METHODS OF PURIFYING ADENO-ASSOCIATED VIRUS (AAV) AND/OR RECOMBINANT ADENO-ASSOCIATED VIRUS (rAAV) AND GRADIENTS AND FLOW-THROUGH BUFFERS THEREFORE

Abstract

A method of purifying a viral vector is provided. The method includes loading a nonionic density gradient into a continuous flow centrifuge rotor; loading a material including the viral vector and a predictable contaminant into the continuous flow centrifuge rotor; rotating the continuous flow centrifuge rotor in a manner sufficient to separate the viral vector from the predictable contaminant and the material in the nonionic density gradient; and unloading the separated viral vector from the continuous flow centrifuge rotor.

What is claimed is: 
     
         1 . A method of purifying a viral vector, comprising:
 loading a nonionic density gradient into a continuous flow centrifuge rotor;   loading a material including the viral vector and a predictable contaminant into the continuous flow centrifuge rotor while the continuous flow centrifuge rotor is rotating so as to separate the viral vector and the predictable contaminant from the material in the nonionic density gradient; and   unloading the separated viral vector and the predicable contaminant from the continuous flow centrifuge rotor.   
     
     
         2 . The method of  claim 1 , wherein the material is a lysate comprising the viral vector. 
     
     
         3 . The method of  claim 1 , wherein the nonionic density gradient comprises iodixanol in a solution with an aqueous buffer. 
     
     
         4 . The method of  claim 3 , wherein the aqueous buffer comprises phosphate-buffered saline (PBS). 
     
     
         5 . The method of  claim 1 , wherein the nonionic density gradient comprises iodixanol in a solution of between 1 and 60 percent weight per volume (% w/v) with an aqueous buffer. 
     
     
         6 . The method of  claim 1 , wherein the nonionic density gradient comprises iodixanol in a solution of between 25 and 50% w/v with an aqueous buffer. 
     
     
         7 . The method of  claim 6 , wherein the iodixanol comprises a two-layer stepped gradient comprising a 25% w/v layer and a 50% w/v layer. 
     
     
         8 . The method of  claim 1 , wherein the viral vector comprises at least one of adeno-associated virus (AAV) and recombinant adeno-associated virus (rAAV). 
     
     
         9 . The method of  claim 1 , wherein the step of loading the nonionic density gradient into the continuous flow centrifuge rotor comprises loading the nonionic density gradient while the continuous flow centrifuge rotor is static or is rotating. 
     
     
         10 . The method of  claim 1 , wherein the step of unloading the separated viral vector and the predicable contaminant from the continuous flow centrifuge rotor comprises unloading while the continuous flow centrifuge rotor is static or is rotating. 
     
     
         11 . The method of  claim 1 , further comprising orienting the nonionic density gradient prior to loading the material into the continuous flow centrifuge rotor. 
     
     
         12 . The method of  claim 1 , wherein the step of loading the nonionic density gradient into the continuous flow centrifuge rotor comprises loading a mixed gradient or a linear gradient. 
     
     
         13 . The method of  claim 1 , further comprising performing a second centrifugation step to remove the predictable contaminant from the separated viral vector. 
     
     
         14 . The method of  claim 13 , wherein the second centrifugation step comprises a continuous or non-continuous centrifugation. 
     
     
         15 . The method of  claim 14 , wherein the second centrifugation step comprises using a gradient with an ionic modifying agent sufficient to remove the predicable contaminant. 
     
     
         16 . A method of purifying a viral vector, comprising:
 loading a nonionic density gradient into a continuous flow centrifuge rotor;   loading a material including the viral vector and a predictable contaminant into the continuous flow centrifuge rotor, while the continuous flow centrifuge rotor is rotating in the presence of a modifying agent so that the viral vector is separated from the material and the predicable contaminant in the nonionic density gradient; and   unloading the separated viral vector from the continuous flow centrifuge rotor.   
     
     
         17 . The method of  claim 16 , wherein the modifying agent is present in a concentration to provide sufficient ionic potential to alter any co-purifying interaction between the predicable contaminant and the viral vector. 
     
     
         18 . The method of  claim 16 , wherein the material is a lysate comprising the viral vector. 
     
     
         19 . The method of  claim 16 , wherein the nonionic density gradient comprises the modifying agent. 
     
     
         20 . The method of  claim 16 , wherein the material comprises the modifying agent. 
     
     
         21 . The method of  claim 16 , wherein the step of rotating the continuous flow centrifuge rotor further comprises rotating while using a flow through buffer, the flow through buffer comprising the modifying agent. 
     
     
         22 . The method of  claim 16 , wherein the modifying agent further comprises an agent selected from the group consisting of caesium chloride (CsCl), potassium chloride (KCl), sodium chloride (NaCl), Percoll®, iohexol, metrizamide, Sucrose, Glycerol, Glucose, potassium Bromide (KBr), and any combinations thereof. 
     
     
         23 . The method of  claim 16 , wherein the modifying agent comprises CsCl at a concentration of up to 40% w/v. 
     
     
         24 . The method of  claim 16 , wherein the modifying agent comprises CsCl at a concentration of 20% w/v. 
     
     
         25 . The method of  claim 16 , wherein the nonionic density gradient comprises iodixanol in a solution with an aqueous buffer. 
     
     
         26 . The method of  claim 25 , wherein the aqueous buffer comprises phosphate-buffered saline (PBS). 
     
     
         27 . The method of  claim 16 , wherein the nonionic density gradient comprises iodixanol in a solution of between 1 and 60 percent weight per volume (% w/v) with an aqueous buffer. 
     
     
         28 . The method of  claim 16 , wherein the nonionic density gradient comprises iodixanol in a solution of between 25 and 50% w/v with an aqueous buffer. 
     
     
         29 . The method of  claim 28 , wherein the iodixanol comprises a two-layer stepped gradient comprising a 25% w/v layer and a 50% w/v layer. 
     
     
         30 . The method of  claim 16 , wherein the viral vector comprises at least one of adeno-associated virus (AAV) and recombinant adeno-associated virus (rAAV). 
     
     
         31 . The method of  claim 16 , wherein the step of loading the nonionic density gradient into the continuous flow centrifuge rotor comprises loading the nonionic density gradient while the continuous flow centrifuge rotor is static or is rotating. 
     
     
         32 . The method of  claim 16 , wherein the step of unloading the separated viral vector and the predicable contaminant from the continuous flow centrifuge rotor comprises unloading while the continuous flow centrifuge rotor is static or is rotating. 
     
     
         33 . The method of  claim 16 , further comprising orienting the nonionic density gradient prior to loading the material into the continuous flow centrifuge rotor. 
     
     
         34 . The method of  claim 16 , wherein the step of loading the nonionic density gradient into the continuous flow centrifuge rotor comprises loading a mixed gradient or a linear gradient.