Heparin enhanced process for separating antihemophilic factor (Factor VIII) and fibronectin from cryoprecipitate

Abstract

A process is provided for preparing fibronectin and antihemophilic factor (Factor VIII) concentrates from a blood plasma cryoprecipitate.

What is claimed is: 
     
       1. A process for producing a Factor VIII concentrate from blood plasma, the process comprising the steps of: (a) obtaining a cryoprecipitate containing Factor VIII from blood plasma;   (b) dissolving the cryoprecipitate in an aqueous solution containing heparin in an amount sufficient to provide a cryoprecipitate/heparin solution containing from about 30 to about 150 units of heparin per milliliter of solution:   (c) adding a sufficient amount of a precipitant consisting essentially of PEG to the cryoprecipitate/heparin solution while maintaining the solution at a temperature of from 20° C. to 30° C. to precipitate protein contaminants, leaving a PEG supernatant containing Factor VIII;   (d) recovering the PEG supernatant; and   (e) recovering Factor VIII from the PEG supernatant.   
     
     
       2. The process of claim 1 wherein the cryoprecipitate/heparin solution contains from about 35 to about 100 units of heparin per milliliter of solution. 
     
     
       3. The process of claim 1 wherein the pH of the solution of step (c) is adjusted to from about 5.9 to about 6.2. 
     
     
       4. The process of claim 1 additionally comprising the steps of: (a) adding PEG to the PEG supernatant to provide a PEG concentration of from about 8% to about 16% to precipitate Factor VIII;   (b) recovering and then washing the Factor VIII precipitate in an aqueous solution having a glycine concentration of from about 0.1M to about 2.6M, a citrate concentration of from about 0M to about 0.5M and containing from about 10 to about 15 units per milliliter of heparin; and   (c) recovering the washed Factor VIII precipitate by centrifugation.   
     
     
       5. A Factor VIII concentrate produced by the process of claim 1. 
     
     
       6. The process of claim 1, wherein the cryoprecipitate/heparin solution contains from at least about 80 to about 150 units of heparin per milliliter of solution and the pH of the solution is adjusted to about 6.3. 
     
     
       7. A process for producing a Factor VIII concentrate from blood plasma, the process comprising the steps of: (a) obtaining a cryoprecipitate containing Factor VIII from the blood plasma;   (b) dissolving the cryoprecipitate in an aqueous solution containing heparin in an amount sufficient to provide a cryoprecipitate/heparin solution containing from about 30 to about 150 units of heparin per milliliter of solution;   (c) adding a precipitant consisting essentially of PEG to the cryoprecipitate/heparin solution, while maintaining the solution at a temperature of from 20° C. to 30° C., to provide a first solution having a PEG concentration of from about 1% to about 5% and adjusting the pH of the solution to from about 5.9 to about 6.2 to thereby precipitate unwanted protein contaminants from the solution to provide a first PEG supernatant containing Factor VIII;   (d) recovering the first PEG supernatant;   (e) adding an additional precipitant consisting essentially of PEG to the first PEG supernatant to bring the PEG concentration to from about 8% to about 16%, while maintaining the solution at a temperature of from about 15° C. to about 30° C., to thereby precipitate Factor VIII from the supernatant;   (f) recovering and then washing the Factor VIII precipitate in an aqueous solution containing (1) glycine at a concentration of from about 1.0M to about 2.6M, (2) citrate, and (3) heparin at a concentration of from about 10 to about 15 units heparin per milliliter of solution, wherein the wash is maintained at less than about 15° C.; and   (g) recovering the washed Factor VIII precipitate.   
     
     
       8. The process of claim 7 wherein during step (c) the first PEG solution is maintained at a temperature of about 25° C. during precipitation of unwanted protein contaminated. 
     
     
       9. The process of claim 7 wherein during step (c) the pH of the first PEG solution is adjusted to about 6.1. 
     
     
       10. A factor VIII concentrate produced by the process of claim 7. 
     
     
       11. The process of claim 7, wherein the cryoprecipitate/heparin solution contains from at least about 80 to about 150 units of heparin per milliliter of solution and the pH of the solution is adjusted to about 6.3. 
     
     
       12. A process for producing a Factor VIII concentrate from blood plasma, the process comprising the steps of: (a) obtaining a cryoprecipitate containing Factor VIII from blood plasma;   (b) dissolving the cryoprecipitate in an aqueous solution containing heparin in an amount sufficient to provide a cryoprecipitate/heparin solution containing from about 30 to about 150 units of heparin per milliliter of solution;   (c) adding a precipitant consisting essentially of PEG to the cryoprecipitate/heparin solution to provide a first solution having a PEG concentration of from about 1% to about 5% and adjusting the pH of the solution to from about 5.9 to about 6.2 to thereby precipitate unwanted protein contaminants from the solution to provide a first PEG supernatant containing Factor VIII;   (d) recovering the first PEG supernatant;   (e) adding additional precipitant consisting essentially of PEG to the first PEG supernatant to bring the PEG concentration to from about 8% to about 16% to thereby precipitate Factor VIII from the supernatant;   (f) recovering and then washing the Factor VIII precipitate in an aqueous solution containing (1) glycine at a concentration of from about 1 to about 2.6M, (2) citrate at a concentration of from about 0.1 to about 0.5M, and (3) heparin at a concentration of from about 10 to about 15 units of heparin per milliliter of solution, wherein the wash is maintained at less than about 15° C.; and   (g) recovering the washed Factor VIII precipitate.   
     
     
       13. A process for producing a Factor VIII concentrate from blood plasma, the process comprising the steps of: (a) obtaining a cryoprecipitate containing Factor VIII from blood plasma;   (b) dissolving the cryoprecipitate in an aqueous solution containing heparin in an amount sufficient to provide a cryoprecipitate/heparin solution containing about 80 units of heparin per milliliter of solution;   (c) adding a precipitant consisting essentially of PEG to the cryoprecipitate/heparin solution to provide a first solution having a PEG concentration of about 3% to 4% and adjusting the pH of the solution to about 6.3 to thereby precipitate unwanted protein contaminants from the solution to provide a first PEG supernatant containing Factor VIII;   (d) recovering the first PEG supernatant;   (e) adding additional precipitant consisting essentially of PEG to the first PEG supernatant to bring the PEG concentration to about 12% to thereby precipitate Factor VIII from the supernatant;   (f) recovering and then washing the Factor VIII precipitate in an aqueous solution containing (1) glycine at a concentration of about 1.6M, (2) citrate at a concentration of about 0.2M, and (3) heparin at a concentration of from about 10 to about 15 units of heparin per milliliter of solution, wherein the wash is maintained at less than about 15° C.; and   (g) recovering the washed Factor VIII precipitate.