Method of purifying therapeutic proteins

Abstract

The present invention relates generally to a method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a solution comprising fibrinogen and/or Factor VIII and/or von Willebrand factor (VWF), the method comprising: (i) passing a feedstock comprising fibrinogen and/or Factor VIII and/or VWF through a hydrophobic charge-induction chromatographic resin under conditions selected such that the plasminogen and/or tissue plasminogen activator and/or other protease(s) is bound to the resin; and (ii) recovering the solution comprising fibrinogen and/or Factor VIII and/or VWF which passes through the resin; wherein the concentration of the plasminogen and/or tissue plasminogen activator and/or protease(s) in the recovered 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 . A method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a solution comprising fibrinogen and/or Factor VIII and/or von Willebrand factor (VWF), the method comprising:
 (i) passing a feedstock comprising fibrinogen and/or Factor VIII and/or VWF through a hydrophobic charge-induction chromatographic resin under conditions selected such that the plasminogen and/or tissue plasminogen activator and/or other protease(s) is bound to the resin; and   (ii) recovering a solution comprising fibrinogen and/or Factor VIII and/or VWF which passes through the resin;   
       wherein the concentration of plasminogen and/or tissue plasminogen activator and/or proteases(s) in the recovered solution is reduced by at least 50% compared to the feedstock. 
     
     
         2 . A method of reducing the level of plasminogen and/or tissue plasminogen activator and/or other protease(s) in a solution comprising fibrinogen and/or Factor VIII and/or von Willebrand factor (VWF), the method comprising:
 (i) passing a feedstock comprising fibrinogen and/or Factor VIII and/or VWF through a first hydrophobic charge-induction chromatographic resin;   (ii) recovering a solution comprising fibrinogen and/or Factor VIII and/or VWF which passes through the first hydrophobic charge-induction chromatographic resin;   (iii) passing the solution that is recovered in step (ii) through a second hydrophobic charge-induction chromatographic resin; and   (iv) recovering a solution comprising fibrinogen and/or Factor VIII and/or VWF which passes through the second hydrophobic charge-induction chromatographic resin;   
       wherein the conditions of the chromatographic steps are such that the plasminogen and/or tissue plasminogen activator and/or other protease(s) is bound to the first and/or second resin, and wherein the concentration of plasminogen and/or tissue plasminogen activator and/or other protease(s) in the solution that is recovered in step (iv) is reduced by at least 50% compared to the feedstock. 
     
     
         3 . The method of  claim 2  wherein the first and second hydrophobic charge-induction chromatographic resins are the same. 
     
     
         4 . The method of  claim 1 , further comprising passing the solution comprising fibrinogen and/or Factor VIII and/or VWF recovered in step (ii) through an anion exchange chromatographic resin. 
     
     
         5 . The method of  claim 2 , further comprising passing the solution comprising fibrinogen and/or Factor VIII and/or VWF recovered in step (ii) and/or step (iv) through an anion exchange chromatographic resin. 
     
     
         6 . The method of  claim 1 , further comprising passing the feedstock comprising fibrinogen and/or Factor VIII and/or VWF through an anion exchange chromatographic resin prior to step (i). 
     
     
         7 . The method of  claim 4 , wherein the anion exchange resin is a strong anion exchange resin. 
     
     
         8 . The method of  claim 4 , wherein the solution comprising the fibrinogen and/or Factor VIII and/or VWF is passed through the anion exchange chromatographic resin in the presence of about 170 mM to about 230 mM NaCl. 
     
     
         9 . The method of  claim 4 , wherein fibrinogen is eluted from the anion exchange chromatographic resin with an elution buffer comprising from about 150 mM to about 300 mM NaCl. 
     
     
         10 . The method of  claim 7 , wherein fibrinogen is eluted from the anion exchange chromatographic resin with an elution buffer comprising a free amino acid at a concentration of about 1-3% w/v. 
     
     
         11 . The method of  claim 10 , wherein the free amino acid is arginine. 
     
     
         12 . The method of  claim 1 , wherein the feedstock comprising fibrinogen and/or Factor VIII and/or VWF is subjected to a viral inactivation step prior to step (i). 
     
     
         13 . The method of  claim 1 , wherein the solution comprising fibrinogen and/or Factor VIII and/or VWF recovered from the hydrophobic charge-induction chromatographic resin in step (ii) is subjected to a viral inactivation step. 
     
     
         14 . The method of  claim 4 , wherein the feedstock or the solution comprising fibrinogen and/or Factor VIII and/or VWF recovered in step (ii) is subjected to a viral inactivation step before it is passed through the anion exchange chromatographic resin. 
     
     
         15 . The method of  claim 11 , wherein the viral inactivation step comprises pasteurisation or treatment with an organic solvent and detergent. 
     
     
         16 . The method of  claim 1 , wherein the feedstock comprising fibrinogen and/or Factor VIII and/or VWF is a solubilised plasma cryoprecipitate. 
     
     
         17 . The method of  claim 1 , wherein, prior to step (i), vitamin K-dependent proteins are removed or reduced from the feedstock. 
     
     
         18 . The method of  claim 17 , wherein the vitamin K-dependent proteins are removed or reduced by precipitating the vitamin K-dependent proteins from the feedstock by adding aluminium hydroxide to the feedstock. 
     
     
         19 . The method of  claim 1 , further comprising, prior to step (i), precipitating the fibrinogen and/or Factor VIII and/or VWF from the feedstock by adding glycine to the feedstock, recovering the precipitated fibrinogen and/or Factor VIII and/or VWF, solubilising the precipitated fibrinogen and/or Factor VIII and/or VWF, wherein the solubilised fibrinogen and/or Factor VIII and/or VWF is passed through the hydrophobic charge-induction chromatographic resin in step (i). 
     
     
         20 . The method of  claim 1 , wherein the feedstock has a pH from about 6.5 to about 8.5. 
     
     
         21 . The method of  claim 1 , wherein the hydrophobic charge-induction chromatographic resin is equilibrated at a pH from about 6.5 to about 8.5 prior to passing the feedstock through the resin. 
     
     
         22 . The method of  claim 1 , wherein the hydrophobic charge-induction chromatographic resin comprises a ligand selected from mercaptoethylpyridine, n-hexylamine and phenylpropylamine. 
     
     
         23 . The method of  claim 22 , wherein the hydrophobic charge-induction chromatographic resin comprises n-hexylamine. 
     
     
         24 . A solution comprising fibrinogen and/or Factor VIII and/or VWF recovered by the method of  claim 1 . 
     
     
         25 . The solution of  claim 24 , wherein at least 80% of the total protein of the solution comprises fibrinogen. 
     
     
         26 . A solution comprising fibrinogen, wherein:
 (a) at least 75% of the total protein of the solution comprises fibrinogen;   (b) less than 50 pg/mg of the total protein comprises tissue plasminogen activator; and   (c) less than 1 μg/mg of the total protein comprises plasminogen.   
     
     
         27 . The solution of  claim 26 , wherein less than 1.5×10 −5  U/mg of the total protein comprises Factor II. 
     
     
         28 . The solution of  claim 26 , wherein:
 (a) at least 90% of the total protein comprises fibrinogen;   (b) less than 50 pg/mg of the total protein comprises tissue plasminogen activator; and   (c) less than 150 ng/mg of the total protein comprises plasminogen.   
     
     
         29 . The solution of  claim 28 , wherein:
 (a) less than 3.5×10 −6  U/mg of the total protein comprises Factor II; and/or   (b) less than 150 μg/mg of the total protein comprises fibronectin.   
     
     
         30 . The solution of  claim 26 , wherein:
 (a) at least 90% of the total protein comprises fibrinogen;   (b) less than 20 pg/mg of the total protein comprises tissue plasminogen activator; and   (c) less than 10 ng/mg of the total protein comprises plasminogen.   
     
     
         31 . The solution of  claim 30 , wherein
 (a) less than 2.7×10 −6  U/mg of the total protein comprises Factor II; and/or   (b) less than 15 μg/mg of the total protein comprises fibronectin.   
     
     
         32 . The solution of  claim 25 , wherein at least 80% of the total protein comprises monomeric fibrinogen. 
     
     
         33 . The solution of  claim 26 , wherein the fibrinogen retains from about 90% to 100% activity after at least 4 weeks in storage at a temperature of about 0° C. to about 8° C. 
     
     
         34 . The solution of  claim 26 , wherein the fibrinogen retains from about 60% to about 70% activity after 5 weeks in storage at a temperature of about 30° C. 
     
     
         35 . A pharmaceutical formulation comprising the solution of  claim 26  and a pharmaceutically acceptable carrier. 
     
     
         36 . The pharmaceutical formulation of  claim 35  having a volume of at least 5 mL and comprising at least 5 mg/mL fibrinogen. 
     
     
         37 . The pharmaceutical formulation of  claim 35  having a volume of at least 5 mL and comprising at least 20 mg/mL fibrinogen. 
     
     
         38 . A method of treating or preventing a condition associated with fibrinogen deficiency, the method comprising administering to a subject in need thereof the solution of  claim 26 . 
     
     
         39 . The method of  claim 38 , wherein the condition is selected from afibrinogenemia, hypofibrinogenemia and dysfibrinogenemia. 
     
     
         40 .- 41 . (canceled) 
     
     
         42 . A fibrin glue comprising the solution of  claim 26 . 
     
     
         43 . A method of producing a stable liquid fibrinogen solution, the method comprising:
 (i) passing a feedstock comprising fibrinogen through a hydrophobic charge-induction chromatographic resin under conditions selected such that plasminogen and/or tissue plasminogen activator and/or other protease(s) are bound to the resin; and   (ii) recovering a solution comprising fibrinogen which passes through the resin;   
       wherein the concentration of plasminogen and/or tissue plasminogen activator and/or proteases(s) in the recovered solution is reduced by at least 50% compared to the feedstock. 
     
     
         44 . A method of producing a stable liquid fibrinogen solution, the method comprising:
 (i) passing a feedstock comprising fibrinogen through a first hydrophobic charge-induction chromatographic resin;   (ii) recovering a solution comprising fibrinogen which passes through the first hydrophobic charge-induction chromatographic resin;   (iii) passing the solution that is recovered in step (ii) through a second hydrophobic charge-induction chromatographic resin; and   (iv) recovering a solution comprising fibrinogen which passes through the second hydrophobic charge-induction chromatographic resin;   
       wherein the conditions of the chromatographic steps are such that plasminogen and/or tissue plasminogen activator and/or other protease(s) are bound to the first and/or second resin, and wherein the concentration of plasminogen and/or tissue plasminogen activator and/or other protease(s) in the solution that is recovered in step (iv) is reduced by at least 50% compared to the feedstock. 
     
     
         45 . The method of  claim 43 , wherein the stable liquid fibrinogen solution retains from about 90% to 100% activity after at least 4 weeks in storage at a temperature of about 0° C. to about 8° C. 
     
     
         46 . The method of  claim 43 , wherein the stable liquid fibrinogen solution retains from about 60% to about 70% activity after at least 5 weeks in storage at a temperature of about 30° C. 
     
     
         47 . A vessel containing at least 5 mL of the stable liquid fibrinogen solution prepared according to  claim 43 , wherein the concentration of fibrinogen is at least 20 mg/mL.