Compositions comprising viruses and methods for concentrating virus preparations

Abstract

A composition is disclosed comprising virus in a formulation comprising a polyhydroxy hydrocarbon buffered to maintain a pH in a range from about 7 to about 8.5 at a temperature in the range from about 2° C. to 27° C. Methods for concentrating and purifying virus preparations are also disclosed.

1 - 28 . (canceled) 
     
     
         29 . A purified adenovirus composition obtained by a method of purifying a virus preparation, the method comprising the steps of:
 (a) subjecting the virus preparation to anion-exchange chromatography, wherein the adenovirus is eluted from an anion-exchange chromatographic medium; and   (b) subjecting the elute from step (a) containing the adenovirus to size exclusion chromatography, wherein the adenovirus is eluted from a size exclusion chromatographic medium.   
     
     
         30 . The adenovirus composition of  claim 29 , wherein the virus preparation is a cell lysate. 
     
     
         31 . The adenovirus composition of  claim 30 , wherein the cell lysate is filtered before step (a). 
     
     
         32 . The adenovirus composition of  claim 30 , wherein the cell lysate is subject to nuclease treatment before step (a). 
     
     
         33 . The adenovirus composition of  claim 29 , wherein the adenovirus is recombinant. 
     
     
         34 . The adenovirus composition of  claim 29 , wherein the anion-exchange medium is DEAE-FRACTOGEL. 
     
     
         35 . The adenovirus composition of  claim 29 , wherein the size exclusion medium is SUPERDEX-200. 
     
     
         36 . The adenovirus composition of  claim 29 , wherein the anion-exchange medium comprises diethylaminoethyl groups on a cross-linked agarose, cellulose, polyacrylamide or polystyrene backbone. 
     
     
         37 . The adenovirus composition of  claim 29 , wherein the size-exclusion medium comprises a cross-linked polysaccharide. 
     
     
         38 . The adenovirus composition of  claim 37 , wherein the cross-linked polysaccharide is a composite of cross-linked agarose and dextran. 
     
     
         39 . The adenovirus composition of  claim 29 , wherein the anion-exchange chromatographic medium is extensively washed before step (a). 
     
     
         40 . The adenovirus composition of  claim 29 , wherein step (b) further comprises eluting the adenovirus from the size-exclusion chromatographic medium into a low-salt buffer by a high-salt elution buffer, wherein the size-exclusion medium is in a column containing a salt gradient which decreases in ionic strength from the top of the column towards the bottom of the column. 
     
     
         41 . The adenovirus composition of  claim 29 , wherein the anion-exchange chromatographic medium or the size exclusion chromatographic medium is contacted with a buffer comprising glycerol, including wash, equilibration, loading and elution buffer. 
     
     
         42 . The adenovirus composition of  claim 29 , wherein step (a) is performed by:
 (i) pre-equilibrating a DEAE-EMD FRACTOGEL 650M column with 5 bed volume (BV) of 0.5 M NaOH/1M NaCl, by 6 BV of 0.1 M HCl/1 M NaCl, and 20 BV of Buffer A, wherein Buffer A is a solution consisting of 265 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 50 mM sodium phosphate at pH 7.5;   (ii) loading the virus preparation to the column and subsequently washing the column with 4 BV of Buffer A, wherein the virus preparation prior to loading has a conductivity equal to that of Buffer A;   (iii) washing the column with 8 BV of 94% Buffer A/6% Buffer B, wherein Buffer B is a solution consisting of 600 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 50 mM sodium phosphate at pH 7.5; and   (iv) eluting the adenovirus with 30 BV of a linear gradient from 94% Buffer A/6% Buffer B to 100% Buffer B.   
     
     
         43 . The adenovirus composition of  claim 29 , wherein step (b) is performed by:
 (i) pre-equilibrating a SUPERDEX-200 column with 0.5 bed volume (BV) of 0.5 M NaOH, 1 BV of H 2 O, and 2 BV of Buffer C, wherein Buffer C is a solution consisting of 130 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 50 mM sodium phosphate at pH 7.5;   (ii) loading the elute from step (a) to the column; and   (iii) eluting the adenovirus with Buffer C.   
     
     
         44 . The adenovirus composition of  claim 29 , wherein step (b) is performed by:
 (i) pre-equilibrating a SUPERDEX-200 column with 0.5 bed volume (BV) of 0.5 M NaOH, 1 BV of H 2 O, and 2 BV of Buffer C, wherein Buffer C is a solution consisting of 130 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 20 mM sodium phosphate at pH 8.0;   (ii) loading to the column 0.15 BV of a linear gradient from 100% Buffer C to 100% Buffer D, wherein Buffer D is a solution consisting of 420 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 20 mM sodium phosphate at pH 8.0;   (iii) loading the elute from step (a) to the column; and   (iv) eluting the adenovirus with Buffer D.   
     
     
         45 . The adenovirus composition of  claim 29 , wherein step (a) is performed by:
 (i) pre-equilibrating a FRACTOGEL EMD DEAE-650M column with Buffer J-1, wherein Buffer J-1 is a solution consisting of 265 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 50 mM sodium phosphate at pH 7.5;   (ii) loading the virus preparation to the column;   (iii) washing the column with 8 BV of 94% Buffer J-1/6% Buffer J-2, wherein Buffer J-2 is a solution consisting of 600 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 50 mM sodium phosphate at pH 7.5; and   (iv) eluting the adenovirus with 30 BV of a linear gradient from 94% Buffer J-1/6% Buffer J-2 to 100% Buffer J-2.   
     
     
         46 . The adenovirus composition of  claim 29 , wherein step (b) is performed by:
 (i) pre-equilibrating a SUPERDEX-200 column with Buffer K-1, wherein Buffer K−1 is a solution consisting of 100 mM NaCl, 2 mM MgCl 2 , 2% (w/v) sucrose, and 20 mM sodium phosphate at pH 8.0;   (ii) loading the elute from step (a) to the column; and   (iii) eluting the adenovirus with Buffer K-1.   
     
     
         47 . The adenovirus composition of  claim 46 , wherein the elute from step (iii) is subsequently filtered through a 0.2 μm hydrophilic DURAPORE membrane at 2-15° C.