than 1/10,000 of the viral particles are empty particles.