Recombinant cell clones having increased stability and methods of making and using the same
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. An isolated recombinant mammalian cell clone stable in serum- and protein-free medium and expressing a recombinant product, wherein said cell clone is stable in serum- and protein-free medium for at least 40 generations with neither selection pressure for a selection marker nor selection for a polypeptide factor that replaces serum components.
2. An isolated recombinant mammalian cell clone as set forth in claim 1 , said cell clone being stable for at least 50 generations.
3. A cell culture comprising at least 90% of stable recombinant mammalian cells expressing a recombinant product, said stable recombinant cells stable for more than 40 generations under serum- and protein-free conditions with neither selection pressure for a selection marker nor selection for a polypeptide factor that replaces serum components.
4. A cell culture as set forth in claim 3 , wherein said stable recombinant cells are stable for more than 50 generations under serum- and protein-free conditions.
5. A cell culture as set forth in claim 3 , wherein said stable recombinant cells contain a sequence encoding for a recombinant polypeptide or recombinant protein.
6. An isolated recombinant CHO cell clone selected with neither selective pressure for a selection marker nor selection for a polypeptide factor that replaces serum components, said cell clone stable in serum- and protein-free medium for at least 40 generations and expressing a recombinant product.
7. An isolated recombinant CHO cell clone as set forth in claim 6 , said cell clone being stable for at least 50 generations.
8. An isolated recombinant CHO cell clone as set forth in claim 6 , wherein said cell clone contains sequences encoding a recombinant polypeptide or said recombinant protein.
9. An isolated recombinant CHO cell clone which has been cultivated in serum- and protein-free medium with neither selective pressure for a selection marker nor selection for a polypeptide factor that replaces serum components for at least 40 generations and which expresses a recombinant product.
10. An isolated recombinant CHO cell clone of claim 9 , wherein the cell clone has been cultivated for at least 50 generations.
11. An isolated recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses Factor VIII as the recombinant product.
12. An isolated recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses Factor IX as the recombinant product.
13. An isolated recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses Factor VII as the recombinant product.
14. An isolated recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses von Willebrand factor (vWF) as the recombinant product.
15. A cell culture as set forth in claim 3 , wherein said stable recombinant cells express Factor VIII as the recombinant product.
16. A cell culture as set forth in claim 3 , wherein said stable recombinant cells express Factor IX as the recombinant product.
17. A cell culture as set forth in claim 3 , wherein said stable recombinant cells express Factor VII as the recombinant product.
18. A cell culture as set forth in claim 3 , wherein said stable recombinant cells express vWF as the recombinant product.
19. An isolated recombinant CHO cell clone of claim 9 , wherein the cell clone expresses Factor VIII as the recombinant product.
20. An isolated recombinant CHO cell clone of claim 9 , wherein the cell clone expresses Factor IX as the recombinant product.
21. An isolated recombinant CHO cell clone of claim 9 , wherein the cell clone expresses Factor VII as the recombinant product.
22. An isolated recombinant CHO cell clone of claim 9 , wherein the cell clone expresses vWF as the recombinant product.