Formulation of sugar solutions for continuous ultracentrifugation for virus purification

Abstract

The present invention provides a method for purification of a virus or virus antigen comprising providing a virus preparation and centrifugation of said virus preparation in a gradient of a sugar established by the addition of two or more buffered sugar layers of different concentration. The method leads to higher yields and reduces unwanted aggregation of the virus or virus antigen by increasing the volume of the peak pool.

1 - 34 . (canceled) 
     
     
         35 . A method for purification of a virus or a virus antigen, comprising
 providing a solution containing a sugar gradient by centrifuging in an ultracentrifuge at least
 (i) a first buffered sugar solution comprising a first physiological buffer and 
 (ii) a second buffered sugar solution having a higher density than the first buffered sugar solution and comprising a second physiological buffer, which is the same or different from the first physiological buffer, 
 wherein the concentration of sugar in the first buffered sugar solution has a sucrose equivalent between 35% to 50% (w/w %) and the concentration of sugar in the second buffered sugar solution has a sucrose equivalent between 50% to 65% (w/w %); 
   adding a virus preparation to the sugar gradient and centrifuging in an ultracentrifuge comprising an ultracentrifuge rotor to obtain a peak pool, and   extracting the peak pool to obtain the virus or viral antigen.   
     
     
         36 . The method of  claim 35 , wherein the peak pool is extracted with a sucrose equivalent between 30% and 54% (w/w %) sucrose. 
     
     
         37 . The method of  claim 35 , wherein at least one buffer has a concentration of between 5 mM to 50 mM. 
     
     
         38 . The method of  claim 35 , wherein the step of centrifuging the virus preparation and buffered sugar solutions is performed with a relative centrifugation of at least 20,000 g. 
     
     
         39 . The method of  claim 35 , wherein the virus preparation is obtained by centrifugation without a preclarifier. 
     
     
         40 . The method of  claim 35 , wherein said first buffered sugar solution comprises a sugar in a concentration range from 40% to 50% (w/w %) sucrose equivalent. 
     
     
         41 . The method of  claim 35 , wherein said second buffered sugar solution comprises a sugar in a concentration range from 52% to 58% (w/w %) sucrose equivalent. 
     
     
         42 . The method of  claim 35 , wherein said virus is an adenovirus. 
     
     
         43 . The method of  claim 35 , wherein said virus preparation comprises cells inoculated with the virus. 
     
     
         44 . The method of  claim 43 , wherein said cells are of an animal cell culture or cell line. 
     
     
         45 . The method of  claim 43 , wherein said cells are epithelial cells. 
     
     
         46 . The method of  claim 35 , wherein at least one of the physiological buffers is an amine buffer. 
     
     
         47 . The method of  claim 46 , wherein at least one of the physiological buffers is a trishydroxymethylaminomethane (TRIS) buffer. 
     
     
         48 . The method of  claim 35 , wherein the provided solution containing a sugar gradient further comprises
 (iii) a third buffered sugar solution having a higher density than the first buffered sugar solution.   
     
     
         49 . A method of preparing a vaccine against a virus or viral antigen, wherein said virus or viral antigen is obtained using the method according to  claim 35 . 
     
     
         50 . A method for purification of a virus or virus antigen comprising
 providing a solution containing a sugar gradient by centrifuging in an ultracentrifuge at least
 (i) a first buffered sugar solution having a density of 1.15 kg/l to 1.23 kg/l and 
 (ii) a second buffered sugar solution having a density of 1.23 kg/l to 1.32 kg/l and which is higher than the density of the first buffered sugar solution, 
   adding a virus or virus antigen preparation to the sugar gradient and centrifuging in an ultracentrifuge comprising an ultracentrifugation rotor to obtain a peak pool, and extracting the peak pool to obtain the virus or virus antigen.   
     
     
         51 . The method of  claim 50 , wherein the peak pool has a density between 1.13 kg/l to 1.25 kg/l. 
     
     
         52 . The method of  claim 50 , wherein said volume ratio of said first buffered sugar solution to said second buffered sugar solution is less than 20:1. 
     
     
         53 . The method of  claim 50 , wherein said virus is an adenovirus. 
     
     
         54 . The method of  claim 50 , wherein the provided solution containing a sugar gradient further comprises
 (iii) a third buffered sugar solution having a higher density than the first buffered sugar solution.