Method for the production of vitamin K-dependent proteins

1. An isolated eukaryotic host cell expressing: a) a first polynucleotide, said polynucleotide comprising a polynucleotide encoding a first propeptide and factor VII in a first expression unit, said first propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 and b) a second polynucleotide, said polynucleotide consisting of a polynucleotide encoding a second propeptide in a second expression unit, said second propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, and wherein said second polynucleotide only encodes the second propeptide.

2. The eukaryotic host cell according to claim 1 , wherein said factor VII is human FVII.

3. The isolated eukaryotic host cell according to claim 1 , wherein said host cell is transfected with a further polynucleotide encoding a gamma-glutamyl carboxylase in an expression unit.

4. The isolated eukaryotic host cell according to claim 1 , wherein said host cell is a mammalian cell.

5. The isolated eukaryotic host cell according to claim 4 , wherein said mammalian cell is a human cell.

6. The isolated eukaryotic host cell according to claim 4 , wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells.

7. The isolated eukaryotic host cell according to claim 1 , wherein said first and second polynucleotides are DNA.

8. The isolated eukaryotic host cell according to claim 1 , wherein said first and second polynucleotides are RNA.

9. A method for producing factor VII comprising a) cultivation of a eukaryotic host cell according to claim 1 in a suitable culture medium under conditions which allow the first polynucleotide and the second polynucleotide to be expressed; and b) isolation of said factor VII from the medium.

10. The method according to claim 9 , wherein said factor VII is human FVII.

11. The method according to claim 9 , wherein said host cell is transfected with a further polynucleotide encoding a gamma-glutamyl carboxylase in an expression unit.

12. The method according to claim 9 , wherein said first expression unit is present on a first vector and said second expression unit is present on a second separate vector.

13. The method according to claim 9 , wherein said first expression unit and said second expression unit are present on the same vector.

14. The method according to claim 9 , wherein said culture medium is a serum free medium.

15. The method according to claim 9 , wherein said host cell is a mammalian cell.

16. The method according to claim 15 , wherein said mammalian cell is a human cell.

17. The method according to claim 15 , wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells.

18. The method according to claim 9 , wherein said first and second polynucleotides are DNA.

19. The method according to claim 9 , wherein said first and second polynucleotides are RNA.

20. A method for producing factor VIIa comprising a) cultivation of a eukaryotic host cell according to claim 1 in a suitable culture medium under conditions which allow the first polynucleotide and the second polynucleotide to be expressed; and b) isolation and activation of factor VII from the medium.

21. The method according to claim 20 , wherein said factor VIIa is human factor VIIa.

22. The method according to claim 20 , wherein said host cell is transfected with a further polynucleotide encoding a gamma-glutamyl carboxylase in an expression unit.

23. The method according to claim 20 , wherein said first expression unit is present on a first vector and said second expression unit is present on a second separate vector.

24. The method according to claim 20 , wherein said first expression unit and said second expression unit are present on the same vector.

25. The method according to claim 20 , wherein said culture medium is a serum free medium.

26. The method according to claim 20 , wherein said host cell is a mammalian cell.

27. The method according to claim 26 , wherein said mammalian cell is a human cell.

28. The method according to claim 26 , wherein said mammalian cell is independently selected from MIK cells, IIEK cells, COS cells, or CHO cells.

29. The method according to claim 20 , wherein said first and second polynucleotides are DNA.

30. The method according to claim 20 , wherein said first and second polynucleotides are RNA.

31. An isolated eukaryotic host cell transfected with: a) a first polynucleotide, said polynucleotide comprising a polynucleotide encoding a first propeptide and factor VII in a first expression unit, said first propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18; and b) a second polynucleotide, said polynucleotide consisting of a polynucleotide encoding a second propeptide in a second expression unit, said second propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

32. The eukaryotic host cell according to claim 31 , wherein said factor VII is human FVII.

33. The isolated eukaryotic host cell according to claim 31 , wherein said host cell is transfected with a further polynucleotide encoding a gamma-glutamyl carboxylase in an expression unit.

34. The isolated eukaryotic host cell according to claim 31 , wherein said host cell is a mammalian cell.

35. The isolated eukaryotic host cell according to claim 34 , wherein said mammalian cell is a human cell.

36. The isolated eukaryotic host cell according to claim 34 , wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells.

37. The isolated eukaryotic host cell according to claim 31 , wherein said first and second polynucleotides are DNA.

38. The isolated eukaryotic host cell according to claim 31 , wherein said first and second polynucleotides are RNA.

39. A method for producing factor VII comprising a) transfection of a eukaryotic host cell with: i) a first polynucleotide, said polynucleotide comprising a polynucleotide encoding a first propeptide and factor VII in a first expression unit, said first propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 and ii) a second polynucleotide, said polynucleotide consisting of a polynucleotide encoding a second propeptide in a second expression unit, said second propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, to produce a co-transfected eukaryotic host cell; b) cultivation in a suitable culture medium of the co-transfected eukaryotic host cell under conditions which allow the first polynucleotide and the second polynucleotide to be expressed; and c) isolation of said factor VII from the medium.

40. The method according to claim 39 , wherein said factor VII is human FVII.

41. The method according to claim 39 , wherein said host cell is transfected with a further polynucleotide encoding a gamma-glutamyl carboxylase in an expression unit.

42. The method according to claim 39 , wherein said first expression unit is present on a first vector and said second expression unit is present on a second separate vector.

43. The method according to claim 39 , wherein said first expression unit and said second expression unit are present on the same vector.

44. The method according to claim 39 , wherein said culture medium is a serum free medium.

45. The method according to claim 39 , wherein said host cell is a mammalian cell.

46. The method according to claim 45 , wherein said mammalian cell is a human cell.

47. The method according to claim 45 , wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells.

48. The method according to claim 39 , wherein said first and second polynucleotides are DNA.

49. The method according to claim 39 , wherein said first and second polynucleotides are RNA.

50. A method for producing factor VIIa comprising a) transfection of a eukaryotic host cell with: i) a first polynucleotide, said polynucleotide comprising a polynucleotide encoding a first propeptide and factor VII in a first expression unit, said first propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 and ii) a second polynucleotide, said polynucleotide consisting of a polynucleotide encoding a second propeptide in a second expression unit, said second propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, to produce a co-transfected eukaryotic host cell; b) cultivation in a suitable culture medium of the co-transfected eukaryotic host cell under conditions which allow the first polynucleotide and the second polynucleotide to be expressed; and c) isolation and activation of said factor VII from the medium.

51. The method according to claim 50 , wherein said factor VIIa is human FVIIa.

52. The method according to claim 50 , wherein said host cell is transfected with a further polynucleotide encoding a gamma-glutamyl carboxylase in an expression unit.

53. The method according to claim 50 , wherein said first expression unit is present on a first vector and said second expression unit is present on a second separate vector.

54. The method according to claim 50 , wherein said first expression unit and said second expression unit are present on the same vector.

55. The method according to claim 50 , wherein said culture medium is a serum free medium.

56. The method according to claim 50 , wherein said host cell is a mammalian cell.

57. The method according to claim 56 , wherein said mammalian cell is a human cell.

58. The method according to claim 56 , wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells.

59. The method according to claim 56 , wherein said first and second polynucleotides are DNA.

60. The method according to claim 50 , wherein said first and second polynucleotides are RNA.

61. A recombinant vector, said vector comprising a first polynucleotide, said polynucleotide comprising a polynucleotide encoding a first propeptide and factor VII in a first expression unit, said first propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 and a second polynucleotide, said polynucleotide consisting of a polynucleotide encoding a second propeptide in a second expression unit, said second propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

62. The recombinant vector according to claim 61 , wherein said factor VII is human FVII.

63. The recombinant vector according to claim 61 , wherein said first and second polynucleotides are DNA.

64. The recombinant vector according to claim 61 , wherein said first and second polynucleotides are RNA.

65. A method for preparing a eukaryotic host cell producing factor VII, comprising transfection of a eukaryotic host cell with a vector of claim 61 .

66. The method of claim 65 , wherein said host cell is further transfected with a polynucleotide encoding a gamma-glutamyl carboxylase.

67. A method for preparing a eukaryotic host cell producing factor VII comprising a) transfection of a eukaryotic host cell with a first polynucleotide, said polynucleotide comprising a polynucleotide encoding a first propeptide and factor VII in a first expression unit, said first propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 and b) transfection with a second polynucleotide, said polynucleotide consisting of a polynucleotide encoding a second propeptide in a second expression unit, said second propeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

68. The method according to claim 67 , wherein the said factor VII is human factor VII.

69. The method according to claim 67 , wherein said host cell is further transfected with a polynucleotide encoding a gamma-glutamyl carboxylase.