Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1986-05-05
1988-09-13
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435 70, 435 91, 4351723, 4352402, 4353171, 435320, 435253, 536 27, 935 14, 935 55, 935 62, 935 70, C12P 2100, C12P 1934, C12N 1500, C12N 120
Patent
active
047709995
DESCRIPTION:
BRIEF SUMMARY
This invention relates generally to the cloning and expression in high yield of Factor IX, and more particularly, to the production of biologically-active Factor IX by means of culturing mammalian cells into media containing vitamin K which Factor IX cDNA has been chromosomally-integrated.
The plasma glycoprotein, Factor IX, plays a critical role in the blood-clotting process. Normally synthesized in the liver, Factor IX requires vitamin K activity for the .gamma.-carboxylation of its 12 amino-terminal glutamic acid residues. A deficiency of Factor IX in the body characterizes a type of hemophilia (type B). Treatment of this disease is presently limited to intravenous tranfusion of human plasma protein concentrates of Factor IX. However, in addition to the practical disadvantages of time and expense, transfusion of blood concentrates involves the risk of transmission of viral hepatitis, acquired immune deficiency syndrome or thromboembolic diseases to the recipient. An alternative method of producing Factor IX, other than extraction from human plasma, is therefore highly desirable.
The application of recombinant DNA techniques to the production of Factor IX has elicited considerable information about the protein. The cDNA coding for human Factor IX has been isolated, characterized, and cloned into expression vectors. See, e.g., K. H. Choo et al, "Molecular Cloning of the Gene for Human Anti-hemophilic Factor IX', Nature, Vol. 299: 178-180 (September 1982) and K. Kurachi et al, "Isolation and Characterization of a cDNA Coding for Human Factor IX," Proc. Natl. Acad. Sci. U.S.A., Vol. 79: 6461-65 (November 1982).
PCT patent application WO No. 84/00360 published Feb. 16, 1984 describes the identification and cloning of a human Factor IX nucleotide sequence or fragments thereof for use primarily as diagnostic probes. This application only prophetically refers to the production of a human Factor IX polypeptide through growth in mammalian tissue culture cells, preferably a hepatoma cell line. The inventions' subsequent article, D. S. Anson et al, "Expression of Active Human Clotting Factor IX from Recombinant DNA Clones in Mammalian Cells" Nature, Vol. 315, pp 683-685 (June 20, 1985), describes the production of very low levels of a purportedly active human Factor IX polypeptide from a transformed rat hepatoma cell line. (See page 685, column 1)
European patent application No. 162,782, published Nov. 11, 1985, refers to construction of a recombinant viral vector containing a sequence coding for human Factor IX. Biologically active human Factor IX in low yields is assertedly obtained from bacterial cells containing the episomally integrated recombinant Factor IX sequence. The influence of viral components on growth of the host cells and glycoprotein of the protein however can result in unreliable and varied batches of protein. [See, also H. de la Salle et al, "Active .gamma.-carboxylated human Factor IX expressed using recombinant DNA techniques," Nature, 316: 268-270 (July 18, 1985)]
Another recent report, S. Busby et al. "Expression of active human Factor IX in transfected cells," Nature, 316: 271-273 (July 18, 1985) also refers to expression of low levels of recombinant Factor IX in BHK cells co-transfected with a neo gene marker.
Even these recent studies therefore demonstrate the continued difficulty in obtaining a stable system for producing high yields of Factor IX in biologically active and reliably consistent form.
In accordance with the present invention it is surprisingly discovered that high yields of biologically active Factor IX protein can be produced by culturing a CHO cell line containing chromosomally integrated Factor IX cDNA and adding heterologous vitamin K to the culture medium for a predetermined time prior to harvesting the polypeptide. Even cells that have not previously been demonstrated to provide .gamma.-carboxylation, can be shown to provide active Factor IX by this method.
Vitamin K is added to the culture medium in the form of K1 or K3. Where K3 is the vitamin of choice, a concentrat
REFERENCES:
Kurachi et al., (1982), Proceedings National Academy Sciences, U.S.A., vol. 79, pp. 6461-6464.
Choo et al., (1982), Nature, vol. 299, pp. 178-180.
Fair et al., (1984, Jul.), Blood, vol. 64--pp. 194-204.
Kaufman et al., (1982), Molecular and Cellular Biology, vol. 2, pp. 1304-1319.
Nucleic Acids Res., vol. 11, pp. 2325-2335, 1983, M. Jaye et al.
Embo J., vol. 3, pp. 1053-1060, 1984, D. Ansen et al.
Nature, vol. 315, pp. 683-685, 1985, D. Ansen et al.
Nature, vol. 316, pp. 268-270, 1985, H. De La Salle et al.
Nature, vol. 316, pp. 271-273, 1985, S. Busby et al.
Proc. Natl. Acad. Sci., U.S.A., vol. 76, pp. 4990-4994, K. Katayama et al.
Kaufman Randal J.
Shoemaker Charles B.
Wasley Louise C.
Bak Mary
Eisen Bruce M.
Genetics Institute Inc.
O'Shaughnessy Brian P.
Seidman S.
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