Collagenase formulations and methods of producing the same

Abstract

Disclosed herein are improved collagenase-containing formulations and methods of preparing the same. The collagenase-containing formulations comprise a collagenase, about 30 mM to about 240 mM of a disaccharide, about 50 mM to about 800 mM of mannitol, and about 6 mM to about 10 mM of a Tris-HCl. Lyophilized and reconstituted formulations are also provided.

1 . A formulation comprising:
 a collagenase;   about 30 mM to about 240 mM of a disaccharide;   about 50 mM to about 800 mM of mannitol; and   about 6 mM to about 10 mM of a Tris-HCl.   
     
     
         2 . The formulation of  claim 1 , wherein the collagenase comprises a collagenase I. 
     
     
         3 . The formulation of  claim 2 , wherein the collagenase I comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         4 . The formulation of  claim 1 , wherein the collagenase comprises a collagenase II. 
     
     
         5 . The formulation of  claim 4 , wherein the collagenase II comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         6 . The formulation of  claim 1 , wherein the collagenase comprises a mixture of collagenase I and collagenase II. 
     
     
         7 . The formulation of  claim 6 , wherein the collagenase I comprises the amino acid sequence of SEQ ID NO: 1 and the collagenase II comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         8 . The formulation of  claim 7 , wherein the collagenase is collagenase  Clostridium histolyticum  (CCH). 
     
     
         9 . The formulation of  claim 1 , wherein the disaccharide comprises sucrose or trehalose. 
     
     
         10 . The formulation of  claim 1 , wherein the pH of the formulation is about 7.8 to about 8.8. 
     
     
         11 . The formulation of  claim 1 , wherein the formulation comprises:
 CCH;   about 60 mM sucrose;   about 225 mM mannitol; and   about 10 mM Tris-HCl,   wherein the formulation has a pH of about 8.5.   
     
     
         12 . The formulation of  claim 1 , further comprising a surfactant comprising polysorbate 20, polysorbate 80, or poloxamer 188. 
     
     
         13 . The formulation of  claim 12 , comprising from about 0.01% to about 2% of the surfactant. 
     
     
         14 . The formulation of  claim 13 , comprising about 0.02% of the surfactant. 
     
     
         15 . The formulation of  claim 1 , wherein the formulation is liquid. 
     
     
         16 . A lyophilized formulation comprising:
 a collagenase;   a disaccharide;   mannitol; and   Tris-HCl.   
     
     
         17 . The lyophilized formulation of  claim 16 , wherein the collagenase comprises a collagenase I. 
     
     
         18 . The lyophilized formulation of  claim 17 , wherein the collagenase I comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         19 . The lyophilized formulation of  claim 16 , wherein the collagenase comprises a collagenase II. 
     
     
         20 . The lyophilized formulation of  claim 19 , wherein the collagenase II comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         21 . The lyophilized formulation of  claim 16 , wherein the collagenase comprises a mixture of collagenase I and collagenase II. 
     
     
         22 . The lyophilized formulation of  claim 21 , wherein the collagenase I comprises the amino acid sequence of SEQ ID NO: 1 and the collagenase II comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         23 . The lyophilized formulation of  claim 21  or  22 , wherein the collagenase is collagenase  Clostridium histolyticum  (CCH). 
     
     
         24 . The lyophilized formulation of  claim 16 , wherein the disaccharide comprises sucrose or trehalose. 
     
     
         25 . The lyophilized formulation of  claim 16 , wherein, prior to lyophilization, the formulation comprises:
 CCH;   60 mM sucrose;   225 mM mannitol; and   10 mM Tris-HCl, and   having a pH of about 8.5.   
     
     
         26 . The lyophilized formulation of  claim 16 , wherein the lyophilized formulation is stable at pressures above 380 μbar. 
     
     
         27 . The lyophilized formulation of  claim 26 , wherein the lyophilized formulation is stable at a pressure of about 4000 μbar. 
     
     
         28 . The lyophilized formulation of  claim 16 , wherein the lyophilized formulation is stable at:
 (a) 2-8° C. for at least 36 months;   (b) 25° C./60% relative humidity for at least 36 months;   (c) 40° C./75% relative humidity for at least 6 months; or   (d) any combination of (a) to (c).   
     
     
         29 . The lyophilized formulation of  claim 16 , wherein the lyophilized formulation is formed by a method comprising:
 freezing the formulation at a temperature between about −25° C. and −55° C. to form a frozen formulation; and   drying the frozen formulation at a temperature between about 25° C. and about 50° C. to form the lyophilized formulation.   
     
     
         30 . The lyophilized formulation of  claim 29 , wherein the lyophilized formulation is formed by a method comprising:
 freezing the formulation at a single temperature between about −25° C. and −55° C. to form a frozen formulation; and   drying the frozen formulation at a single temperature between about 25° C. and about 50° C. to form the lyophilized formulation.   
     
     
         31 . The lyophilized formulation of  claim 16 , wherein the lyophilized formulation is formed by a lyophilization method that is performed for less than 72 hours. 
     
     
         32 . The lyophilized formulation of  claim 16 , wherein the lyophilized formulation is formed by a lyophilization method that is performed at a pressure of between about 380 μbar to about 4000 μbar. 
     
     
         33 . The lyophilized formulation of  claim 16 , in a unit-dose vial, multi-dose vial, cartridge, or syringe. 
     
     
         34 . A reconstituted formulation comprising:
 a collagenase;   a disaccharide;   mannitol;   Tris-HCl;   calcium chloride; and   sodium chloride.   
     
     
         35 . The reconstituted formulation of  claim 34 , wherein the collagenase is collagenase  Clostridium histolyticum  (CCH). 
     
     
         36 . The reconstituted formulation of  claim 35 , wherein the disaccharide comprises sucrose or trehalose. 
     
     
         37 . The reconstituted formulation of  claim 34 , wherein the reconstituted formulation is isotonic to human blood. 
     
     
         38 . A kit comprising:
 a container comprising the lyophilized formulation of  claim 16 ; and   a container comprising a sterile diluent comprising calcium chloride and sodium chloride.