Processes for Preparing a Polypeptide

Abstract

The present invention relates to an improved process for preparing a polypeptide or pharmaceutically acceptable salt thereof comprising L-tyrosine, L-alanine, L-glutamate, and L-lysine. The polypeptide or pharmaceutically acceptable salt thereof is preferably glatiramer acetate. The process comprises: (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected L-glutamate and N-carboxyanhydride of a protected L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide; (b) admixing an acid with the protected polypeptide formed in Step (a) and a solvent, to form a product; and (c) admixing a substance selected from the group consisting of an alkali or alkaline earth metal hydroxide, a carbonate, a hydrogencarbonate, and mixtures thereof, with the product formed in Step (b), and a solvent or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof.

1 - 31 . (canceled) 
   
   
       32 . A process for preparing a polypeptide comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceutically acceptable salt thereof, wherein said process comprises treating a protected polypeptide with an aqueous solution of an alkali or alkaline earth metal hydroxide to form a polypeptide or a pharmaceutically acceptable salt thereof. 
   
   
       33 . A process for preparing a polypeptide comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a pharmaceutically acceptable salt thereof, wherein said process comprises:
 (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected L-glutamate and N-carboxyanhydride of a protected L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide; and   (b) adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected polypeptide formed in Step (a) to form a polypeptide or a pharmaceutically acceptable salt thereof.   
   
   
       34 . A process for preparing glatiramer acetate comprising:
 (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a γ-benzyl L-glutamate and N-carboxyanhydride of N ε -trifluoroacetyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected glatiramer;   (b) adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected glatiramer formed in Step (a) to form a glatiramer; and   (c) treating the glatiramer with acetic acid to form glatiramer acetate.   
   
   
       35 . The process according to  claim 33 , wherein Step (b) is conducted at a temperature of from about −78° C. to about 40° C. 
   
   
       36 . The process according to  claim 35 , wherein Step (b), is conducted at a temperature of from about −25° C. to about 30° C. 
   
   
       37 . The process according to  claim 36 , wherein Step (b), is conducted at a temperature of from about −10° C. to about 10° C. 
   
   
       38 . The process according to  claim 37 , wherein Step (b), is conducted at a temperature of from about 0° C. 
   
   
       39 . The process according to  claim 32  wherein the pH is from about 13 to about 14. 
   
   
       40 . The process according to  claim 32  which additionally comprises a buffer. 
   
   
       41 . The process according to  claim 40 , wherein the buffer is an acetate buffer and the pH is from about 8 to about 12. 
   
   
       42 . The process according to  claim 32 , wherein the alkali or alkaline earth metal hydroxide is selected from the group consisting of calcium hydroxide, lithium hydroxide, magnesium hydroxide, potassium hydroxide, sodium hydroxide and mixtures thereof. 
   
   
       43 . The process according to  claim 42 , wherein the alkali or alkaline earth metal hydroxide is sodium hydroxide. 
   
   
       44 . The process according to  claim 32 , wherein the alkali or alkaline earth metal hydroxide is present in an amount of from about 0.1 wt. % to about 400 wt. %, based on the total weight of the polypeptide or pharmaceutically acceptable salt thereof. 
   
   
       45 . The process according to  claim 44 , wherein the alkali or alkaline earth metal hydroxide is present in an amount of from about 10 wt. % to about 300 wt. %, based on the total weight of the polypeptide or pharmaceutically acceptable salt thereof. 
   
   
       46 . The process according to  claim 45 , wherein the alkali or alkaline earth metal hydroxide is present in an amount of from about 140 wt. % to about 260 wt. %, based on the total weight of the polypeptide or pharmaceutically acceptable salt thereof. 
   
   
       47 . A polypeptide or a pharmaceutically acceptable salt thereof which is prepared by a process comprising treating a protected polypeptide with an aqueous solution of an alkali or alkaline earth metal hydroxide to form the polypeptide or pharmaceutically acceptable salt thereof, wherein said polypeptide comprises L-tyrosine, L-alanine, L-glutamate and L-lysine. 
   
   
       48 . A polypeptide or a pharmaceutically acceptable salt thereof which is prepared by a process comprising:
 (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected L-glutamate and N-carboxyanhydride of a protected L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide; and   (b) adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected polypeptide formed in Step (a) to form a polypeptide or a pharmaceutically acceptable salt thereof.   
   
   
       49 . Glatiramer acetate which is prepared by a process comprising
 (a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a γ-benzyl L-glutamate and N-carboxyanhydride of N ε -trifluoroacetyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected glatiramer;   (b) adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected glatiramer formed in Step (a) to form a glatiramer; and   (c) treating the glatiramer formed in Step (b) with acetic acid to form glatiramer acetate.   
   
   
       50 - 77 . (canceled)