Process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or a mixture of both proteins by means of ion-exchange chromatography

Abstract

A process for the preparation in pure form of the protease activating blood clotting factor VII and/or its proenzyme is described in which these are obtained from biological fluids or those obtained in the case of preparation by genetic engineering a,) by anion- or cation-exchange chromatography at a pH below the isoelectric point of the protein to be isolated or b) by a combination of anion- or cation-exchange chromatography with affinity chromatography and/or fractional precipitation at pHs of between 2.5 and 9.0, preferably between 2.5 and 7.2, the affinity chromatography being carried out using calcium phosphate/hydroxyapatite a hydrophobic matrix, a chelate matrix, a matrix which is coated with an immobilized monoclonal or polyclonal antibody directed against the protein to be isolated, or its F(ab) or F(ab) 2 fragments. A pharmaceutical preparation and a reagent are moreover described which contain the said protease and its proenzyme.

1 . A process for the preparation in pure form of the protease activating blood clotting factor VII and/or its proenzyme, wherein they are obtained from biological fluids or those obtained in the case of preparation by genetic engineering 
 a) by anion- and/or cation-exchange chromatography at a pH below the isoelectric point of the protein to be isolated or    b) by a combination of anion- or cation-exchange chromatography with affinity chromatography and/or fractional precipitation at pHs of between 2.5 and 9.0, preferably between 2.5 and 7.2, the affinity-chromatography separation processes being carried out using 
 calcium phosphate/hydroxyapatite  
 a hydrophobic matrix,  
 a chelate matrix,  
 a matrix which is coated with an immobilized monoclonal or polyclonal antibody directed against the protein to be isolated, or its F(ab) or F(ab) 2  fragments.  
   
     
     
         2 . The process as claimed in  claim 1 , wherein the preparation in pure form is carried out in the presence of one or more protein stabilizers which are selected from 
 solubilizers, preferably hydroxyproline,    detergents, preferably Tween® or Triton®,    proteins, preferably albumin, gelatin, fibronectin, vitronectin or similar proteins,    reductants, preferably dithiothreitol, mercaptoethanol or cystienee, and/or    proteinase inhibitors such as aprotinin, α-2-antiplasmin, C1-esterase inhibitor, the inter-α-trypsin inhibitor, the antithrombinIII/heparin or synthetic inhibitors.    
     
     
         3 . The process as claimed in  claim 2 , wherein, in the preparation in pure form, further protein stabilizers which are employed are 
 complexing agents of divalent ions, preferably EGTA, EDTA or citrate and/or    divalent ions, preferably calcium ions, and/or    amino acids, preferably glutamate, arginine, lysine or glycine, and/or    sugars, preferably glucose, arabinose, mannose or mannitol, and/or    alcohols, preferably ethylene glycol or polyethylene glycol.    
     
     
         4 . The process as claimed in claims  1 , wherein, for the chromatographic separation of the protease activating factor VII from its proenzyme by means of stepwise elution, a substance is immobilized on the support material which has bonds of different strength to the protease on the one hand and to the proenzyme on the other hand, and the different eluates are then collected separately from one another and the respective protein is isolated from them.  
     
     
         5 . The process as claimed in  claim 1 , wherein the fractional precipitation of the protease and/or its proenzyme from its solution is carried out by addition of 
 polyethylene glycol from a concentration of at least 10% by weight or    ammonium sulfate from a concentration of at least 15% by weight.    
     
     
         6 . A pharmaceutical preparation, which comprises the protease activating blood clotting factor VII and/or its proenzyme together with one or more protein stabilizers as set forth in claims  2  and  3 , for assisting blood clotting in the case of a tendency to bleeding, in the case of absence of factors of the endogenous clotting pathway, as FEIBA or for the prophylaxis and/or therapy of syndromes associated with thrombotic complications, in inherited or acquired deficiency states of the protease or its proenzyme, for assisting wound healing alone or as a constituent of a fibrin adhesive, a web or in combination with growth factors, for subcutaneous or intramuscular, intravenous or topical treatment.  
     
     
         7 . The use of the pharmaceutical preparation of  claim 6  for the coating of surfaces of articles, consisting of plastic or metals, to be implanted in the body, such as synthetic heart valves, blood vessels or cannulas inserted for taking blood or for artificial feeding.  
     
     
         8 . A reagent comprising the protease activating blood clotting factor VII and/or its proenzyme together with one or more protein stabilizers as set forth in claims  2  and  3  for use in biological test systems and for antigen detection.