Factor VII glycoforms

Abstract

The present invention provides preparations of Factor VIIa polypeptides or Factor VIIa-related polypeptides that exhibit predetermined glycoform patterns. The preparations of the invention exhibit improved functional properties and are useful for treating Factor VII-mediated conditions.

1 - 24 . (canceled)  
     
     
         25 . A preparation comprising a plurality of Factor VII polypeptides or Factor VII-related polypeptides produced in Chinese Hamster Ovary (CHO) cells in the absence of serum, wherein said cells are adapted to grow in the absence of serum and are cultured in the absence of serum both in the growth phase and in the production phase.  
     
     
         26 . A preparation as defined in  claim 25 , wherein the Factor VII polypeptides are selected from the group consisting of: human S52A-Factor VII, human S60A-Factor VII, human Factor VII that has been proteolytically cleaved between residues 290 and 291; human Factor VII that has been proteolytically cleaved between residues 315 and 316; and Factor VII that has been oxidized, wherein wild-type human Factor VII has the sequence of SEQ ID NO:3.  
     
     
         27 . A preparation as defined in  claim 25 , wherein the human Factor VII-related polypeptides are selected from the group consisting of: R152E-Factor VII, S344A-Factor VII, FFR-Factor VII, and Factor VIIa lacking the Gla domain, wherein wild-type human Factor VII has the sequence of SEQ ID NO:3.  
     
     
         28 . A preparation as defined in  claim 25 , wherein the preparation exhibits a bioavailability that is at least 110% of the bioavailability of a reference preparation.  
     
     
         29 . A preparation as defined in  claim 28 , wherein the preparation exhibits a bioavailability that is at least 120% of the bioavailability of a reference preparation.  
     
     
         30 . A preparation as defined in  claim 29 , wherein the preparation exhibits a bioavailability that is at least 130% of the bioavailability of a reference preparation.  
     
     
         31 . A preparation as defined in  claim 30 , wherein the preparation exhibits a bioavailability that is at least 140% of the bioavailability of a reference preparation.  
     
     
         32 . A preparation as defined in  claim 25 , wherein the preparation exhibits tissue factor-independent thrombin generating activity that is at least 110% that of a reference preparation.  
     
     
         33 . A preparation as defined in  claim 25 , wherein the preparation exhibits a bioavailability that is at least 110% of the bioavailability of a reference preparation, wherein less than about 93% of the oligosaccharide chains in the reference preparation comprise at least one sialic acid moiety.  
     
     
         34 . A method for producing a preparation comprising Factor VII polypeptides or Factor VII-related polypeptides having a predetermined pattern of N-linked glycosylation, said method comprising culturing a Chinese Hamster Ovary (CHO) cell that has been adapted to grow in the absence of serum in a medium lacking serum for both growth and production phases.  
     
     
         35 . A pharmaceutical formulation comprising a preparation as defined in  claim 25  and a pharmaceutically acceptable carrier or adjuvant.  
     
     
         36 . A method for treating a Factor VII-responsive syndrome, the method comprising administering a pharmaceutical formulation as defined in  claim 35  to a patient in need of such treatment, under conditions that result in a decrease in bleeding and/or an increase in blood clotting.  
     
     
         37 . A method as defined in  claim 36 , wherein the syndrome is selected from the group consisting of haemophilia A, haemophilia B, Factor XI deficiency, Factor VII deficiency, thrombocytopenia, von Willebrand's disease, presence of a clotting factor inhibitor, surgery, trauma, and anticoagulant therapy.  
     
     
         38 . A method for decreasing bleeding and/or increasing clotting, the method comprising administering a pharmaceutical formulation as defined in  claim 35  to a patient in need of such treatment, under conditions that result in a decrease in bleeding and/or an increase in blood clotting.  
     
     
         39 . A preparation comprising a plurality of Factor VII polypeptides or Factor VII-related polypeptides produced in Chinese Hamster Ovary (CHO) cells in the absence of animal-derived components, wherein said cells are adapted to grow in the absence of animal-derived components and are cultured in the absence of animal-derived components both in the growth phase and in the production phase.  
     
     
         40 . A preparation as defined in  claim 39 , wherein the preparation exhibits a bioavailability that is at least 110% of the bioavailability of a reference preparation, wherein the oligosaccharides of the reference preparation lack fucose linked α1−>3 to an antennary N-acetylglucosamine.  
     
     
         41 . A preparation as defined in  claim 40 , wherein the preparation exhibits a bioavailability that is at least 120% of the bioavailability of a reference preparation.  
     
     
         42 . A preparation as defined in  claim 41 , wherein the preparation exhibits a bioavailability that is at least 130% of the bioavailability of a reference preparation.  
     
     
         43 . A preparation as defined in  claim 42 , wherein the preparation exhibits a bioavailability that is at least 140% of the bioavailability of a reference preparation.  
     
     
         44 . A preparation as defined in  claim 39 , wherein the preparation exhibits tissue factor-independent thrombin generating activity that is at least 110% that of a reference preparation.  
     
     
         45 . A preparation as defined in  claim 44 , wherein the preparation exhibits a bioavailability that is at least 110% of the bioavailability of a reference preparation, wherein less than about 93% of the oligosaccharide chains in the reference preparation comprise at least one sialic acid moiety.  
     
     
         46 . A method for producing a preparation comprising Factor VII polypeptides or Factor VII-related polypeptides having a predetermined pattern of N-linked glycosylation, said method comprising culturing a Chinese Hamster Ovary (CHO) cell that has been adapted to grow in the absence of animal-derived components in a medium lacking animal-derived components for both growth and production phases.  
     
     
         47 . A pharmaceutical formulation comprising a preparation as defined in  claim 46  and a pharmaceutically acceptable carrier or adjuvant.  
     
     
         48 . A method for treating a Factor VII-responsive syndrome, the method comprising administering a pharmaceutical formulation as defined in  claim 47  to a patient in need of such treatment, under conditions that result in a decrease in bleeding and/or an increase in blood clotting.  
     
     
         49 . A method as defined in  claim 48 , wherein the syndrome is selected from the group consisting of haemophilia A, haemophilia B, Factor XI deficiency, Factor VII deficiency, thrombocytopenia, von Willebrand's disease, presence of a clotting factor inhibitor, surgery, trauma, and anticoagulant therapy.  
     
     
         50 . A method for decreasing bleeding and/or increasing clotting, the method comprising administering a pharmaceutical formulation as defined in  claim 47  to a patient in need of such treatment, under conditions that result in a decrease in bleeding and/or an increase in blood clotting, wherein the formulation comprises Factor VII polypeptides.