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 method for preparing a recombinant product, the method comprising:
culturing a stable recombinant mammalian cell clone that produces a recombinant product, wherein the 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 pressure for an amplification marker in the cell clone, and expressing the recombinant product in serum- and protein-free medium so as to obtain a cell culture; and
recovering the expressed recombinant product from the cell culture, wherein the recombinant product is selected from the group consisting of: Factor VIII, Factor IX, Factor VII, and von Willebrand Factor (vWF).
10. The method according to claim 9, wherein the stable recombinant mammalian cell clone is a CHO cell clone.
11. The method according to claim 9, wherein the serum- and protein-free medium 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.
12. The method according to claim 9, wherein the serum- and protein-free medium comprises soybean peptone having a molecular weight of ≤1000 Dalton.
13. The method according to claim 9 further comprising
providing a stable recombinant mammalian cell clone that expresses a recombinant product and is stable in serum- and protein-free medium for at least 40 generations with neither selection pressure for a selection marker nor selection pressure for an amplification marker in the cell clone.
14. The method of claim 9, wherein the stable recombinant mammalian cell clone is produced by:
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 to obtain a stable recombinant mammalian cell clone.