Production of poliovirus at high titers for vaccine production

Abstract

Described is a process for producing poliovirus, the process comprising: a) providing a serum-free suspension culture of cells, which are primary human retina cells that have been immortalized by expression of adenovirus E1 sequences, b) infecting the cells with poliovirus, at a cell density of between 2×10 6 cells/ml and 150×10 6 cells/ml, and c) harvesting poliovirus at a time of between 12 and 48 hours after infection.

What is claimed is: 
     
         1 . A process for producing poliovirus, the process comprising the steps of:
 a) providing a serum-free suspension culture of cells, which are primary human retina (HER) cells that have been immortalized by expression of adenovirus E1 sequences,   b) infecting the cells with poliovirus, at a cell density of between 2×10 6  cells/ml and 150×10 6  cells/ml, and   c) harvesting poliovirus at a time of between 12 and 48 hours after infection.   
     
     
         2 . The process of  claim 1 , wherein the HER cells are PER.C6® cells. 
     
     
         3 . The process of  claim 1 , wherein the infecting and/or virus propagation is performed at a temperature of between 34° C. and 36° C. 
     
     
         4 . The process of  claim 1 , wherein the infecting is performed at a cell density of between 5×10 6  cells/ml and 20×10 6  cells/ml. 
     
     
         5 . The process of  claim 1 , wherein the infecting is performed at a multiplicity of infection (MOI) of between one (1) and three (3). 
     
     
         6 . The process of  claim 1 , wherein the harvesting poliovirus is performed at a time of between 18 and 30 hours after infection. 
     
     
         7 . The process of  claim 1 , wherein the poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3. 
     
     
         8 . The process of  claim 7 , wherein the poliovirus is poliovirus type 1 strain Mahoney, poliovirus type 2 strain MEF, or poliovirus type 3 strain Saukett. 
     
     
         9 . The process of  claim 7 , wherein the poliovirus is an attenuated poliovirus. 
     
     
         10 . A process for producing a polio vaccine, comprising a process according to  claim 1 , further comprising purifying, optionally inactivating, and formulating the harvested poliovirus to obtain a polio vaccine. 
     
     
         11 . A poliovirus bulk useful for preparation of a polio vaccine, the poliovirus bulk being produced by the process of  claim 1  and comprising culture medium and poliovirus at a titer of at least 10 9.4  TCID50/ml. 
     
     
         12 . A polio vaccine free of non-human protein, produced by the process of  claim 10 . 
     
     
         13 . A process for obtaining a poliovirus preparation in cell culture at a titer of at least about 10 9.4  CCID 50 /ml, the process comprising the steps of:
 a) providing a serum-free suspension culture of cells, which are human fetal retina (HER) cells that have been immortalized by expression of adenovirus E1 sequences,   b) infecting the cells with poliovirus, at a cell density of between 2×10 6  cells/ml and 150×10 6  cells/ml, and   c) harvesting poliovirus at a time of between 12 and 48 hours after infection to obtain the poliovirus preparation having a titer of at least about 10 9.4  CCID 50 /ml.   
     
     
         14 . The process of  claim 2 , wherein the infecting and/or virus propagation is performed at a temperature of between 34° C. and 36° C. 
     
     
         15 . The process of  claim 2 , wherein the infecting is performed at a cell density of between 5×10 6  cells/ml and 20×10 6  cells/ml. 
     
     
         16 . The process of  claim 1 , wherein infecting is performed at a cell density of around 10×10 6  cells/ml. 
     
     
         17 . The process of  claim 2 , wherein infecting is performed at a cell density of around 10×10 6  cells/ml. 
     
     
         18 . The process of  claim 1 , wherein infecting is performed at a multiplicity of infection (MOI) of around 2. 
     
     
         19 . The process of  claim 2 , wherein infecting is performed at a multiplicity of infection (MOI) of between 1 and 3. 
     
     
         20 . The process of  claim 2 , wherein harvesting poliovirus is performed at a time of between 18 and 30 hours after infection. 
     
     
         21 . The process of  claim 4 , wherein harvesting poliovirus is performed at a time of between 18 and 30 hours after infection. 
     
     
         22 . The process of  claim 5 , wherein harvesting poliovirus is performed at a time of between 18 and 30 hours after infection. 
     
     
         23 . The process of  claim 1 , wherein harvesting poliovirus is performed at a time of around 24 hours after infection. 
     
     
         24 . The process of  claim 2 , wherein harvesting poliovirus is performed at a time of around 24 hours after infection. 
     
     
         25 . The process of  claim 4 , wherein harvesting poliovirus is performed at a time of around 24 hours after infection. 
     
     
         26 . The process of  claim 2 , wherein poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3. 
     
     
         27 . The process of  claim 3 , wherein poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3. 
     
     
         28 . The process of  claim 4 , wherein poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3. 
     
     
         29 . The process of  claim 5 , wherein poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3. 
     
     
         30 . The process of  claim 2 , wherein poliovirus is poliovirus type 1 strain Mahoney, poliovirus type 2 strain MEF, or poliovirus type 3 strain Saukett. 
     
     
         31 . The process of  claim 3 , wherein the poliovirus is poliovirus type 1 strain Mahoney, poliovirus type 2 strain MEF, or poliovirus type 3 strain Saukett. 
     
     
         32 . The process of  claim 4 , wherein the poliovirus is poliovirus type 1 strain Mahoney, poliovirus type 2 strain MEF, or poliovirus type 3 strain Saukett. 
     
     
         33 . The process of  claim 5 , wherein the poliovirus is poliovirus type 1 strain Mahoney, poliovirus type 2 strain MEF, or poliovirus type 3 strain Saukett. 
     
     
         34 . The process of  claim 2 , wherein the poliovirus is an attenuated poliovirus. 
     
     
         35 . The process of  claim 4 , wherein the poliovirus is an attenuated poliovirus. 
     
     
         36 . The process of  claim 1 , wherein the poliovirus is a Sabin strain. 
     
     
         37 . The process of  claim 2 , wherein the poliovirus is a Sabin strain. 
     
     
         38 . The process of  claim 10 , wherein infecting and/or virus propagation is performed at a temperature of between 34° C. and 36° C. 
     
     
         39 . The process of  claim 10 , wherein infecting is performed at a cell density of between 5×10 6  cells/ml and 20×10 6  cells/ml. 
     
     
         40 . The process of  claim 10 , wherein infecting is performed at a multiplicity of infection (MOI) of between 1 and 3. 
     
     
         41 . The process of  claim 10 , wherein harvesting poliovirus is performed at a time of between 18 and 30 hours after infection. 
     
     
         42 . The process of  claim 10 , wherein poliovirus is poliovirus type 1, poliovirus type 2 or poliovirus type 3. 
     
     
         43 . The process of  claim 10 , wherein the poliovirus is an attenuated poliovirus. 
     
     
         44 . The process of  claim 13 , wherein the poliovirus preparation has a titer of at least about 10 9.8  CCID 50 /ml. 
     
     
         45 . The process of  claim 13 , wherein the poliovirus preparation has a titer of at least about 10 10  CCID 50 /ml.