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.

What is claimed is: 
     
       1. A method for obtaining a an isolated stable recombinant mammalian cell clone that produces a recombinant product and is stable under production conditions in serum- and protein-free medium for at least 40 generations, the method comprising:
 providing a recombinant original mammalian cell clone, wherein the recombinant original mammalian cell clone has a selection marker and an amplification marker, 
 cultivating the recombinant original cell clone on serum-containing medium, 
 adapting the cells to serum- and protein-free medium with neither without selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components, 
 testing the cell culture after adaptation for stable product-producers with neither without selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components, and 
 cloningisolating a stable product-producer-cell clone in serum- and protein-free conditions with neitherwithout selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components. 
 
     
     
       2. The method according to  claim 1 , wherein the stable product-producer cell clone obtained is present in isolated form after the step of cloning. 
     
     
       3. The method according to  claim 1 , wherein the recombinant cell clone comprises a nucleic acid encoding a recombinant polypeptide or protein. 
     
     
       4. The method according to  claim 1 , wherein the recombinant product is Factor VIII. 
     
     
       5. The method according to  claim 1 , wherein the recombinant product is Factor IX. 
     
     
       6. The method according to  claim 1 , wherein the recombinant product is Factor VII. 
     
     
       7. The method according to  claim 1 , wherein the recombinant product is von Willebrand factor (vWF). 
     
     
       8. The method according to  claim 1 , wherein the original mammalian cell clone is a CHO cell clone. 
     
     
       9. A method for preparing a recombinant protein, the method comprising:
 culturing the stable recombinant mammalian cell clone obtained by the method of claim 1 and expressing the recombinant protein in serum- and protein-free media so as to obtain a cell culture; and   recovering the expressed recombinant protein from the cell culture.   
     
     
       10. The method according to claim 9, wherein the recombinant protein is Factor VIII. 
     
     
       11. The method according to claim 9, wherein the recombinant protein is Factor IX. 
     
     
       12. The method according to claim 9, wherein the recombinant protein is Factor VII. 
     
     
       13. The method according to claim 9, wherein the recombinant protein is von Willebrand factor (vWF). 
     
     
       14. The method according to claim 9, wherein the original mammalian cell clone is a CHO cell clone. 
     
     
       15. The method according to claim 9, wherein the serum- and protein-free media comprises one or more amino acids selected from the group consisting of: L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine. 
     
     
       16. The method according to claim 9, wherein the serum- and protein free media comprises soybean peptone having a molecular weight of ≦1000 Dalton.