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. A stable recombinant mammalian cell clone, obtainable by a method comprising:
providing a recombinant original individual mammalian cell clone, wherein the original individual mammalian cell clone has a selection marker,
cultivating the recombinant original cell clone on serum-containing medium,
adapting the cells to serum- and protein-free medium with neither 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 selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components, and
cloning a stable product-producer-cell clone in serum- and protein-free conditions with neither selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components;
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, with neither selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components.
2. The stable recombinant mammalian cell clone according to claim 1 , wherein the clone is present in isolated form after the step of cloning.
3. The stable recombinant mammalian cell clone according to claim 1 , wherein the cell clone comprises a nucleic acid encoding a recombinant polypeptide or protein.
4. A cell culture obtainable by cultivating a stable recombinant cell clone of claim 1 .
5. The cell culture of claim 4 , wherein the cell culture contains at least 90% stable recombinant cell clones.
6. The cell culture according to claim 4 , wherein the stable recombinant cells comprise a nucleic acid encoding a recombinant polypeptide or protein.
7. The cell culture according to claim 4 , wherein the stable recombinant cells are immobilised on a microcarrier.
8. The stable recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses Factor VIII as the recombinant product.
9. The stable recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses Factor IX as the recombinant product.
10. The stable recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses Factor VII as the recombinant product.
11. The stable recombinant mammalian cell clone of claim 1 , wherein the recombinant cell clone expresses von Willebrand factor (vWF) as the recombinant product.
12. The cell culture of claim 4 , wherein the stable recombinant cell clone expresses Factor VIII as the recombinant product.
13. The cell culture of claim 4 , wherein the stable recombinant cell clone expresses Factor IX as the recombinant product.
14. The cell culture of claim 4 , wherein the stable recombinant cell clone expresses Factor VII as the recombinant product.
15. The cell culture of claim 4 , wherein the stable recombinant cell clone expresses vWF as the recombinant product.