Novel method for the protection and purification of adenoviral vectors

Abstract

The present invention relates to improved methods for producing adenovirus compositions wherein host cells are grown in a bioreactor and purified by size partitioning purification to provide purified adenovirus compositions.

1 . A method for removing contaminants from a virus-containing composition comprising obtaining an aqueous composition comprising a selected virus and undesirable contaminants and subjecting the aqueous composition to size partitioning purification using a size partitioning membrane having partitioning pores that retain virus and permit the passage of contaminants therethrough to remove contaminants and thereby provide a purified virus composition.  
     
     
         2 . The method of  claim 1 , wherein the virus is adenovirus, lentivirus, adenoassociated virus, retrovirus, or herpes virus.  
     
     
         3 . A method of producing purified adenovirus composition comprising: 
 a) growing host cells in a medium;    b) providing nutrients to said host cells;    c) infecting said host cells with an adenovirus;    d) lysing said host cells to provide a cell lysate comprising adenovirus; and    e) purifying adenovirus from said lysate by size partitioning purification utilizing a size partitioning membrane to provide a purified adenovirus composition.    
     
     
         4 . The method of  claim 1  or  3  wherein the size partitioning membrane is a dialysis membrane.  
     
     
         5 . The membrane of  claim 1  or  3  wherein the size partitioning membrane is a porous filter  
     
     
         6 . The method of  claim 1  or  3 , wherein the size partitioning membrane is in a tangential flow filtration device.  
     
     
         7 . The method of  claim 1  or  3  wherein the size partitioning membrane has a pore size of less than about 0.08 microns.  
     
     
         8 . The method of  claim 5  whrein the size partitioning membrane has a pore size of less than about 0.08 microns and greater than about 0.0001 microns.  
     
     
         9 . The method of  claim 5  wherein the size partitioning membrane has a pore size of less than about 0.05 microns and greater than about 0.0001 microns.  
     
     
         10 . The method of  claim 5  wherein the size partitioning membrane has a pore size of less than about 0.02 microns and greater than about 0.0001 microns.  
     
     
         11 . The method of  claim 5  wherein the size partitioning membrane has a pore size of less than about 0.01 microns and greater than about 0.0001 microns.  
     
     
         12 . The method of  claim 1  or  3 , wherein the virus is purified to a pharmaceutically acceptable degree without the use of ion exchange chromatography.  
     
     
         13 . The method of  claim 1  or  3 , wherein said medium is a serum-free medium and said host cells are capable of growing in serum-free media.  
     
     
         14 . The method of  claim 13  wherein said host cells have been adapted for growth in serum-free media by a sequential decrease in the fetal bovine serum content of the growth media.  
     
     
         15 . The method of  claim 3 , wherein said host cells are 293 cells.  
     
     
         16 . The method of  claim 3  wherein the lysis step is carried out by a process that includes hypotonic solution, hypertonic solution, impinging jet, microfluidization, solid shear, detergent, liquid shear, high pressure extrusion, autolysis or sonication.  
     
     
         17 . The method of  claim 16  wherein the cells are lysed by detergent lysis.  
     
     
         18 . The method of  claim 16  wherein the cells are lysed by detergent Thesit®, NP-40®, Tween-20®, Brij-58®, Triton X-100® or octyl glucoside.  
     
     
         19 . The method of  claim 17  wherein said detergent is present in the lysis solution at a concentration of about 1% (w/v).  
     
     
         20 . The method of  claim 3 , wherein the host cells are grown at least part of the time in a perfusion chamber, a bioreactor, a flexible bed platform or by fed batch.  
     
     
         21 . The method of  claim 1  wherein the purified adenovirus composition has a purity of less than 10 nanograms of contaminating DNA per 1 milliliter dose.  
     
     
         22 . The method of  claim 3 , wherein said adenovirus comprises an adenoviral vector encoding an exogenous gene construct.  
     
     
         23 . The method of  claim 21 , wherein said gene construct is operatively linked to a promoter.  
     
     
         24 . The method of  claim 23 , wherein said promoter is SV40 IE, RSV LTR, β-actin, CMV IE, adenovirus major late, polyoma F9-1, or tyrosinase.  
     
     
         25 . The method of  claim 22 , wherein said exogenous gene construct encodes a therapeutic gene.  
     
     
         26 . The method of  claim 22 , wherein said therapeutic gene encodes antisense ras, antisense myc, antisense raf, antisense erb, antisense src, antisense fins, antisense jun, antisense trk, antisense ret, antisense gsp, antisense hst, antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, thymidine kinase or p53.  
     
     
         27 . The method of  claim 22 , wherein said therapeutic gene encodes p53.  
     
     
         28 . The method of  claim 22 , wherein said adenovirus is a replication-incompetent adenovirus.  
     
     
         29 . The method of  claim 22 , wherein the adenovirus is lacking at least a portion of the E1-region.  
     
     
         30 . The method of  claim 22 , wherein the adenovirus is lacking at least a portion of the E1A and/or E1B region.  
     
     
         31 . The method of  claim 22 , wherein said host cells are capable of complementing replication.  
     
     
         32 . The method of  claim 22  wherein the cells are perfused with a gluconse containing media at a rate to provide a glucose concentration higher than 0.5 g/L.  
     
     
         33 . The method of  claim 22  wherein the cells are perfused with a glucose containing media at a rate to provide a glucose concentration of between about 0.7 and 1.7 g/L.  
     
     
         34 . The method of  claim 1  or  3 , wherein said cell lysate is treated with Benzonas® or Pulmozym®.  
     
     
         35 . The method of  claim 1  or  3 , wherein said cells are grown as a cell suspension culture.  
     
     
         36 . The method of  claim 1  or  3 , wherein said cells are grown as an anchorage-dependent culture.  
     
     
         37 . The method of  claim 3 , wherein at least 5×10 15  viral particles are obtained from a single culture preparation.  
     
     
         38 . The method of  claim 37 , wherein at least 1×10 16  viral particles are obtained.  
     
     
         39 . A purified adenovirus composition produced according to the method of claims  1  or  3 .