Purification of Factor Xlll Polypeptides From Biological Materials

Abstract

The invention relates to a method for purifying a factor XIII polypeptide from a biological material, the method comprising subjecting the material to sequential chromatography on an anion-exchange matrix and a hydrophobic interaction matrix.

1 . A method for purifying a factor XIII polypeptide from a biological material comprising subjecting the material to sequential chromatography on an anion-exchange matrix and a hydrophobic interaction matrix wherein no precipitation step to produce a factor XIII polypeptide-containing precipitate is performed. 
     
     
         2 . A method according to  claim 1 , wherein the factor XIII polypeptide is a recombinant factor XIII. 
     
     
         3 . A method according to  claim 2 , wherein the factor XIII polypeptide is human factor XIII. 
     
     
         4 . A method according to  claim 2 , wherein the factor XIII polypeptide is a dimer of A subunits. 
     
     
         5 . A method according to  claim 4 , wherein the factor XIII polypeptide is a dimer of human A subunits. 
     
     
         6 . A method according to  claim 1 , wherein the biological material is a biological fluid. 
     
     
         7 . A method according to  claim 6 , wherein the biological fluid is the supernatant of a cell lysate. 
     
     
         8 . A method according to  claim 7 , wherein the biological fluid is the supernatant of a yeast cell lysate. 
     
     
         9 . A method for purifying a factor XIII polypeptide from a biological material comprising:
 (a) subjecting a biological material comprising a factor XIII polypeptide to chromatography on a first anion-exchange chromatographic material, said chromatography comprising:
 (i) applying said biological material to said first anion-exchange chromatographic material; 
 (ii) eluting unbound material from the first anion-exchange chromatographic material with a buffer A comprising one or more stabilizing agents selected from sugars, alcohols, and alditols which are capable of increasing the stability of the factor XIII polypeptide, which buffer A is suitable for eluting material not bound to the first anion-exchange chromatographic material; and 
 (iii) eluting said factor XIII polypeptide from the first anion-exchange chromatographic material by gradient-elution with buffer A′, which buffer A′ is suitable for eluting said factor XIII polypeptide from said first anion-exchange chromatographic material; and 
   (b) subjecting the eluate from step (iii), or a fluid prepared by use of the eluate from step (iii), to chromatography using a hydrophobic interaction chromatographic material, said chromatography comprising:   (iv) applying the eluate from step (iii), or a fluid prepared by use of the eluate from step (iii), to said hydrophobic interaction chromatographic material;
 (v) eluting unbound material from the chromatographic material with buffer B, which buffer B is suitable for eluting material not bound to the hydrophobic interaction chromatographic material; and 
 (vi) eluting said factor XIII polypeptide from said chromatographic material by gradient-elution with buffer B′, which buffer B′ is suitable for eluting factor XIII from said hydrophobic interaction chromatographic material. 
   
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . A method according to  claim 9 , wherein the stabilizing agent is a sugar, a C 3 -C 8 -alcohol, or an alditol. 
     
     
         13 . A method according to  claim 12 , wherein the stabilizing agent is a polyalcohol. 
     
     
         14 . A method according to  claim 12 , wherein the stabilizing agent is selected from the group consisting of glycerol, propylene glycol, propan-1,3-diol, propyl alcohol, and isopropyl alcohol. 
     
     
         15 . A method according to  claim 14 , wherein the stabilizing agent is selected from the group consisting of glycerol, propylene glycol, and propan-1,3-diol. 
     
     
         16 . A method according to  claim 15 , wherein said stabilizing agent is present in a concentration of from about 5% (v/v) to about 50% (v/v). 
     
     
         17 . A method according to  claim 16 , wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 50% (v/v). 
     
     
         18 . A method according to  claim 17 , wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 20% (v/v). 
     
     
         19 . A method according to  claim 18 , wherein said stabilizing agent is present in a concentration of about 10% (v/v). 
     
     
         20 . A method according to  claim 18 , wherein said stabilizing agent is present in a concentration of about 20% (v/v). 
     
     
         21 . A method according to  claim 9 , wherein the pH of buffer A is between about 6.5 and about 9. 
     
     
         22 . A method according to  claim 21 , wherein the pH of buffer A is between about 7 and about 9. 
     
     
         23 . A method according to  claim 22 , wherein the pH of buffer A is about 8. 
     
     
         24 . A method according to  claim 9 , wherein buffer A has a conductivity of less than about 2 mS/cm. 
     
     
         25 . A method according to  claim 9 , wherein no precipitation to form a crystalline precipitate of the factor XIII polypeptide is performed between step (iii) and step (iv). 
     
     
         26 . A method according to  claim 9 , wherein no precipitation step using sodium acetate to form a crystalline precipitate of the factor XIII polypeptide is performed between step (iii) and step (iv). 
     
     
         27 . A method according to  claim 9 , wherein the hydrophobic interaction chromatographic material uses phenyl as the ligand. 
     
     
         28 . A method according to  claim 27 , wherein the hydrophobic interaction chromatographic material is Source™ 15Phe. 
     
     
         29 . A method according to  claim 27 , wherein the hydrophobic interaction chromatographic material is Phenyl Sepharose™ 6 Fast Flow High Substitution. 
     
     
         30 . A method according to  claim 27 , wherein the hydrophobic interaction chromatographic material is Phenyl Sepharose™ High Performance High Substitution. 
     
     
         31 . A method according to  claim 9 , wherein the pH of buffer B is from about 6 to about 8. 
     
     
         32 . A method according to  claim 31 , wherein the pH of buffer B is about 7.5. 
     
     
         33 . A method according to  claim 9 , wherein buffer B has a conductivity of more than 25 mS/cm. 
     
     
         34 . A method according to  claim 33 , wherein buffer B has a conductivity of at most 50 mS/cm. 
     
     
         35 . A method according to  claim 9 , wherein the eluate from stage (vi), or a fluid prepared by use of the eluate from stage (vi), is treated by use of a method comprising a step of:
 (1) addition of one or more stabilizing agents which are capable of increasing the stability of the factor XIII polypeptide in an amount effective to significantly improve the stability thereof, and/or   (2) adjusting the pH of the eluate from stage (vi), or a fluid prepared by use of the eluate from stage (vi), to a pH between about 7 and about 8.   
     
     
         36 . A method according to  claim 35 , wherein the stabilizing agent used in step (1) is a sugar, an alcohol, or an alditol. 
     
     
         37 . A method according to  claim 36 , wherein the stabilizing agent used in step (1) is a sugar, a C 3 -C 8 -alcohol, or an alditol. 
     
     
         38 . A method according to  claim 37 , wherein the stabilizing agent used in step (1) is a polyalcohol. 
     
     
         39 . A method according to  claim 37 , wherein the stabilizing agent used in step (1) is selected from the group consisting of glycerol, propylene glycol, propan-1,3-diol, propyl alcohol, and isopropyl alcohol. 
     
     
         40 . A method according to  claim 39 , wherein the stabilizing agent used in step (1) is selected from the group consisting of glycerol, propylene glycol, and propan-1,3-diol. 
     
     
         41 . A method according to  claim 40 , wherein the stabilizing agent used in step (1) is added to a concentration of from about 5% (v/v) to about 50% (v/v). 
     
     
         42 . A method according to  claim 41 , wherein the stabilizing agent used in step (1) is added to a concentration of from about 10% (v/v) to about 50% (v/v). 
     
     
         43 . A method according to  claim 42 , wherein the stabilizing agent used in step (1) is added to a concentration of from about 10% (v/v) to about 20% (v/v). 
     
     
         44 . A method according to  claim 35  wherein the pH of the eluate from stage (vi), or of a fluid prepared by use of the eluate from stage (vi), is adjusted in step (2) to a pH between about 7 and about 8. 
     
     
         45 . A method according to  claim 44 , wherein the pH of the eluate from stage (vi), or of a fluid prepared by use of the eluate from stage (vi), is adjusted in step (2) to a pH of about 7.5. 
     
     
         46 . A method according to  claim 9 , wherein no precipitation step to produce a factor XIII polypeptide-containing precipitate is performed after step (vi). 
     
     
         47 . A method according to  claim 46 , wherein the method further comprises a step of subjecting the eluate from the hydrophobic interaction chromatography material, or a material prepared by use of the eluate from the hydrophobic interaction chromatography material, to chromatography on a second anion-exchange chromatography matrix. 
     
     
         48 . A method according to  claim 9 , wherein the method further comprises a step of:
 subjecting the eluate from stage (vi), or a fluid prepared by use of the eluate from stage (vi), to chromatography on a second anion-exchange chromatographic material, said chromatography comprising:   (vii) applying the eluate from stage (vi), or a fluid prepared by use of the eluate from stage (vi), to said second anion-exchange chromatographic material;   (viii) eluting unbound material from the second anion-exchange chromatographic material with buffer C, which buffer C is suitable for eluting material not bound to the second anion-exchange chromatographic material; and   (ix) eluting said factor XIII polypeptide from the second anion-exchange chromatographic material with buffer C′, wherein buffer C′ is suitable for eluting factor XIII polypeptides which bind to the second anion-exchange chromatographic material in step (viii).   
     
     
         49 . A method according to  claim 48 , wherein buffer C and/or buffer C′ comprises one or more stabilizing agents which are capable of increasing the stability of the factor XIII polypeptide. 
     
     
         50 . A method according to  claim 49 , wherein buffer C and/or buffer C′ comprises a stabilizing agent, which stabilizing agent is a sugar, an alcohol, or an alditol. 
     
     
         51 . A method according to  claim 50 , wherein buffer C and/or buffer C′ comprises a stabilizing agent, which stabilizing agent is a sugar, a C 3 -C 8 -alcohol, or an alditol. 
     
     
         52 . A method according to  claim 51 , wherein buffer C and/or buffer C′ comprises a stabilizing agent, which stabilizing agent is a polyalcohol. 
     
     
         53 . A method according to  claim 51 , wherein buffer C and/or buffer C′ comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1,3-diol, propyl alcohol, and isopropyl alcohol. 
     
     
         54 . A method according to  claim 53 , wherein buffer C and/or buffer C′ comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol, and propan-1,3-diol. 
     
     
         55 . A method according to  claim 54 , wherein said stabilizing agent is present in a concentration of from about 5% (v/v) to about 50% (v/v). 
     
     
         56 . A method according to  claim 55 , wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 50% (v/v). 
     
     
         57 . A method according to  claim 56 , wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 20% (v/v). 
     
     
         58 . A method according to  claim 57 , wherein said stabilizing agent is present in a concentration of about 10% (v/v). 
     
     
         59 . A method according to  claim 57 , wherein said stabilizing agent is present in a concentration of about 20% (v/v). 
     
     
         60 . A method according to  claim 48 , wherein buffer C and/or buffer C′ has a pH of about 7.5. 
     
     
         61 . A method according to  claim 48 , wherein the conductivity of the eluate from step (ix) containing the factor XIII polypeptide is adjusted to about 10 mS/cm. 
     
     
         62 . A method according to  claim 48 , wherein the pH of the eluate from step (ix) containing the factor XIII polypeptide is adjusted to about 7.5. 
     
     
         63 . A method according to  claim 54 , wherein no precipitation step to produce a factor XIII polypeptide-containing precipitate is performed after step (ix). 
     
     
         64 . A method according to  claim 12 , wherein no precipitation step to produce a factor XIII polypeptide-containing precipitate is performed. 
     
     
         65 - 71 . (canceled) 
     
     
         72 . A pharmaceutical composition comprising a factor XIII polypeptide prepared by use a method according to  claim 9 . 
     
     
         73 . A pharmaceutical composition according to  claim 72 , wherein the pharmaceutical composition is an aqueous composition and comprises 20% (v/v) propylene glycol. 
     
     
         74 . A pharmaceutical composition according to  claim 72 , wherein the pharmaceutical composition is an aqueous composition and comprises 1.5% (w/v) sucrose and 3.5% (w/v) mannitol. 
     
     
         75 . A method of reducing blood loss in a patient comprising administering to the patient an amount of a composition according to  claim 72  that is effective to reduce blood loss. 
     
     
         76 . A method of reducing the incidence of intraventricular hemorrhage in a premature infant comprising administering to the infant an amount of a composition according to  claim 72  that is effective to prevent hemorrhage. 
     
     
         77 . A method of reducing surgery-related blood loss in a patient during or after surgery comprising administering to the patient an amount of a composition according to  claim 72  that is effective to reduce blood loss during or after surgery. 
     
     
         78 . A method of treating hemophilia A comprising administering to a hemophilia A patient an amount of a composition according to  claim 72  that is effective for treating hemophilia A. 
     
     
         79 . A method of treating hemophilia B comprising administering to a hemophilia B patient an amount of a composition according to  claim 72  that is effective for treating hemophilia B. 
     
     
         80 . A method of treating a platelet disorder comprising administering to a platelet disorder patient an amount of a composition according to  claim 72  that is effective for treating platelet disorders. 
     
     
         81 - 88 . (canceled) 
     
     
         89 . A method according to  claim 35 , wherein no precipitation step to produce a factor XIII polypeptide-containing precipitate is performed after step (vi). 
     
     
         90 . A method according to  claim 15 , wherein no precipitation step to produce a factor XIII polypeptide-containing precipitate is performed after step (vi).