Method for the production and purification of adenoviral vectors

Abstract

The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production with cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.

1 - 43 . (canceled) 
     
     
         44 . A method for producing a purified adeno-associated virus composition comprising:
 a) growing host cells, wherein the host cells encode adeno-associated virus cap and rep genes;   b) providing nutrients to the host cells by perfusion or through a fed-batch process;   c) infecting the host cells with an adenovirus and an adeno-associated virus;   d) lysing the host cells to provide a cell lysate comprising adeno-associated virus; and   e) purifying adeno-associated virus from the lysate to provide a purified adeno-associated virus composition.   
     
     
         45 . The method of  claim 44 , further comprising subjecting the lysate to a clarification step. 
     
     
         46 . The method of  claim 44 , further comprising subjecting the lysate to a concentration step. 
     
     
         47 . The method of  claim 44 , further comprising subjecting the lysate to a diafiltration step. 
     
     
         48 . The method of  claim 44 , further comprising treating the lysate with a nuclease. 
     
     
         49 . The method of  claim 48 , wherein the nuclease is BENZONASE™. 
     
     
         50 . The method of  claim 48 , wherein the nuclease is PULMOZYME™. 
     
     
         51 . The method of  claim 44 , wherein purifying the adeno-associated virus from the lysate comprises using at least one chromatography step. 
     
     
         52 . The method of  claim 51 , wherein purifying the adeno-associated virus from the lysate comprises using at least two chromatography steps. 
     
     
         53 . The method of  claim 52 , wherein purifying the adeno-associated virus from the lysate comprises using at least three chromatography steps. 
     
     
         54 . A method for producing a purified adeno-associated virus composition comprising:
 a) growing host cells, wherein the host cells encode adeno-associated virus cap and rep genes;   b) providing nutrients to the host cells by perfusion or through a fed-batch process;   c) infecting the host cells with an adeno-associated virus and a plasmid encoding at least a portion of an adenoviral gene that assists with adeno-associated virus replication;   d) lysing the host cells to provide a cell lysate comprising adeno-associated virus; and   e) purifying adeno-associated virus from the lysate to provide a purified adeno-associated virus composition.   
     
     
         55 . The method of  claim 54 , wherein the adenovirus gene is E1, E2, E4, or E5. 
     
     
         56 . The method of  claim 54 , further comprising subjecting the lysate to a clarification step. 
     
     
         57 . The method of  claim 54 , further comprising subjecting the lysate to a concentration step. 
     
     
         58 . The method of  claim 54 , further comprising subjecting the lysate to a diafiltration step. 
     
     
         59 . The method of  claim 54 , further comprising treating the lysate with a nuclease. 
     
     
         60 . The method of  claim 59 , wherein the nuclease is BENZONASE™. 
     
     
         61 . The method of  claim 59 , wherein the nuclease is PULMOZYME™. 
     
     
         62 . The method of  claim 54 , wherein purifying the adeno-associated virus from the lysate comprises using at least one chromatography step. 
     
     
         63 . The method of  claim 62 , wherein purifying the adeno-associated virus from the lysate comprises using at least two chromatography steps. 
     
     
         64 . The method of  claim 63 , wherein purifying the adeno-associated virus from the lysate comprises using at least three chromatography steps.