US2012276079A1PendingUtilityA1

Method of producing recombinant vitamin k dependent proteins

53
Individually held — no corporate assignee on recordPriority: Oct 30, 2009Filed: Apr 30, 2012Published: Nov 1, 2012
Est. expiryOct 30, 2029(~3.3 yrs left)· nominal 20-yr term from priority
Inventors:William N. DrohanMarian J. DrohanMichael J. Griffith
C12N 9/64A61K 38/36C07K 14/745A61P 7/04C12P 21/02
53
PatentIndex Score
0
Cited by
0
References
0
Claims

Abstract

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.

Claims

exact text as granted — not AI-modified
1 . A cell line which produces recombinant vitamin K dependent protein having at least 20% biological activity, wherein the cell line does not contain heterologous genetic material encoding proteins involved in the post-translation modification of vitamin K dependent proteins. 
     
     
         2 . The cell line of  claim 1 , wherein the cell line is a mammalian cell line. 
     
     
         3 . The cell line of  claim 2 , wherein the mammalian cell line is Chinese Hamster Ovary. 
     
     
         4 . The cell line of  claim 1 , which has not been subjected to selection with methotrexate. 
     
     
         5 . The cell line 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. 
     
     
         6 . The cell line of  claim 5 , wherein the Vitamin K dependent protein is Factor VII/VIIa or Factor IX. 
     
     
         7 . The cell line of  claims 1 , comprising a gene encoding the Vitamin K dependent protein which is operably linked to a Chinese hamster elongation factor 1 (CHEF-1) promoter. 
     
     
         8 . A method of producing a recombinant biologically active Vitamin K dependent protein 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;   (b) performing at least one round of cloning and screening to identify cell clones which produce at least 10 mg/L of the Vitamin K dependent protein which is at least 10% biologically active;   (c) optionally, repeating the cell cloning of step (b) one or more times to identify single cells producing more than 10 mg/L of the Vitamin K dependent protein which is at least 10% biologically active; and   (d) harvesting the Vitamin K dependent protein.   
     
     
         9 . The method of  claim 8 , 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. 
     
     
         10 . The method of  claim 9 , wherein the Vitamin K dependent Protein is Factor IX. 
     
     
         11 . The method of  claim 10 , wherein the cloning is limit dilution cloning. 
     
     
         12 . The method of  claim 9 , wherein the Vitamin K dependent Protein is Factor VII. 
     
     
         13 . The method of  claim 12 , wherein the cloning is semi-solid matrix cloning. 
     
     
         14 . The method of  claim 8 , wherein the promoter is a Chinese hamster elongation factor 1 (CHEF-1) promoter. 
     
     
         15 . The method of  claim 8 , further comprising preselecting cells producing at least 10 mg/L Vitamin K dependent protein antigen before step (b). 
     
     
         16 . The method of  claim 8 , wherein the cell line is cultured in a media that includes vitamin K. 
     
     
         17 . The method of  claim 8 , wherein the cell line is cultured in a media that does not include vitamin K. 
     
     
         18 . The method of  claim 8 , wherein at least 20% of the Vitamin K dependent protein is biologically active. 
     
     
         19 . The method of  claim 8 , wherein at least 30% of the Vitamin K dependent protein is biologically active. 
     
     
         20 . The method of  claim 8 , wherein at least 40% of the Vitamin K dependent protein is biologically active. 
     
     
         21 . The method of  claim 8 , wherein at least 58% of the Vitamin K dependent protein is biologically active. 
     
     
         22 . The method of  claim 8 , wherein at least 70% of the Vitamin K dependent protein is biologically active. 
     
     
         23 . The method of  claim 8 , wherein at least 85% of the Vitamin K dependent protein is biologically active. 
     
     
         24 . The method of  claims 8 , wherein at least 95% of the Vitamin K dependent protein is biologically active. 
     
     
         25 . The method of  claims 8 , wherein at least 99% of the Vitamin K dependent protein is biologically active 
     
     
         26 . The method of  claim 8 , wherein the vitamin K dependent protein is produced in an amount of at least 20 mg/L. 
     
     
         27 . The method of any of  claim 8 , wherein the vitamin K dependent protein is produced in an amount of at least 30 mg/L. 
     
     
         28 . The method of  claim 8 , wherein the vitamin K dependent protein is produced in an amount of at least 40 mg/L. 
     
     
         29 . The method of  claim 8 , wherein the mammalian cells are Chinese Hamster Ovary Cells. 
     
     
         30 . A recombinant Factor IX protein produced by the method of  claim 8 . 
     
     
         31 . A recombinant Factor VII protein produced by the method of  claim 8 . 
     
     
         32 . A pharmaceutical composition comprising the Factor IX protein of  claim 30  or the Factor VII protein of  claim 31 . 
     
     
         33 . A kit comprising the recombinant Factor IX protein of  claim 30  or the Factor VII protein of  claim 31 . 
     
     
         34 . A method of treating hemophilia which comprises administering an effective amount of the pharmaceutical composition of  claim 32  to a patient in need thereof. 
     
     
         35 . A recombinant Factor IX protein having at least 10% biological activity. 
     
     
         36 . The recombinant Factor IX protein of  claim 35 , having at least 20% biological activity. 
     
     
         37 . The recombinant Factor IX protein of  claim 35 , having at least 30% biological activity. 
     
     
         38 . The recombinant Factor IX protein of  claim 35 , having at least 40% biological activity. 
     
     
         39 . The recombinant Factor IX protein of  claim 35 , having at least 58% biological activity. 
     
     
         40 . A recombinant Factor VII/VIIa protein having at least 70% biological activity. 
     
     
         41 . A recombinant Factor VII/VIIa protein having at least 85% biological activity. 
     
     
         42 . A recombinant Factor VII/VIIa protein having at least 99% biological activity. 
     
     
         43 . A cell line which produces recombinant vitamin K dependent protein having at least 20% biological activity, wherein the cell line is generated by:
 (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; and   (b) performing at least one round of cloning and screening to identify cell clones which produce at least 10 mg/L of the Vitamin K dependent protein which is at least 10% biologically active.   
     
     
         44 . The method of  claim 43 , wherein the cloning is limit dilution cloning or semi-solid-matrix cloning. 
     
     
         45 . A pharmaceutical composition comprising the Factor VII protein of  claim 31 .

Join the waitlist — get patent alerts

Track US2012276079A1 — get alerts on status changes and closely related new filings.

We store only your email — no account needed. See our privacy policy.