Recombinant cell clones having increased stability and methods of making and using the same
View Patent ↗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 obtaining a 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,
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.
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 stable product-producer-cell clone that produces a recombinant product obtained by 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 to create a cell culture,
adapting the cell culture to serum- and protein-free medium with neither selection pressure for the selection marker nor selection pressure for the amplification marker,
testing the cell culture after adaptation for stable product-producers with neither selection pressure for the selection marker nor selection pressure for the amplification marker, and
isolating a stable product-producer-cell clone in serum- and protein-free conditions with neither selection pressure for the selection marker nor selection pressure for the amplification marker.
10. The stable product-producer-cell clone according to claim 9, wherein the recombinant product is Factor VIII.
11. The stable product-producer-cell clone according to claim 9, wherein the recombinant product is Factor IX.
12. The stable product-producer-cell clone according to claim 9, wherein the recombinant product is Factor VII.
13. The stable product-producer-cell clone according to claim 9, wherein the recombinant product is von Willebrand factor (vWF).
14. The stable product-producer-cell clone according to claim 9, wherein the stable recombinant mammalian cell clone is a CHO cell clone.
15. A cell culture comprising a stable product-producer-cell clone that produces a recombinant product, wherein the cell clone is obtained by 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 to create a cell culture,
adapting the cell culture to serum- and protein-free medium with neither selection pressure for the selection marker nor selection pressure for the amplification marker,
testing the cell culture after adaptation for stable product-producers with neither selection pressure for the selection marker nor selection pressure for the amplification marker,
isolating a stable product-producer-cell clone in serum- and protein-free conditions with neither selection pressure for the selection marker nor selection pressure for the amplification marker, and
culturing the stable product-producer-cell clone in a cell culture free of serum- or protein-containing additives of human or animal origin.
16. The cell culture according to claim 15, wherein the recombinant product is Factor VIII.
17. The cell culture according to claim 15, wherein the recombinant product is Factor IX.
18. The cell culture according to claim 15, wherein the recombinant product is Factor VII.
19. The cell culture according to claim 15, wherein the recombinant product is von Willebrand factor (vWF).