Methods of adenovirus purification

Abstract

A process for purifying virus particles, especially recombinant adenovirus vector particles, is presented. The process relies on various combinations of cell lysis, detergent-based precipitation of host cell contaminants away from the virus, depth filtration or centrifugation, ultrafiltration, nuclease digestion and chromatography to robustly and economically produce highly purified product. This process results in contaminating DNA levels which are consistently below detectable levels.

1 - 2 . (canceled)  
     
     
         3 . A method of purifying adenovirus particles from a cell culture preparation which comprises: 
 a) lysing host cells within the cell culture preparation;    b) selectively precipitating impurity DNA away from the adenovirus particles by adding a selective precipitation agent to the post-lysis host cell culture medium, wherein the selective precipitation agent is selected from the group consisting of: a quartnery ammonium compound, a cationic detergent and a poly amine;    c) clarifying the cell culture medium; and    d) recovering purified adenovirus particles from the cell culture medium; wherein at least 80% of impurity DNA is precipitated away from medium containing the virus particles.    
     
     
         4 . (canceled)  
     
     
         5 . The method of purifying adenovirus particles of  claim 3 , further comprising: 
 e conducting ultrafiltration on the adenovirus-containing medium of step d; and,    f) recovering concentrated, purified, adenovirus particles.    
     
     
         6 . (canceled)  
     
     
         7 . The method of  claim 3  wherein step c) is performed by a method selected from the group consisting of depth filtration, dead end filtration, microfiltration, centrifugation or a combination thereof.  
     
     
         8 . The method of  claim 3  wherein step c) is performed by depth filtration.  
     
     
         9 . The method of  claim 3  wherein step c) is performed by the combination of centrifugation and depth filtration.  
     
     
         10 . The method of  claim 5  wherein the purified adenovirus particles of step f are subjected to cation exchange chromatography.  
     
     
         11 . (canceled)  
     
     
         12 . The method of  claim 3  wherein the clarified medium of step c) is treated with a nuclease.  
     
     
         13 . The method of  claim 3 , which further comprises: 
 e) subjecting-the adenovirus-containing medium recovered from step d) to anion exchange chromatography;    f) diafiltering the adenovirus containing eluate recovered from of step e, and    g) recovering concentrated purified adenovirus.    
     
     
         14 - 25 . (canceled)  
     
     
         26 . A method of purifying adenovirus particles from a cell culture preparation which comprises: 
 a) lysing host cells within the cell culture preparation;    b) selectively precipitating impurity DNA away from the adenovirus particles by adding a selective precipitation agent to the post-lysis host cell culture medium, wherein the selective precipitation agent is selected from the group consisting of: a monoalkyltrimethylammonium salt, a monoalkyldimethylbenzylammonium salt, a dialkyldimethylammonium salt, a heteroaromatic ammonium salt, a bis-quarternary ammonium salt and a polymeric quaternary ammonium salt;    c) clarifying the cell culture medium; and    d) recovering purified adenovirus particles from the cell culture medium; wherein at least 80% of impurity DNA is precipitated away from medium containing the virus particles.    
     
     
         27 . The method of  claim 26 , further comprising: 
 e) conducting ultrafiltration on the adenovirus-containing medium of step d; and 6    f) recovering concentrated, purified, adenovirus particles.    
     
     
         28 . The method of  claim 26  wherein step c) is performed by a method selected from the group consisting of depth filtration, dead end filtration, microfiltration, centrifugation or a combination thereof.  
     
     
         29 . The method of  claim 26  wherein step c) is performed by depth filtration.  
     
     
         30 . The method of  claim 26  wherein step c) is performed by the combination of centrifugation and depth filtration.  
     
     
         31 . The method of  claim 27  wherein the purified adenovirus particles of step f) are subjected to cation exchange chromatography.  
     
     
         32 . The method of  claim 26  wherein the clarified medium of step c) is treated with a nuclease.  
     
     
         33 . The method of  claim 26 , which further comprises: 
 e) subjecting-the adenovirus-containing medium recovered from step d) to anion exchange chromatography;    f) diafiltering the adenovirus containing eluate recovered from of step e, and    g) recovering concentrated, purified adenovirus.    
     
     
         34 . The method of  claim 26  which further comprises subjecting medium comprising the purified adenovirus particles to anion exchange chromatography wherein said anion exchange chromatography employs a buffer comprising a detergent.  
     
     
         35 . The method of  claim 34  wherein the detergent is a non-ionic surfactant.  
     
     
         36 . The method of  claim 35  wherein the detergent is polysorbate-80 (PS-80).  
     
     
         37 . The method of  claim 36  wherein the concentration of PS-80 is 0.1%.