Method of producing biologically active vitamin K dependent proteins by recombinant methods
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.
1 . 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 promoter and at least one gene encoding a processing factor(s) operably linked to at least one promoter, either simultaneously or sequentially; and
harvesting the vitamin K dependent protein product,
wherein at least one of the promoters is a Chinese hamster elongation factor 1-α (CHEF1) promoter, and
whereby the cell produces biologically active vitamin K dependent protein product in an amount of at least about 15 mg/L.
2 . The method of claim 1 , wherein the vitamin K dependent protein product is selected from the group consisting of Factor II, Factor VII, Factor IX, Factor X, Protein C and Protein S.
3 . The method of claim 2 , wherein the vitamin K dependent protein is Factor Ix.
4 . The method of claim 2 , wherein the vitamin K dependent protein is Factor VII.
5 . The method of claim 1 , wherein the processing factor is a nucleic acid selected from the group consisting of paired basic amino acid converting enzyme (PACE), vitamin K dependent epoxide reductase (VKOR), vitamin K dependent γ-glutamyl carboxylase (VKGC) and combinations thereof operably linked to one or more promoter(s).
6 . The method of claim 5 , wherein the processing factor proteins comprise VKOR and VKGC.
7 . The method of claim 5 , wherein at least one of the genes is overexpressed.
8 . The method of claim 7 , wherein the overexpressed gene is operably linked to a Chinese hamster elongation factor 1-α(CHEF1) promoter.
9 . The method of claim 1 , 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.
10 . The method of claim 1 , wherein at least 25% of the vitamin K dependent protein is biologically active.
11 . The method of claim 10 , wherein at least 50% of the vitamin K dependent protein is biologically active.
12 . The method of claim 11 , wherein at least 80% of the vitamin K dependent protein is biologically active.
13 . The method of claim 1 , wherein the mammalian cell is selected from the group consisting of CHO cells and HEK 293 cells.
14 . The method of claim 1 , wherein the biologically active vitamin K dependent protein is produced in an amount of at least about 20 mg/L.
15 . The method of claim 14 , wherein the biologically active vitamin K dependent protein is produced in an amount of at least about 30 mg/L.
16 . The method of claim 15 , wherein the biologically active vitamin K dependent protein is produced in an amount of at least about 50 mg/L.
17 . 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 promoter and at least one gene encoding a processing factor(s) operably linked to at least one promoter:
performing a first selection for cells which express high levels of the vitamin K dependent protein product or the processing factor(s);
cloning the selected cells;
performing a second selection for cells which express high levels of the vitamin K dependent protein product or the processing factor(s);
amplifying the cloned cells; and
harvesting the vitamin K dependent protein product,
whereby the cell produces biologically active vitamin K dependent protein product in an amount of at least about 15 mg/L.
18 . The method of claim 17 , wherein the transfecting steps with the gene(s) encoding the processing factor(s) are performed before the transfecting steps with the gene encoding the vitamin K dependent protein.
19 . The method of claim 17 , wherein the transfecting steps with the gene encoding the vitamin K dependent protein are performed before transfecting steps with the gene(s) encoding the processing factor(s).
20 . The method of claim 17 , wherein the mammalian cell is selected for expression of endogenous levels of one or more processing factors before transfection.
21 . A recombinant mammalian cell comprising a gene for a vitamin K dependent protein operably linked to a promoter and a gene for at least one processing factor operably linked to at least one promoter, wherein at least one of the promoters is a Chinese hamster elongation factor 1-α (CHEF1) promoter, and wherein the expression of the protein(s) encoded by the gene for at least one processing factor(s) in the cell facilitates the production of biologically active vitamin K dependent protein in an amount of at least about 15 mg/L.
22 . The recombinant mammalian cell of claim 21 , wherein the vitamin K dependent protein is selected from the group consisting of Factor II, Factor VII, Factor IX, Factor X, Protein C and Protein S.
23 . The recombinant mammalian cell of claim 22 , wherein the vitamin K dependent protein is Factor IX or Factor VII.
24 . The recombinant mammalian cell of claim 21 wherein the processing factor is a gene which produces a processing gene product selected from the group consisting of PACE, VKOR, VKGC, and combinations thereof, operably linked to one or more promoter(s) for expression in said cell.
25 . The recombinant mammalian cell of claim 21 , wherein the processing factors comprise VKOR and VKGC.
26 . The recombinant mammalian cell of claim 24 , wherein at least one of the processing gene products is expressed at a higher level than observed in normal, nontransfected cells of the same line.
27 . The recombinant mammalian cell of claim 26 , wherein the overexpressed gene product is operably linked to a Chinese hamster elongation factor 1-α (CHEF1) promoter.
28 . The recombinant mammalian cell of claim 21 which is a CHO cell or HEK293 cell.
29 . A recombinant Factor IX protein produced by the method of claim 1 .
30 . A pharmaceutical composition comprising the Factor IX protein of claim 29 .
31 . A kit comprising the recombinant Factor IX protein of claim 29 .
32 . A method of treating hemophilia which comprises administering an effective amount of the pharmaceutical composition of claim 30 to a patient in need thereof.
33 . A method of selecting the cell of claim 21 , which comprises selecting for variants of a specific tissue culture cell line that contain naturally occurring processing factor(s) capable of producing a vitamin K dependent protein composed of amino acids that are posttranslationally modified to contain at least 25% of the sulfation and at least 25% of the phosphorylation levels present in the corresponding plasma-derived vitamin K dependent protein.
34 . The method of claim 33 , wherein the vitamin K dependent protein is Factor IX.
35 . 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 promoter;
(b) selecting for cells which express high levels of the vitamin K dependent protein product;
(c) transfecting the selected cells with one or more processing factor(s) 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) amplifying the cloned cells; and
(h) harvesting the product from the cloned cells in an amount of at least 15 mg/L recombinant biologically active vitamin K dependent protein.