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.

What is claimed is:  
     
         1 . A composition 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.  
     
     
         2 . The composition of  claim 1  wherein the virus is a recombinant virus.  
     
     
         3 . The composition of  claim 1  wherein the polyhydroxy hydrocarbon is glycerol.  
     
     
         4 . The composition of  claim 1  further comprising a disaccharide.  
     
     
         5 . The composition of  claim 1  wherein the composition comprises a buffer system comprising sodium phosphate monobasic dihydrate in a concentration of about 0.5 to 10 mg/mL and tromethamine in a concentration of about 0.5 to 10 mg/mL.  
     
     
         6 . The composition of  claim 1  further comprising a divalent metal salt in a concentration of about 0.1 to 1 mg/mL.  
     
     
         7 . The composition of  claim 1  which further comprises a diluent comprising water.  
     
     
         8 . The composition of  claim 1  wherein the virus is present in a concentration of about 1×10 9  to 1×10 13  particles/mL.  
     
     
         9 . The composition of  claim 1  wherein the virus is adenovirus.  
     
     
         10 . The composition of  claim 2  wherein the recombinant virus comprises a wild-type p53 gene.  
     
     
         11 . The composition of  claim 10  wherein the recombinant virus is A/C/N153.  
     
     
         12 . The composition of  claim 1  wherein the virus is adenovirus; the polyhydroxy hydrocarbon is glycerol; the buffer system maintains the pH in a range from about 7.3 to about 7.9 at a temperature ranging from 20° C. to 27° C.; and the composition further comprises a disaccharide.  
     
     
         13 . The composition of  claim 12  wherein the adenovirus is A/C/N/53; the disaccharide is sucrose; the buffer system comprises sodium phosphate monobasic dihydrate and tromethamine; and the composition further comprises a divalent metal salt and water.  
     
     
         14 . A method for concentrating a virus preparation comprising: 
 (a) adding a polyhydroxy hydrocarbon to a virus preparation to a final polyhydroxy hydrocarbon concentration of about 20% or more; and    (b) subjecting the virus preparation to a filtration process wherein the concentration of virus is increased by applying pressure to the preparation such that diluent is removed from the virus preparation through a filter while the virus is retained.    
     
     
         15 . The method of  claim 14  wherein the filtration process comprises ultrafiltration.  
     
     
         16 . The method of  claim 14  wherein the filtration process comprises tangential flow filtration.  
     
     
         17 . A method of purifying a virus preparation comprising: 
 (a) subjecting the virus preparation to anion-exchange chromatography, wherein the virus is eluted as a virus preparation product from an anion-exchange chromatographic medium;    (b) adding a polyhydroxy hydrocarbon to the virus preparation product of step (a) so that the concentration of polyhydroxy hydrocarbon in the preparation reaches a level of about 25% or more; and    (c) increasing the concentration of virus in the virus preparation product of step (b) by applying pressure to the preparation such that diluent is removed from the virus preparation through a filter while the virus is retained; and    (d) subjecting the concentrated virus preparation product of step (c) to one or more additional processing steps.    
     
     
         18 . The method of  claim 14  wherein the virus is a recombinant virus.  
     
     
         19 . The method of  claim 14  wherein the virus is a recombinant virus carrying a therapeutic transgene for use in gene therapy.  
     
     
         20 . The method of  claim 17  wherein an additional processing step comprises size exclusion chromatography.  
     
     
         21 . The method of  claim 17  wherein the process of step (c) comprises tangential flow filtration.  
     
     
         22 . The method of  claim 21  wherein the process of step (c) is carried out using apparatus comprising a Pellicon XL filtration system.  
     
     
         23 . A virus preparation concentrated by the method of  claim 14 .  
     
     
         24 . A virus preparation purified by the method of  claim 17 .  
     
     
         25 . A method for concentrating a virus preparation comprising: 
 (a) centrifuging a composition which comprises a first layer comprising a polyhydroxy hydrocarbon in a concentration of 35% to 80% (v/v), the first layer overlaid with a second layer comprising a polyhydroxy hydrocarbon in a concentration of 5% to 30% (v/v), the second layer overlaid with a third layer comprising virus; and    (b) recovering the virus from the first layer.    
     
     
         26 . The composition of  claim 1 , wherein the composition is subjected to an additional processing step of agitation, followed by microfiltration, for the prevention of particulate formation during storage of the composition.  
     
     
         27 . The composition of  claim 1 , wherein the composition is subjected to one or more freeze/thaw cycles, followed by agitation, and then microfiltration, for the prevention of particulate formation during storage of the composition.  
     
     
         28 . The composition of  claim 12  further comprising a monovalent metal salt in a concentration of about 0.6 to 10.0 mg/mL.