METHOD OF PRODUCING BIOLOGICALLY ACTIVE VITAMIN K DEPENDENT PROTEINS BY RECOMBINANT METHODS

1 - 47 . (canceled)

48 . A method of producing a recombinant biologically active vitamin K dependent protein product, comprising the steps of:

transfecting a mammalian cell with a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and at least two genes,

wherein the at least two genes comprise a gene encoding vitamin K dependent epoxide reductase (VKOR) and a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC); wherein each of said at least two genes is operably linked to a promoter; and

harvesting the vitamin K dependent protein product,

whereby the cell produces biologically active vitamin K dependent protein product.

49 . The method of claim 48 , wherein the vitamin K dependent protein product is selected from the group consisting of Factor II, Factor VIII, Factor IX, Factor X, Protein C and Protein S.

50 . The method of claim 49 , wherein the vitamin K dependent protein is Factor IX.

51 . The method of claim 49 , wherein the vitamin K dependent protein is Factor VIII.

52 . The method of claim 48 , wherein the processing factors further comprise a gene encoding paired basic amino acid converting enzyme (PACE) operably liked to a promoter.

53 . The method of claim 48 , wherein at least about 75% of the glutamic acid residues within the gla-domain of the biologically active vitamin K dependent protein product are gamma carboxylated.

54 . The method of claim 48 , wherein at least 50% of the vitamin K dependent protein is biologically active.

55 . The method of claim 48 , wherein the mammalian cell is selected from the group consisting of CHO cells and HEK 293 cells.

56 . A method of producing a recombinant biologically active vitamin K dependent protein product, comprising the steps of:

transfecting a mammalian cell with a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter;

transfecting the mammalian cell with at least two genes, wherein the at least two comprise a gene encoding vitamin K dependent epoxide reductase (VKOR) and a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC); wherein each of said at least two genes is operably linked to a promoter;

performing a first selection for cells which express high levels of the vitamin K dependent protein product or the processing factors;

cloning the selected cells;

performing a second selection for cells which express high levels of the vitamin K dependent protein product or the processing factors;

growing the cloned cells; and

harvesting the vitamin K dependent protein product,

whereby the cell produces vitamin K dependent protein product.

57 . The method of claim 56 , wherein the step of transfecting with the genes encoding the processing factors are performed before the step of transfecting with the gene encoding the vitamin K dependent protein.

58 . The method of claim 56 , wherein the step of transfecting with the gene encoding the vitamin K dependent protein is performed before the step of transfecting with the genes encoding the processing factors.

59 . The method of claim 56 , wherein the mammalian cell is selected for expression of endogenous levels of one or more processing factors before transfection.

60 . A method of producing a recombinant biologically active vitamin K dependent protein product, comprising the steps of:

(a) transfecting a mammalian cell with a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter;

(b) selecting for cells which express high levels of the vitamin K dependent protein product;

(c) transfecting the selected cells with at least two genes wherein the at least two genes comprise a gene encoding the vitamin K dependent epoxide reductase (VKOR) and a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC); wherein each of said at least two genes is operably linked to a promoter,

(d) repeating step (b);

(e) optionally, repeating steps (a) and/or (c) followed by (b);

(f) cloning the selected cells;

(g) growing the cloned cells; and

(h) harvesting the product from the cloned cells, whereby the cell produces biologically active vitamin K dependent protein.

61 . A method of producing a recombinant biologically active vitamin K dependent protein product, comprising the steps of:

transfecting a mammalian cell with a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter and at least two genes, wherein the at least two genes comprise a gene encoding vitamin K dependent epoxide reductase (VKOR) and a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC) and wherein each of said at least two genes is operably linked to a promoter, and harvesting the vitamin K dependent protein product,

wherein the cell produces biologically active vitamin K dependent protein in an amount that is at least about 10 mg/L.

62 . The method of claim 61 , wherein the biologically active vitamin K dependent protein is produced in an amount of at least about 20 mg/L.

63 . The method of claim 61 , wherein the biologically active vitamin K dependent protein is produced in an amount of at least about 30 mg/L.

64 . The method of claim 61 , wherein the biologically active vitamin K dependent protein is produced in an amount of at least about 50 mg/L.

65 . The method of claim 48 , wherein at least 70% of the vitamin K dependent protein is biologically active.

66 . The method of claim 48 , wherein at least 80% of the vitamin K dependent protein is biologically active.

67 . The method of claim 56 or 60 , wherein at least 50% of the vitamin K dependent protein is biologically active.

68 . The method of claim 56 or 60 , wherein at least 70% of the vitamin K dependent protein is biologically active.

69 . The method of claim 56 or 60 , wherein at least 80% of the vitamin K dependent protein is biologically active.

70 . A method of producing a recombinant biologically active vitamin K dependent protein product, comprising the steps of:

transfecting a mammalian cell with a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter and at least two genes, wherein the at least two genes comprise a gene encoding vitamin K dependent epoxide reductase (VKOR) and a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC) wherein each of the at least two genes are operably linked to a promoter; and harvesting the vitamin K dependent protein product,

and subsequently re-transfecting the cells with a gene encoding VKOR operably linked to a promoter,

whereby the cell produces biologically active vitamin K dependent protein.

71 . The method of any one of claims 48 , 56 , 60 , 61 and 70 , wherein the gene encoding VKOR and the gene encoding VKGC are operably linked to the Chinese Hamster elongation factor 1-α (CHEF1) promoter.

72 . The method of any one of claims 48 , 56 , 60 , 61 and 70 , wherein the gene encoding VKOR and the gene encoding VKGC are operably linked to at least one promoter that is different than said Chinese hamster elongation factor 1-α (CHEF1) promoter.

73 . The method of any one of claims 48 , 56 , 60 , 61 and 70 , wherein the gene encoding VKOR and the gene encoding VKGC are operably linked to different promoters.

74 . The method of any one of claims 48 , 56 , 60 , 61 and 70 , wherein the gene encoding VKOR and the gene encoding VKGC are operably linked to the same promoter.