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

• US 6,475,725 B1
• Filed: 06/02/1999
• Issued: 11/05/2002
• Est. Priority Date: 06/20/1997
• Status: Expired due to Term

• Current Assignee: Baxalta GmbH (Shire plc), Baxalta Incorporated (Shire plc)
• Original Assignee: Baxter AG (Baxter International Inc.)
• Inventors: Reiter, Manfred, Dorner, Friedrich, Mundt, Wolfgang
• Primary Examiner(s): Guzo, David
• Application Number:
  US09/324,612
• Time in Patent Office: 1,252 Days
• Field of Search: 435/325, 435/366, 435/358, 435/352, 435/69.1, 435/69.6, 435/383, 435/404, 435/6, 435/455
• US Class Current: 435/6.12
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2500/50 Soluble polymers, e.g. polyethyleneglycol [PEG]
• CPC Class Codes: C12N 2500/76 from plants
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 5/0075 using microcarriers

Abstract

Disclosed are 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.

Claims

1. A method for preparing a recombinant factor VIII protein or polypeptide having factor VIII activity from a CHO cell culture wherein said CHO cells have been transformed to express recombinant factor VIII, comprising the steps of

multiplying a CHO recombinant cell clone stable in serum- and protein-free medium, wherein purified, ultrafiltered soybean peptide is added to the medium, 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.

2. A Method of production of a recombinant product under serum and protein-free conditions on a large technical scale, comprising the steps of:

providing an isolated, stable recombinant cell clone obtainable by cultivating a recombinant original cell clone in serum-containing medium, adapting the cells on serum- and protein free medium, assaying said cell clone for stable producers of said recombinant product and cloning of a stable product—

producing cell clone under serum- and protein-free conditions,

multiplying said stable recombinant cell clone in serum- and protein-free medium, so to obtain a cell culture,

producing the said cell culture containing stable cells in a bioreactor, and

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

wherein a serum- and protein-free medium is used which contains one or more amino acids selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine.

3. The Method according to claim 2, wherein a serum- and protein-free medium is used which comprises synthetic minimal-medium containing a yeast or soybean extract.

4. The Method according to claim 2, wherein the recombinant factor VIII protein or a recombinant polypeptide with factor VIII activity is produced as recombinant product.

5. Method according to claim 2, wherein recombinant von Willebrand factor (vWF)-protein or a recombinant polypeptide with factor vWF—

activity as a recombinant product is produced.

6. Method according to claim 2, wherein cleaned ultra-filtrated soybean peptone is added to the medium.

7. Method according to claim 2, wherein the medium contains cyclodextrin or a derivative thereof and/or a protease inhibitor.

8. Method according to claim 3, wherein the soybean extract is a purified ultra-filtrated soybean peptone.

9. A serum- and protein-free synthetic medium comprised of minimum medium, soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine, and sodium selenite, and wherein said medium additionally contains a mixture of amino acids selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine.

10. A serum- and protein-free synthetic medium comprised of minimum medium, purified and ultrafiltrated soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine and sodium selenite.

11. A serum- and protein-free synthetic medium comprised of minimum medium, purified and ultrafiltrated soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine and sodium selenite, wherein said medium additionally contains a mixture of amino acids selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline and L-tryptophan.

12. A serum- or protein-free medium comprising at least one amino acid selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine and purified, ultrafiltrated soybean peptone.

13. The medium of claim 12, wherein said soybean peptone has an average molecular weight of <

1000 Dalton.

14. The medium of claim 12, wherein said amino acid is L-asparagine.

15. The medium of claim 12, wherein said amino acid is L-cysteine.

16. The medium of claim 12, wherein said amino acid is L-cystine.

17. The medium of claim 12, wherein said amino acid is L-proline.

18. The medium of claim 12, wherein said amino acid is L-tryptophan.

19. The medium of claim 12, wherein said amino acid is L-glutamine.

20. The medium of claim 13, wherein said soybean peptone has an average molecular weight of <

500 Dalton.

21. The medium of claim 13, wherein said soybean peptone has an average molecular weight of <

350 Dalton.

22. The medium of claim 13, wherein said soybean peptone has an average molecular weight of 350 Dalton.

23. The medium of claim 14, wherein said L-asparagine is added in an amount of between 1 mg/l and 100 mg/l.

24. The medium of claim 23, wherein said L-asparagine is added in an amount of 20 mg/l.

25. The medium of claim 15, wherein said L-cysteine is added in an amount of between 1 mg/l and 100 mg/l.

26. The medium of claim 25, wherein said L-cysteine is added in an amount of 15 mg/l.

27. The medium of claim 16, wherein said L-cystine is added in an amount of between 1 mg/l and 100 mg/l.

28. The medium of claim 27, wherein said L-cystine is added in an amount of 20 mg/l.

29. The medium of claim 17, wherein said L-proline is added in an amount of between 1 mg/l and 150 mg/l.

30. The medium of claim 29, wherein said L-proline is added in an amount of 35 mg/l.

31. The medium of claim 18, wherein said L-tryptophan is added in an amount of between 1 mg/l and 100 mg/l.

32. The medium of claim 31, wherein said L-tryptophan is added in an amount of 20 mg/l.

33. The medium of claim 19, wherein said L-glutamine is added in an amount of between 50 mg/l and 1000 mg/l.

34. The medium of claim 33, wherein said L-glutamine is added in an amount of 240 mg/l.