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 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.