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 . A method for large-scale production of Factor VII or a Factor VII-related polypeptide, comprising the steps of: 
 (i) propagating a large-scale culture of mammalian cells in medium lacking animal-derived components until the large-scale culture cells reach a second predetermined density, said large-scale culture having been created by a method comprising: 
 inoculating mammalian cells expressing Factor VII or a Factor VII-related polypeptide into a seed culture vessel containing medium lacking animal-derived components;  
 propagating the inoculated cells at least until the cells have reached a first predetermined density to form a seed culture, transferring the seed culture to a large-scale culture vessel containing medium lacking animal derived components to form said large-scale culture;  
   (ii) maintaining the large-scale culture in medium lacking animal-derived components under conditions appropriate for Factor VII expression, thereby causing the cells to produce Factor VII or a Factor VII-related polypeptide, and    (iii) recovering the produced Factor VII or Factor VII-related polypeptide from the maintained culture.    
     
     
         2 . A method as defined in  claim 1 , wherein said cells are CHO cells.  
     
     
         3 . A method as defined in  claim 1 , wherein said Factor VII has a glycosylation pattern different from both Factor VII produced in vivo and Factor VII produced in BHK cells.  
     
     
         4 . A method as defined in  claim 1 , wherein said seed culture has been transferred to and propagated in one or more intermediate size vessels of progressively larger size prior to being transferred to said large scale vessel.  
     
     
         5 . A method as defined in  claim 1 , wherein the cells have been rendered suspension culture competent prior to being inoculated into the seed vessel.  
     
     
         6 . A method as defined in  claim 1 , wherein the cells have been adapted to grow in medium lacking animal-derived components prior to said inoculation.  
     
     
         7 . A method as defined in  claim 1 , wherein the large-scale culture is a macrocarrier culture.  
     
     
         8 . A method as defined in  claim 7 , wherein the large-scale culture is a macroporous carrier culture, said macroporous carrier bearing a positive charge.  
     
     
         9 . A method as defined in  claim 1 , wherein the maintaining step comprises regularly harvesting a portion of the supernatant of said large-scale culture and replacing it with fresh medium lacking animal-derived components.  
     
     
         10 . A method as defined in  claim 1 , wherein the maintaining step comprises sedimentation of the cell-containing carriers prior to said harvesting.  
     
     
         11 . A method as defined in  claim 1 , wherein the maintaining step comprises cooling the culture to a pre-determined temperature before the sedimentation.  
     
     
         12 . A method as defined in  claim 1 , wherein the maintaining step comprises feeding said cells with glucose.  
     
     
         13 . A method as defined in  claim 12 , wherein feeding comprises pulse feeding from 1 to 4 times per 24-hour period.  
     
     
         14 . A method as defined in  claim 12 , wherein said feeding comprises gradual or continuous introduction of glucose into the large scale culture.  
     
     
         15 . A method for large-scale production of Factor VII or a Factor VII-related polypeptide, comprising the steps of: 
 (i) maintaining a large-scale culture of mammalian cells having a second predetermined density in medium lacking animal-derived components under conditions appropriate for Factor VII expression, thereby causing the cells to produce Factor VII or a Factor VII-related polypeptide, said large-scale culture having been created by a method comprising: 
 inoculating mammalian cells expressing Factor VII or a Factor VII-related polypeptide into a seed culture vessel containing medium lacking animal-derived components;  
 propagating the inoculated cells at least until the cells have reached a first predetermined density to form a seed culture, transferring the seed culture to a large-scale culture vessel containing medium lacking animal derived components to form said large-scale culture; and  
   (ii) recovering produced Factor VII or Factor VII-related polypeptide from the maintained culture.    
     
     
         16 . A method for large-scale production of Factor VII or a Factor VII-related polypeptide, comprising the steps of: 
 (i) maintaining a large-scale culture of mammalian cells having a second predetermined density in medium lacking animal-derived components under conditions appropriate for Factor VII expression, thereby causing the cells to produce Factor VII or a Factor VII-related polypeptide, said large-scale culture having been created by a method comprising: 
 inoculating mammalian cells expressing Factor VII or a Factor VII-related polypeptide into a seed culture vessel containing medium lacking animal-derived components;  
 propagating the inoculated cells at least until the cells have reached a first predetermined density to form a seed culture, and  
   (ii) transferring the seed culture to a large-scale culture vessel containing medium lacking animal derived components to form said large-scale culture.    
     
     
         17 . A Factor VII or Factor VII-related polypeptide produced by a method as defined in  claim 1 .  
     
     
         18 . A Factor VII or Factor VII-related polypeptide produced by a method as defined in  claim 15 .  
     
     
         19 . A Factor VII or Factor VII-related polypeptide produced by a method as defined in  claim 16 .  
     
     
         20 . A preparation comprising a plurality of Factor VII or Factor VII-related polypeptides expressed by recombinant BHK or CHO cells in the presence of media lacking animal-derived components (serum-free Factor VII), wherein the Factor VII or Factor VII-related polypeptides comprise N-linked oligosaccharides chains and the oligosaccharides exhibit a glycoform pattern differing from that of the same Factor VII or Factor VII-related polypeptide expressed by the same cells in the presence of serum (serum-raised Factor VII) and from that of Factor VII purified from human plasma (native Factor VII) and wherein a percentage of oligosaccharide chains in said preparation comprise at least one sialic acid moiety, said percentage being higher than that observed in serum-raised Factor VII preparations and lower than the corresponding percentage in native Factor VII preparations, said serum-free Factor VII preparation having a higher bioavailability than the bioavailability of a serum-raised Factor VII preparation.  
     
     
         21 . A preparation as defined in  claim 20 , wherein said serum-free Factor VII glycoform pattern exhibits an additional difference from that of Factor VII expressed by the same cells in the presence of serum (serum-raised Factor VII) and from that of Factor VII purified from human plasma (native Factor VII), said additional difference comprising one or more of the following: 
 (i) percentage of the oligosaccharide chains having a neutral charge, wherein the percentage of oligosaccharide chains of serum-free Factor VII having a neutral charge is lower than that of serum-raised Factor VII and higher than that of native Factor VII;    (ii) percentage of the oligosaccharide chains comprising at least one terminal galactose residue, wherein the percentage of oligosaccharide chains of serum-free Factor VII having a at least one terminal galactose residue is lower than that of serum-raised Factor VII and higher than that of native Factor VII;    (iii) percentage of the oligosaccharide chains comprising at least one terminal N-acetylgalactosamine residue, wherein the percentage of oligosaccharide chains of serum-free Factor VII having a at least one terminal N-acetyl galactose residue is lower than that of serum-raised Factor VII and higher than that of native Factor VII; and    (iv) percentage of the oligosaccharide chains comprising at least one uncapped antenna, wherein the percentage of oligosaccharide chains of serum-free Factor VII comprising at least one uncapped antenna is lower than that of serum-raised Factor VII and higher than that of native Factor VII.    
     
     
         22 . A pharmaceutical formulation comprising a polypeptide as defined in  claim 17  and a pharmaceutically acceptable carrier or adjuvant.  
     
     
         23 . A method for treating a Factor VII-responsive syndrome, the method comprising administering a pharmaceutical formulation as defined in  claim 22  to a patient in need of such treatment, under conditions that result in a decrease in bleeding and/or an increase in blood clotting.  
     
     
         24 . A method as defined in  claim 23 , 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.