Method of producing biologically active vitamin k dependent proteins by recombinant methods
AssigneesCnj Holdings Inc, The University of North Carolina at Chapel Hill
InventorsWilliam N. Drohan, Michael J. Griffith, John R. Taylor, Darrel W. Stafford
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Abstract
The invention relates to commercially viable methods for producing biologically active vitamin K dependent proteins, particularly Factor IX. Factor IX is produced at a level of at least about 15 mg/L and is at least 25% biologically active. The method relies upon co-expression of one or more of paired basic amino acid converting enzyme (PACE), vitamin K dependent epoxide reductase (VKOR) and vitamin K dependent γ-glutamyl carboxylase (VKGC) at a preferred ratio so that the vitamin K dependent protein is efficiently produced and processed by a recombinant cell.
Claims46
1-35. (canceled)
A method of producing a high level of recombinant biologically active vitamin K dependent protein comprising:
- (a) transfecting mammalian cells with (i) a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, (ii) a gene encoding vitamin K dependent epoxide reductase (VKOR), and (iii) a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC);
- and
- (b) harvesting recombinant vitamin K dependent protein produced by the mammalian cells;
- wherein the mammalian cells produce at least about 15 mg/L of the recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A method of producing a mammalian cell that produces a high level of recombinant biologically active vitamin K dependent protein comprising transfecting a mammalian cell with:
- (i) a gene encoding a vitamin K dependent protein,
- (ii) a gene encoding vitamin K dependent epoxide reductase (VKOR), and
- (iii) a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC);
- wherein the gene encoding the vitamin K dependent protein is operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
- wherein the cell produces at least about 15 mg/L of a recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A mammalian cell that produces a high level of recombinant biologically active vitamin K dependent protein, wherein the cell comprises:
- (i) a gene encoding a vitamin K dependent protein,
- (ii) a gene encoding vitamin K dependent epoxide reductase (VKOR), and
- (iii) a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC);
- wherein the gene encoding the vitamin K dependent protein is operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
- wherein the cell produces at least about 15 mg/L of a recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A method of producing a high level of recombinant biologically active vitamin K dependent protein comprising:
- (a) transfecting mammalian cells with (i) a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and, (ii) a gene encoding vitamin K dependent epoxide reductase (VKOR) operably linked to a CHEF1 promoter;
- and
- (b) harvesting recombinant vitamin K dependent protein produced by the mammalian cells;
- wherein the mammalian cells produce at least about 15 mg/L of the recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A method of producing a high level of recombinant biologically active vitamin K dependent protein comprising:
- (a) transfecting mammalian cells with (i) a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and, (ii) a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC) operably linked to a CHEF1 promoter;
- and
- (b) harvesting recombinant vitamin K dependent protein produced by the mammalian cells;
- wherein the mammalian cells produce at least about 15 mg/L of the recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A method of producing a mammalian cell that produces a high level of recombinant biologically active vitamin K dependent protein comprising transfecting a mammalian cell with:
- (i) a gene encoding a vitamin K dependent protein, and
- (ii) a gene encoding vitamin K dependent epoxide reductase (VKOR);
- wherein the genes encoding the vitamin K dependent protein and VKOR are operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
- wherein the cell produces at least about 15 mg/L of a recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A method of producing a mammalian cell that produces a high level of recombinant biologically active vitamin K dependent protein comprising transfecting a mammalian cell with:
- (i) a gene encoding a vitamin K dependent protein, and
- (ii) a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC);
- wherein the genes encoding the vitamin K dependent protein and VKGC are operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
- wherein the cell produces at least about 15 mg/L of a recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A mammalian cell that produces a high level of recombinant biologically active vitamin K dependent protein, wherein the cell comprises:
- (i) a gene encoding a vitamin K dependent protein, and
- (ii) a gene encoding vitamin K dependent epoxide reductase (VKOR);
- wherein the genes encoding the vitamin K dependent protein and VKOR are operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
- wherein the cell produces at least about 15 mg/L of a recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
A mammalian cell that produces a high level of recombinant biologically active vitamin K dependent protein, wherein the cell comprises:
- (i) a gene encoding a vitamin K dependent protein, and
- (ii) a gene encoding vitamin K dependent γ-glutamyl carboxylase (VKGC);
- wherein the genes encoding the vitamin K dependent protein and VKGC are operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
- wherein the cell produces at least about 15 mg/L of a recombinant vitamin K dependent protein, and wherein at least 60% of the recombinant vitamin K dependent protein is biologically active.
Dependencies are inferred from the claim text.