METHOD OF PURIFYING THERAPEUTIC PROTEINS
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.