Virus Purification Methods

Abstract

The invention provides a method for the purification of a virus from a host cell, the method comprising the steps of: a) culturing host cells, b) infecting the host cells with a virus, c) treating the cell culture with nuclease, d) lysing the host cells to provide a lysate comprising the virus. The virus is preferably a recombinant adenovirus. The invention further provides a method for the purification of a recombinant virus expressing a heterologous protein that is capable of binding nucleic acid, comprising the steps of: a) culturing host cells, b) infecting the host cells with the recombinant virus, c) lysing the host cells to provide a lysate comprising the recombinant virus, d) subjecting the recombinant virus to anion exchange chromatography and size exclusion chromatography, characterized in that the virus-containing mixture is buffer exchanged at least once with a solution comprising at least 2 M NaCl, or another salt providing an equivalent ionic strength.

1 . A method for the purification of a virus from a host cell, said method comprising in the given order the steps of: 
 a) culturing host cells that are infected with a virus,    b) adding nuclease to the cell culture, and    c) lysing said host cells to provide a lysate comprising the virus.    
     
     
         2 . The method according to  claim 1 , said method further comprising: 
 d) clarification of the lysate.    
     
     
         3 . The method according to  claim 1 , said method further comprising: 
 e) purifying the virus with at least one chromatography step.    
     
     
         4 . The method according to  claim 1 , wherein said virus is a recombinant adenovirus.  
     
     
         5 . The method A method according to  claim 1 , wherein the nuclease of step b) is BENZONASE®.  
     
     
         6 . The method according to  claim 1 , wherein step c) of lysing the host cells is performed with a detergent.  
     
     
         7 . The method according to  claim 6 , wherein the detergent is TRITON® X-100.  
     
     
         8 . The method according to  claim 2 , wherein step d) comprises depth filtration and membrane filtration.  
     
     
         9 . The method according to  claim 8 , wherein the membrane filtration is performed using a combination of 0.8 μm and 0.45 μm filters.  
     
     
         10 . The method according to  claim 3 , wherein prior to step e) the clarified lysate is subjected to ultrafiltration and/or diafiltration.  
     
     
         11 . The method according to  claim 10 , wherein the clarified lysate that is subjected to diafiltration is exchanged against a solution comprising 0.8-2.0 M NaCl, preferably about 1 M NaCl, or another salt providing an equivalent ionic strength.  
     
     
         12 . The method according to  claim 3 , wherein step e) comprises anion exchange chromatography.  
     
     
         13 . The method according to  claim 12 , wherein said anion exchange chromatography is performed using a charged filter comprising anion exchange groups.  
     
     
         14 . The method according to  claim 3 , wherein step e) comprises size exclusion chromatography.  
     
     
         15 . The method according to  claim 3 , wherein step e) comprises: 
 e,i) anion exchange chromatography, and    e,ii) size exclusion chromatography.    
     
     
         16 . The method according to  claim 15 , wherein the mixture containing the recombinant adenovirus is buffer exchanged with a solution comprising at least 2 M NaCl, or another salt providing an equivalent ionic strength, between said steps of anion exchange chromatography and size exclusion chromatography.  
     
     
         17 . The method according to  claim 2 , wherein buffers used in steps d) and subsequent steps are free of detergent, magnesium_chloride and sucrose.  
     
     
         18 . A method for the purification of a virus that is able to lyse host cells, said method comprising the steps of: 
 a) culturing host cells comprising said virus able to lyse host cells,    b) harvesting virus following their release into culture fluid without addition of an external lysis factor, characterized in that a nuclease is added to the culture before 95% of the host cells has been lysed.    
     
     
         19 . A method for the production of a virus comprising a nucleic acid sequence coding for a nucleoprotein of a hemorrhagic fever virus, comprising the steps of: 
 a) culturing host cells that have been infected with said virus,    b) subjecting said culture of host cells comprising said virus to lysis of the host cells to provide a lysate comprising said virus,    c) subjecting the virus to anion exchange chromatography, characterized in that after anion exchange chromatography the virus containing mixture is buffer exchanged with a solution comprising at least 1 M NaCl, or another salt providing an equivalent ionic strength and/or with a solution comprising at least 1% of a detergent.    
     
     
         20 . The method according to  claim 19 , wherein the virus containing mixture is buffer exchanged at least once with a solution comprising at least 1 M NaCl, or another salt providing an equivalent ionic strength.  
     
     
         21 . The method according to  claim 19 , wherein said virus is a recombinant adenovirus.  
     
     
         22 . The method according to  claim 19 , wherein said hemorrhagic fever virus is Ebola_virus.  
     
     
         23 . The method according to  claim 20 , wherein said solution comprises at least 1.5 M NaCl, or another salt providing an equivalent ionic strength.  
     
     
         24 . The method according to  claim 23 , wherein said solution comprises at least 2 M NaCl, or another salt providing an equivalent ionic strength.  
     
     
         25 . The method according to  claim 24 , wherein said solution comprises at least 3 M NaCl, or another salt providing an equivalent ionic strength.  
     
     
         26 . The method according to  claim 25 , wherein said solution comprises about 5 M NaCl, or another salt providing an equivalent ionic strength.  
     
     
         27 . The method according to  claim 27 , further comprising filtering the virus containing mixture that is buffer exchanged through a hydrophilic filter with a pore size of 1.2 μm or less.  
     
     
         28 . The method according to  claim 27 , wherein said pore size is about 0.45 μm or about 0.22 μm.  
     
     
         29 . The method according to  claim 19 , further comprising subjecting the virus containing mixture that is buffer exchanged to size exclusion chromatography.  
     
     
         30 . A method for removing free adenovirus proteins from a recombinant adenovirus preparation, comprising the step of: subjecting a recombinant adenovirus preparation comprising free adenovirus proteins to a charged filter that contains anion exchange groups.  
     
     
         31 . The method according to  claim 30 , wherein said recombinant adenovirus preparation comprises a subgroup B recombinant adenovirus.  
     
     
         32 . The method according to  claim 30 , wherein said recombinant adenovirus is an Ad35 recombinant adenovirus.  
     
     
         33 . The method according to  claim 2 , said method further comprising: 
 e) further purifying the virus with at least one chromatography step.    
     
     
         34 . The method according to  claim 3 , wherein any buffers used in step e) and subsequent steps are free of detergent, magnesium chloride and sucrose.