Method of producing recombinant vitamin K dependent proteins
Methods for producing cell lines with high levels of biologically active recombinant vitamin K dependent proteins are described. The transfected cell lines do not include heterologous genes for processing enzymes and are not subject to selection pressure such as methotrexate resistance. Cell lines producing Factor VII/VIIa and Factor IX are described. These cell lines can be used for isolation of Factor VII/VIIa and/or Factor IX for treatment of Hemophilia.
1. A method of producing a vitamin K dependent protein, the method comprising the steps of: (a) transfecting a population of mammalian cells with a gene encoding the vitamin K dependent protein operably linked to a promoter, wherein the mammalian cells do not contain heterologous genetic material encoding proteins involved in the post-translation modification of the vitamin K dependent protein; (b) performing at least one round of cloning and screening to identify cells which produce at least 30 mg/L of the vitamin K dependent protein, wherein at least 10% of the vitamin K dependent protein is biologically active; (c) optionally, repeating step (b) one or more times; and (d) harvesting the vitamin K dependent protein.
2. The method of claim 1 , 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.
3. The method of claim 2 , wherein the vitamin K dependent protein is Factor IX or Factor VII.
4. The method of claim 1 , wherein in step (b) the cloning is limit dilution cloning or semi-solid matrix cloning.
5. The method of claim 1 , wherein the promoter is a Chinese hamster elongation factor 1 (CHEF-1) promoter.
6. The method of claim 1 , further comprising the step of preselecting cells producing at least 10 mg/L vitamin K dependent protein before step (b).
7. The method of claim 1 , wherein the cells are cultured in a media that includes vitamin K.
8. The method of claim 1 , wherein the cells are cultured in a media that does not include vitamin K.
9. The method of claim 1 , wherein in step (b) at least 20% of the vitamin K dependent protein is biologically active.
10. The method of claim 9 , wherein at least 30% of the vitamin K dependent protein is biologically active.
11. The method of claim 10 , wherein at least 40% of the vitamin K dependent protein is biologically active.
12. The method of claim 1 , wherein the mammalian cells are Chinese hamster ovary cells.
13. A method of producing a vitamin K dependent protein, the method comprising the steps of: (a) transfecting a population of mammalian cells with a gene encoding the vitamin K dependent protein operably linked to a Chinese hamster elongation factor 1 (CHEF-1) promoter, wherein the mammalian cells do not contain heterologous genetic material encoding proteins involved in the post-translation modification of the vitamin K dependent protein; and (b) performing at least one round of cloning and screening to identify cells which produce at least 30 mg/L of the vitamin K dependent protein, wherein at least 10% of the vitamin K dependent protein is biologically active.
14. The method of claim 13 , wherein the cloning is limit dilution cloning or semi-solid-matrix cloning.