Recombinant cell clones having increased stability and methods of making and using the same

1. A method for preparing a recombinant polypeptide in a cell culture, comprising: multiplying an isolated stable mammalian recombinant cell clone that has a selection marker and contains a sequence encoding the recombinant polypeptide in a serum- and protein-free medium comprising a mixture of amino acids containing L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine, wherein the stable cell clone is cultured with neither selective pressure for the selection marker nor selection for a polypeptide factor that replaces serum components, preparing a cell culture containing the stable cell clone in a bioreactor, and harvesting the recombinant polypeptide from the culture supernatant.

2. A method of claim 1 , wherein L-asparagine, L-cysteine, L-cystine, or L-tryptophan in the mixture is present from about 1 mg/l to about 100 mg/l.

3. A method of claim 1 , wherein L-proline in the mixture is present from about 1 mg/l to about 150 mg/l.

4. A method of claim 1 , wherein L-glutamine in the mixture is present from about 50 mg/l to about 1000 mg/l.

5. A method of producing a recombinant product under serum- and protein-free conditions on a large technical scale comprising the steps of:

providing an isolated, stable recombinant original cell clone that contains a selection marker, said original cell clone being stable in serum- and protein-free medium for at least 40 generations and expressing a recombinant product,

multiplying said original stable cell clone in serum- and protein-free medium with neither selective pressure for the selection marker nor selection for a polypeptide factor that replaces serum components so as to obtain a cell culture,

culturing said cell culture containing stable cells in a bioreactor, thereby obtaining said recombinant product, and

harvesting said recombinant product from a supernatant of said cell culture.

6. A method of claim 5 , wherein said original cell clone is stable in serum- and protein-free medium for at least 50 generations.

7. A method of claim 5 , wherein said serum- and protein-free medium is a synthetic minimum medium and further comprises ultrafiltrated soybean peptone.

8. A method of producing a recombinant polypeptide in a cell culture comprising the steps of:

multiplying an isolated stable starting cell clone containing a selection marker expressing a recombinant polypeptide in serum- and protein-free medium with neither selective pressure for the selection marker nor selection for a polypeptide factor that replaces serum components so as to obtain a cell culture,

culturing said cell culture containing stable cells in a bioreactor so as to obtain a cell culture, and

harvesting said recombinant polypeptide from a supernatant of said cell culture.

9. A method of claim 8 , wherein the serum- and protein-free medium comprises minimum medium and ultrafiltrated soybean peptone.

10. The method of claim 1 , wherein the recombinant polypeptide is Factor VIII.

11. The method of claim 1 , wherein the recombinant polypeptide is Factor IX.

12. The method of claim 1 , wherein the recombinant polypeptide is Factor VII.

13. The method of claim 1 , wherein the recombinant polypeptide is von Willebrand factor (vWF).

14. The method of claim 5 , wherein the recombinant product is Factor VIII.

15. The method of claim 5 , wherein the recombinant product is Factor IX.

16. The method of claim 5 , wherein the recombinant product is Factor VII.

17. The method of claim 5 , wherein the recombinant product is vWF.

18. The method of claim 8 , wherein the recombinant polypeptide is Factor VIII.

19. The method of claim 8 , wherein the recombinant polypeptide is Factor IX.

20. The method of claim 8 , wherein the recombinant polypeptide is Factor VII.

21. The method of claim 8 , wherein the recombinant polypeptide is von Willebrand factor (vWF).