Recombinant cell clones having increased stability and methods of making and using the same

Abstract

Disclosed are a stable recombinant cell clones which are stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.

1 . A method for producing a stable recombinant cell clone, which is stable under production conditions in serum- and protein-free medium for at least 40 generations, the method comprising: 
 providing a recombinant original cell clone,    cultivating the recombinant original cell clone on serum-containing medium,    adapting the cells to serum- and protein-free medium in the absence of a selection pressure,    testing the cell culture after adaptation for stable product-producers in the absence of a selection pressure and    cloning a stable product-producer-cell clone in serum- and protein-free conditions in the absence of a selection pressure.    
     
     
         2 . The method according to  claim 1 , wherein the stable cell clone obtained is present in isolated form after the step of cloning.  
     
     
         3 . The method according to  claim 1 , wherein the recombinant original cell clone is a mammalian cell.  
     
     
         4 . The method according to  claim 3 , wherein the mammal cell is selected from the group consisting of: a recombinant CHO cell and a recombinant BHK cell.  
     
     
         5 . The method according to  claim 1 , wherein the recombinant cell clone comprises a nucleic acid encoding a recombinant polypeptide or protein.  
     
     
         6 . The method according to  claim 5 , wherein the recombinant protein is a blood factor selected from the group consisting of: Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C or an activated form of one of the factors, and vWF, and a combination thereof.  
     
     
         7 . The method according to  claim 1 , wherein the original recombinant cell clone is a CHO cell expressing a recombinant polypeptide selected from the group consisting of: von Willebrand factor, Factor VIII, Factor IX, Factor II, and a combination thereof.  
     
     
         8 . A stable recombinant cell clone, obtainable by a method according to of  claim 1 , wherein the cell clone is stable for at least 40 generations and expresses a recombinant product under production conditions in serum- and protein-free medium, in the absence of a selection pressure.  
     
     
         9 . The cell clone according to  claim 8 , wherein the clone is present in isolated form after the step of cloning.  
     
     
         10 . The cell clone according to  claim 8 , wherein the original recombinant cell clone is a recombinant mammal cell.  
     
     
         11 . The cell clone according to  claim 10 , wherein the mammal cell is selected from the group consisting of: a recombinant CHO cell and a recombinant BHK cell.  
     
     
         12 . The cell clone according to  claim 8 , wherein the cell comprises a nucleic acid encoding a recombinant polypeptide or protein.  
     
     
         13 . The cell clone according to  claim 12 , wherein the recombinant protein is a blood factor selected from the group consisting of: Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C or an activated form of one of the factors, vWF, and a combination thereof.  
     
     
         14 . The cell clone according to  claim 12 , wherein the recombinant protein is Factor VIII.  
     
     
         15 . The cell clone according to  claim 12 , wherein the cell clone is a recombinant CHO cell.  
     
     
         16 . The cell clone according to  claim 15 , wherein the cell clone expresses a recombinant polypeptide selected from the group consisting of: von Willebrand factor, Factor VIII, Factor IX, Factor II, and a combination thereof.  
     
     
         17 . The cell clone according to  claim 15 , wherein the cell clone co-expresses Factor VIII and vWF.  
     
     
         18 . A cell culture obtainable by cultivating a stable recombinant cell clone according to  claim 12 .  
     
     
         19 . The cell culture of  claim 18 , wherein the cell culture contains at least 90% stable recombinant cell clones.  
     
     
         20 . The cell culture according to  claim 19 , wherein the stable recombinant cell clones are mammalian cells.  
     
     
         21 . The cell culture according to  claim 20 , wherein the mammalian cells are recombinant CHO cells.  
     
     
         22 . The cell culture according to  claim 21 , wherein the CHO cells are selected from the group consisting of: CHO-DHFR cells and CHO-K1 cells.  
     
     
         23 . The cell culture according to  claim 18 , wherein the stable recombinant cells comprise a nucleic acid encoding a recombinant polypeptide or protein.  
     
     
         24 . The cell culture according to  claim 18 , wherein the stable recombinant cells are immobilised on a microsupport.  
     
     
         25 . A method for industrial production of a recombinant product under serum- and protein-free conditions, the method comprising: 
 providing an isolated, stable recombinant cell clone according to  claim 8 ,    propagating the stable cell clone in serum- and protein-free medium from the initial clone up to the cell culture in the absence of a selection pressure,    producing the cell culture containing stable cells in the bioreactor in the absence of a selection pressure, and    harvesting the recombinant product from the culture supernatant.    
     
     
         26 . The method according to  claim 25 , wherein the serum- and protein-free medium is a synthetic minimal medium comprising an extract selected from the group consisting of: a yeast extract and a soy extract.  
     
     
         27 . The method according to  claim 25 , wherein the medium contains cyclodextrin or a derivative thereof.  
     
     
         28 . The method according to  claim 25 , wherein the serum- and protein-free medium contains a protease inhibitor.  
     
     
         29 . The method according to  claim 25 , wherein the recombinant product is a protein.