Method of purifying therapeutic proteins

Abstract

The present invention relates generally to a method of reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease(s) in a solution comprising at least one protein selected from the group consisting of fibrinogen, Factor VIII and von Willebrand factor (VWF), the method comprising: (i) passing a feedstock comprising at least one protein selected from the group consisting of fibrinogen, Factor VIII and VWF through a hydrophobic charge-induction chromatographic resin under conditions selected such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease(s) present in the feedstock is bound to the resin; and (ii) recovering a solution comprising the at least one protein selected from the group consisting of fibrinogen, Factor VIII and VWF which passes through the resin, wherein the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease(s) in the solution is reduced by at least 50% compared to the feedstock. Also provided are solutions and pharmaceutical formulations comprising the fibrinogen and/or Factor VIII and/or VWF recovered by such methods, and uses thereof.

1 - 37 . (canceled) 
     
     
         38 . A method of treating or preventing a condition associated with fibrinogen deficiency, the method comprising administering to a subject in need thereof a solution comprising fibrinogen, recovered by the method of:
 (i) applying a feedstock comprising fibrinogen to a hydrophobic charge-induction chromatographic resin equilibrated at a pH from 6.5 to 8.5, wherein upon application to the resin, the proteases bind to the resin while the fibrinogen passes through the resin in the unbound flow-through fraction; and   (ii) recovering a solution comprising the fibrinogen which passes through the resin;   wherein the concentration of proteases in the recovered solution is reduced by at least 50% compared to the feedstock.   
     
     
         39 . The method of  claim 38 , wherein the condition is selected from the group consisting of afibrinogenemia, hypofibrinogenemia and dysfibrinogenemia. 
     
     
         40 - 48 . (canceled) 
     
     
         49 . The method of  claim 38 , wherein the solution comprises at least 80% total protein of fibrinogen. 
     
     
         50 . The method of  claim 38 , wherein the solution comprises:
 (a) at least 75% total protein of fibrinogen;   (b) less than 50 pg/mg total protein of tissue plasminogen activator; and/or   (c) less than 1 pg/mg total protein of plasminogen.   
     
     
         51 . The method of  claim 50 , wherein the solution further comprises less than 1.5×10 −5  U/mg total protein of Factor II. 
     
     
         52 . The method of  claim 38 , wherein the solution comprises:
 (a) at least 90% total protein of fibrinogen;   (b) less than 50 pg/mg total protein of tissue plasminogen activator; and/or   (c) less than 150 ng/mg total protein of plasminogen.   
     
     
         53 . The method of  claim 52 , wherein the solution further comprises:
 (a) less than 3.5×10 −6  U/mg total protein of Factor II; and/or   (b) less than 150 pg/mg total protein of fibronectin.   
     
     
         54 . The method of  claim 38 , wherein the solution comprises:
 (a) at least 90% total protein of fibrinogen;   (b) less than 20 pg/mg total protein of tissue plasminogen activator; and/or   (c) less than 10 ng/mg total protein of plasminogen.   
     
     
         55 . The method of  claim 54 , wherein the solution further comprises:
 (a) less than 2.7×10 −6  U/mg total protein of Factor II; and/or   (b) less than 15 pg/mg total protein of fibronectin.   
     
     
         56 . The method of  claim 38 , wherein the solution comprises at least 80% total protein of monomeric fibrinogen. 
     
     
         57 . The method of  claim 38 , wherein the fibrinogen retains from about 90% to 100% activity after at least 4 weeks in storage of the solution at a temperature of about 0° C. to about 8° C. 
     
     
         58 . The method of  claim 38 , wherein the fibrinogen retains from about 60% to about 70% activity after 5 weeks in storage of the solution at a temperature of about 30° C.