Formulation, Solubilization, Purification, and Refolding of Tissue Factor Pathway Inhibitor

Abstract

Compositions are described that are suitable for formulating TFPI. Solubilizers and stabilizers facilitate the preparation of pharmaceutically acceptable compositions of TFPI at various concentrations.

1 . A method of increasing the solubility of a polypeptide selected from the group consisting of (i) human TFPI having SEQ ID No.: 1, (ii) ala-human TFPI having SEQ ED No.: 1 and having one further amino acid which is an ammo-terminal alanine, and (iii) muteins of (i) or (ii) having from 1 to 5 amino acid substitutions, comprising the step of:
 preparing an aqueous composition comprising the polypeptide and a charged polymer.   
     
     
         2 . The method of  claim 1  wherein the charged polymer is selected from the group consisting of dextran sulfate, glycosaminoglycan, heparin, polyaspartate, polyglutamate, agaropectin, alginic acid, carboxymethyl cellulose, polyphosphate, polyethyleneimine, polyethyleneimine cellulose, DEAE dextran, polylysine and polyarginine. 
     
     
         3 . The method of  claim 2  wherein the charged polymer is polyphosphate. 
     
     
         4 . The method of  claim 1  wherein the aqueous composition comprises at least about 0.5 mg/ml of the polypeptide. 
     
     
         5 . The method of  claim 3  wherein the polyphosphate is added to a final concentration of from 1 to 20 mg/ml. 
     
     
         6 . The method of  claim 3  wherein the aqueous composition comprises 3 M urea and 50 mM Tris at a pH of 10.5. 
     
     
         7 . The method of  claim 3  wherein the aqueous composition comprises at least about 0.5 mg/ml polypeptide, 4 mg/ml polyphosphate, 0.1 mM cysteine, 0.05 mM cystine, and 50 mM Tris at a pH of about 10.5. 
     
     
         8 . The method of  claim 3  wherein polyphosphate is added to a final concentration providing a weight ratio of the polypeptide to polyphosphate of from about 2 to 1 to about 1 to 8. 
     
     
         9 . The method of  claim 8  wherein, prior to adding polyphosphate, the polypeptide is present in a solution comprising 6 M urea, 125 mM sodium chloride, and 20 mM sodium phosphate buffer, said solution having a pH of about 7.4. 
     
     
         10 . The method of  claim 8  further comprising the step of:
 removing low molecular weight solutes from the composition comprising the polypeptide and polyphosphate, to form an aqueous composition comprising the polypeptide and polyphosphate essentially free of other solutes, wherein the concentration of the polypeptide in the composition is greater than 0.5 mg/ml.   
     
     
         11 . The method of  claim 1  wherein the polypeptide is present in an insoluble form prior to preparing the aqueous composition. 
     
     
         12 . The method of  claim 11  wherein the insoluble form is an inclusion body. 
     
     
         13 . The method of  claim 1  wherein the aqueous composition further comprises a chaotrope. 
     
     
         14 . The method of  claim 1  further comprising the step, prior to preparing the aqueous composition, of:
 applying the polypeptide to a solid support.   
     
     
         15 . The method of  claim 14  wherein the aqueous composition is formed by adding polyphosphate in a concentration gradient to effect selective elution of the polypeptide from the solid support. 
     
     
         16 . The method of  claim 14  wherein the solid support is an ion exchange resin. 
     
     
         17 . The method of  claim 16  wherein the resin has a negative net charge. 
     
     
         18 . The method of  claim 16  wherein the resin has a positive net charge. 
     
     
         19 . The method of  claim 1  further comprising the step, after the step of preparing the aqueous composition, of:
 applying the polypeptide to a solid support.   
     
     
         20 . The method of  claim 19  wherein the solid support is an ion exchange resin. 
     
     
         21 . The method of  claim 20  wherein the resin has a net negative charge. 
     
     
         22 . The method of  claim 20  wherein the resin has a positive net charge. 
     
     
         23 . The method of  claim 1  wherein the polypeptide is ala-human TFPI having SEQ ID No.: 1 and having one further amino acid which is an amino-terminal alanine. 
     
     
         24 . A method to aid in refolding a polypeptide selected from the group consisting of (i) human TFPI having SEQ ED No.: 1, (ii) ala-human TFPI having SEQ ID No.: 1 and having one further amino acid which is an amino-terminal alanine, and (iii) muteins of (i) or (ii) having from 1 to 5 amino acid substitutions, comprising the step of:
 preparing an aqueous composition comprising the polypeptide and a charged polymer.   
     
     
         25 . The method of  claim 24  wherein the charged polymer is selected from the group consisting of dextran sulfate, glycosaminoglycan, heparin, polyaspartate, polyglutamate, agaropectin, alginic acid, carboxymethyl cellulose, polyphosphate, polyemyleneimine, polyemyleneimine cellulose, DEAE dextran, polylysine and polyarginine. 
     
     
         26 . The method of  claim 25  wherein the charged polymer is polyphosphate. 
     
     
         27 . The method of  claim 24  wherein the solution further comprises urea. 
     
     
         28 . The method of  claim 24  wherein the solution is essentially free of urea. 
     
     
         29 . The method of  claim 24  wherein the solution has a pH of from about 9 to at least about 11. 
     
     
         30 . The method of  claim 24  wherein the polypeptide is allowed to refold for at least about 72 hours. 
     
     
         31 . The method of  claim 24  wherein the refolding process is terminated after from about 72 hours to about 120 hours by lowering the pH of the solution to 5.9+/−0.1. 
     
     
         32 . The method of  claim 27  wherein refolding occurs in a solution comprising about 1 mg/ml polypeptide, about 2 mg/ml polyphosphate, 3 M urea, 0.1 mM cysteine, and 50 mM Tris, having a pH of about 10.5. 
     
     
         33 . The method of  claim 28  wherein refolding occurs in a solution comprising about 0.5 mg/ml polypeptide, about 4 mg/ml polyphosphate, 0.1 mM cysteine, 0.05 mM cystine, and 50 mM Tris, having a pH of about 10.5. 
     
     
         34 . A method to aid in purification of a polypeptide selected from the group consisting of (i) full length, properly folded human TFPI having SEQ ID No.: 1; (ii) full length, properly folded ala-human TFPI having SEQ ID No.: 1 and having one further amino acid which is an ammo-terminal alanine; and (iii) a full length, properly folded mutein selected from the group consisting of muteins having from 1 to 5 amino acid substitutions of (i) or (ii); the method comprising the step of:
 adding a second charged polymer to a composition comprising said polypeptide and a first charged polymer, said second charged polymer bearing a net charge which is opposite to the net charge of said first charged polymer, wherein the amount of said second charged polymer added is sufficient to essentially neutralize said first charged polymer.   
     
     
         35 . The method of  claim 34  wherein the polypeptide is full length, properly folded human TFPI having SEQ ID No.: 1. 
     
     
         36 . The method of  claim 34  wherein the polypeptide is full length, properly folded ala-human TFPI having SEQ ID No.: 1 and having one further amino acid which is an amino-terminal alanine. 
     
     
         37 . The method of  claim 34  wherein the first charged polymer is polyphosphate. 
     
     
         38 . The method of  claim 37  wherein the second charged polymer is polyethyleneimine.