Method for the production and purification of retroviral 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 .- 69 . (canceled) 
   
   
       70 . A method for the purification of a retrovirus comprising the steps of:
 a) growing host cells in substantially serum-free media;   b) infecting said host cells with a retrovirus;   c) harvesting retroviral particles to produce a crude retrovirus composition;   d) concentrating said crude retrovirus composition;   e) exchanging buffer of said crude retrovirus composition; and   f) reducing the concentration of contaminating nucleic acids in said crude retrovirus composition.   
   
   
       71 . The method of  claim 70 , wherein said host cells are adapted for growth in substantially serum-free media. 
   
   
       72 . The method of  claim 70 , further comprising isolating a retroviral particle from said crude retrovirus composition using chromatography. 
   
   
       73 . The method of  claim 70 , wherein said exchanging buffer involves a diafiltration step. 
   
   
       74 . The method of  claim 71 , wherein said substantially serum-free media comprises a fetal bovine serum content of less than 0.03%. 
   
   
       75 . The method of  claim 70 , wherein the retrovirus comprises a heterologous nucleic acid sequence of interest. 
   
   
       76 . The method of  claim 70 , wherein the host cells comprise a packaging cell line containing the gag, pol and env genes of a retrovirus. 
   
   
       77 . A method for the purification of a retrovirus comprising the steps of:
 a) growing host cells;   b) infecting said host cells with a retrovirus;   c) harvesting retroviral particles to produce a crude retrovirus composition;   d) concentrating said crude retrovirus composition;   e) exchanging buffer of said crude retrovirus composition; and   f) reducing the concentration of contaminating nucleic acids in said crude retrovirus composition.   
   
   
       78 . The method of  claim 77 , further comprising isolating a retroviral particle from said crude retrovirus composition using chromatography. 
   
   
       79 . The method of  claim 77 , wherein the retrovirus comprises a heterologous nucleic acid sequence of interest. 
   
   
       80 . The method of  claim 77 , wherein the host cells comprise a packaging cell line containing the gag, pol and env genes of a retrovirus. 
   
   
       81 . The method of  claim 77 , wherein said crude retrovirus composition is treated with Benzonase® or Pulmozyme®. 
   
   
       82 . The method of  claim 77 , further comprising a concentration step employing membrane filtration. 
   
   
       83 . The method of  claim 77 , further comprising a concentration step employing membrane filtration, preferably tangential flow filtration. 
   
   
       84 . The method of  claim 77 , wherein the step of infecting the host cells with a retrovirus comprises introducing into the host cells a recombinant plasmid containing a human cDNA together with retrovirus LTR and Y sequences. 
   
   
       85 . A method for the purification of a retrovirus comprising the steps of:
 a) growing host cells in media at a low perfusion rate;   b) infecting said host cells with a retrovirus;   c) harvesting retroviral particles to produce a crude retrovirus composition;   d) concentrating said crude retrovirus composition;   e) exchanging buffer of said crude retrovirus composition; and   f) reducing the concentration of contaminating nucleic acids in said crude retrovirus composition.   
   
   
       86 . The method of  claim 85 , further comprising isolating a retroviral particle from said crude retrovirus composition using chromatography. 
   
   
       87 . The method of  claim 85 , wherein the retrovirus comprises a heterologous nucleic acid sequence of interest. 
   
   
       88 . The method of  claim 85 , wherein said crude retrovirus composition is treated with Benzonase® or Pulmozyme®.