Processes for preparing a polypeptide

Abstract

The present invention relates to processes 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.

1 . 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: 
 (i) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of protected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide, wherein the protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures thereof; and    (ii) adding an acid to the protected polypeptide formed in Step (i) to form a polypeptide or a pharmaceutically acceptable salt thereof, wherein said acid cleaves the γ-p-methoxybenzyl group from the glutamate moiety and the N-t-butoxycarbonyl group from the lysine moiety.    
   
   
       2 . 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 protected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected glatiramer, wherein the protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate, and mixtures thereof;    (b) adding an acid to the protected glatiramer formed in Step (a) to form a glatiramer, wherein said acid cleaves the γ-p-methoxybenzyl group from the glutamate moiety and the N-t-butoxycarbonyl group from the lysine moiety; and    (c) treating the glatiramer formed in Step (b) with acetic acid to form glatiramer acetate.    
   
   
       3 . The process according to  claim 1 , wherein the acid is selected from the group consisting of acetic acid, hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane sulfonic acid, trifluoromethane sulfonic acid, phosphoric acid, trifluroacetic acid, sulfuric acid and mixtures thereof.  
   
   
       4 . The process according to  claim 3 , wherein the acid is a mixture of acetic acid and hydrochloric acid.  
   
   
       5 . The process according to  claim 3 , wherein the acid is a mixture of acetic acid and hydrogen bromide.  
   
   
       6 . The process according to  claim 3 , wherein the acid is a mixture of acetic acid and sulfuric acid.  
   
   
       7 . The process according to  claim 3 , wherein the acid is trifluroacetic acid.  
   
   
       8 . The process according to  claim 2 , wherein the glatiramer acetate has a weight average molecular weight of about 2 kDa to about 30 kDa.  
   
   
       9 . The process according to  claim 8 , wherein the glatiramer acetate has a weight average molecular weight of about 4.7 kDa to about 11 kDa.  
   
   
       10 . The process according to  claim 9 , wherein the glatiramer acetate has a weight average molecular weight of about 7 kDa to about 10 kDa.  
   
   
       11 . The process according to  claim 1 , wherein the polypeptide is substantially free of polypeptide fragments having a molecular weight of greater than about 40 kDa.  
   
   
       12 . The process according to  claim 1 , wherein the polypeptide is substantially free of polypeptide fragments having a molecular weight of less than about 2 kDa.  
   
   
       13 . The process according to  claim 1 , wherein Step (ii) is conducted at a temperature of from about 10° C. to about 40° C.  
   
   
       14 . The process according to  claim 13 , wherein Step (ii), is conducted at a temperature of from about 15° C. to about 30° C.  
   
   
       15 . The process according to  claim 14 , wherein Step (ii), is conducted at a temperature of about 22° C. to about 25° C.  
   
   
       16 . The process according to  claim 1 , wherein the initiator is diethylamine.  
   
   
       17 . The process according to  claim 1 , wherein the polar aprotic solvent is selected from the group consisting of tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide, 1,4-dioxane, dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxide, dichloromethane and mixtures thereof.  
   
   
       18 . The process according to  claim 17 , wherein the polar aprotic solvent is 1,4-dioxane.  
   
   
       19 . The process according to  claim 17 , wherein the polar aprotic solvent is tetrahydrofuran.  
   
   
       20 . The process according to  claim 1 , wherein the acid is present in an amount of from about 0.1 weight percent (wt. %) to about 100 wt. %, based on the total weight of the polypeptide or pharmaceutically acceptable salt thereof.  
   
   
       21 . The process according to  claim 20 , wherein the acid is present in an amount of from about 1 wt. % to about 10 wt. %, based on the total weight of the polypeptide or pharmaceutically acceptable salt thereof.  
   
   
       22 . The process according to  claim 21 , wherein the acid is present in an amount of from about 2 wt. % to about 6 wt. %, based on the total weight of the polypeptide or pharmaceutically acceptable salt thereof.  
   
   
       23 . A polypeptide or a pharmaceutically acceptable salt thereof which is prepared by a process comprising: 
 (i) polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of protected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected polypeptide, wherein the protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures thereof; and    (ii) adding an acid to the protected polypeptide formed in Step (i) to form a polypeptide or a pharmaceutically acceptable salt thereof, wherein said acid cleaves the γ-p-methoxybenzyl group from the glutamate moiety and the N-t-butoxycarbonyl group from the lysine moiety.    
   
   
       24 . 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 protected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected glatiramer, wherein the protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate, and mixtures thereof;    (b) adding an acid to the protected glatiramer formed in Step (a) to form a glatiramer, wherein said acid cleaves the γ-p-methoxybenzyl group from the glutamate moiety and the N-t-butoxycarbonyl group from the lysine moiety; and    (c) treating the glatiramer formed in Step (b) with acetic acid to form glatiramer acetate.    
   
   
       25 . The glatiramer acetate which is prepared according to the process of  claim 24 , wherein the glatiramer having a weight average molecular weight of less than 2 kDa is removed prior to Step (c).  
   
   
       26 . The glatiramer acetate according to  claim 25 , wherein the glatiramer having a weight average molecular weight of less than 2 kDa is removed by a method selected from the group consisting of dialysis and diafiltration.  
   
   
       27 . The glatiramer acetate according to  claim 26 , wherein the dialysis is initially conducted in water followed by dialysis in an aqueous acetic acid solution.  
   
   
       28 . The glatiramer acetate which is prepared according to the process of  claim 24 , wherein the process additionally comprises a lyophilization step after treating the glatiramer with acetic acid.  
   
   
       29 . A method for treating a patient having an inflammatory, non-autoimmune central nervous system disease or alleviating the symptoms of such a disease, comprising administering a polypeptide or a pharmaceutically acceptable salt thereof prepared according to the process of  claim 1  to the patient in an amount and for a duration of time effective to treat the inflammatory, non-autoimmune central nervous system disease.  
   
   
       30 . A method for treating a patient having an inflammatory, non-autoimmune central nervous system disease or alleviating the symptoms of such a disease, comprising administering glatiramer acetate prepared according to the process of  claim 2  to the patient in an amount and for a duration of time effective to treat the inflammatory, non-autoimmune central nervous system disease.  
   
   
       31 . The method according to  claim 30 , wherein the non-autoimmune central nervous system disease is multiple sclerosis.  
   
   
       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) 1  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) 1  adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected polypeptide formed in Step (a) 1  to form a polypeptide or a pharmaceutically acceptable salt thereof.    
   
   
       34 . A process for preparing glatiramer acetate comprising: 
 (a) 1′  polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a γ-benzyl L-glutamate 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) 1 ′ adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected glatiramer formed in Step (a) 1 ′ to form a glatiramer; and    (c) 1 ′ treating the glatiramer with acetic acid to form glatiramer acetate.    
   
   
       35 . The process according to  claim 33 , wherein Step (b) 1  is conducted at a temperature of from about −78° C. to about 40° C.  
   
   
       36 . The process according to  claim 35 , wherein Step (b) 1 , is conducted at a temperature of from about −25° C. to about 30° C.  
   
   
       37 . The process according to  claim 36 , wherein Step (b) 1 , is conducted at a temperature of from about −10° C. to about 10° C.  
   
   
       38 . The process according to  claim 37 , wherein Step (b) 1 , 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) 1  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) 1  adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected polypeptide formed in Step (a) 1  to form a polypeptide or a pharmaceutically acceptable salt thereof.    
   
   
       49 . Glatiramer acetate which is prepared by a process comprising 
 (a) 1′  polymerizing a mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of a γ-benzyl L-glutamate 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) 1 ′ adding an aqueous solution of an alkali or alkaline earth metal hydroxide to the protected glatiramer formed in Step (a) 1 ′ to form a glatiramer; and    (c) 1 ′ treating the glatiramer formed in Step (b) 1 ′ with acetic acid to form glatiramer acetate.    
   
   
       50 . 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) 2  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) 2  admixing an acid with the protected polypeptide formed in Step (a) 2  and a solvent, to form a product; and    (c) 2  admixing a substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia, and mixtures thereof, with the product formed in Step (b) 2 , and water or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof.    
   
   
       51 . 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) 2  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) 2  admixing an acid with a solution or suspension comprising the protected polypeptide formed in Step (a) 2  and a solvent, to form a product; and    (c) 2  admixing a substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia, and mixtures thereof, with the product formed in Step (b) 2′ , and water or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof.    
   
   
       52 . The process according to  claim 50 , wherein the protected L-lysine is N ε -trifluoroacetyl L-lysine.  
   
   
       53 . The process according to  claim 50 , wherein the protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures thereof.  
   
   
       54 . The process according to  claim 53 , wherein the protected L-glutamate is γ-benzyl L-glutamate.  
   
   
       55 . A process for preparing glatiramer acetate comprising: 
 (a) 2′  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 glatiramer, wherein said protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures thereof;    (b) 2′  admixing an acid with the protected glatiramer formed in Step (a) 2′  and a solvent, to form a product;    (c) 2′  admixing a substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia, and mixtures thereof, with the product formed in Step (b) 2′ , and water or a mixture of a solvent and water, to form a deprotected glatiramer; and    (d) 2 ′ treating the deprotected glatiramer formed in Step (C) 2′  with acetic acid to form glatiramer acetate.    
   
   
       56 . The process according to  claim 50 , wherein the solvent used in Step (b) 2  is selected from the group consisting of polar protic solvents, polar aprotic solvents and mixtures thereof.  
   
   
       57 . The process according to  claim 56 , wherein the solvent is selected from the group consisting of acetic acid, tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide, 1,4-dioxane, dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxide and dichloromethane.  
   
   
       58 . The process according to  claim 57 , wherein the solvent is tetrahydrofuran.  
   
   
       59 . The process according to  claim 57 , wherein the solvent is acetic acid.  
   
   
       60 . The process according to  claim 50 , wherein the substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia and mixtures thereof, is present in an amount of from about 1 -fold (wt.) to about 1,000-fold (wt), based on the total weight of the product of Step (b) 2  which is used in Step (c) 2 .  
   
   
       61 . The process according to  claim 60 , wherein the substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia and mixtures thereof, is present in an amount of from about 10-fold (wt.) to about 500-fold (wt).  
   
   
       62 . The process according to  claim 61 , wherein the substance selected from the group consisting of diisopropylamine, isopropylamine, ammonia and mixtures thereof, is present in an amount of from about 50-fold (wt.) to about 150-fold (wt).  
   
   
       63 . 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) 3  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) 3  admixing an acid with the protected polypeptide formed in Step (a) 3  and a solvent, to form a product; and    (c) 3  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) 3 , and a solvent or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof.    
   
   
       64 . 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) 3  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) 3  admixing an acid with a mixture comprising the protected polypeptide formed in Step (a) 3  and a solvent, to form a product; and    (c) 3  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) 3 , and a solvent or a mixture of a solvent and water, to form a deprotected polypeptide or a pharmaceutically acceptable salt thereof.    
   
   
       65 . The process according to  claim 63 , wherein the protected L-lysine is N ε -trifluoroacetyl L-lysine.  
   
   
       66 . The process according to  claim 63 , wherein the protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures thereof.  
   
   
       67 . A process for preparing glatiramer acetate comprising: 
 (a) 3′  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 N ε -trifluoroacetyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a protected glatiramer, wherein said protected L-glutamate is selected from the group consisting of γ-p-methoxybenzyl L-glutamate, γ-benzyl L-glutamate and mixtures thereof;    (b) 3 ′ admixing an acid with a mixture comprising the protected glatiramer formed in Step (a) 3  and a solvent, to form a product;    (c) 3 ′ admixing a substance selected from the group consisting of an alkali or alkaline earth metal hydroxide, a carbonate, a hydrogencarbonate, and mixtures thereof, with a mixture comprising the product formed in Step (b) 3 ′, and a solvent or a mixture of a solvent and water, to form a deprotected glatiramer; and    (d) 3′  treating the deprotected glatiramer formed in Step (c) 3 ′ with acetic acid to form glatiramer acetate.    
   
   
       68 . The process according to  claim 63 , wherein the solvent used in Step (b) 3  is selected from the group consisting of polar protic solvents, polar aprotic solvents and mixtures thereof.  
   
   
       69 . The process according to  claim 68 , wherein the solvent is selected from the group consisting of acetic acid, tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide, 1,4-dioxane, dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxide and dichloromethane.  
   
   
       70 . The process according to  claim 69 , wherein the solvent is tetrahydrofuran.  
   
   
       71 . The process according to  claim 69 , wherein the solvent is acetic acid.  
   
   
       72 . The process according to  claim 63 , wherein the substance selected from the group consisting of an alkali or alkaline earth metal hydroxide, a carbonate, a hydrogencarbonate, and mixtures thereof, is present in an amount of from about 0.1 wt. % to about 10 wt. %, based on the total weight of solvent or mixture of a solvent and water, which is used in Step (c) 3 .  
   
   
       73 . The process according to  claim 72  wherein the substance selected from the group consisting of an alkali or alkaline earth metal hydroxide, a carbonate, a hydrogencarbonate and mixtures thereof, is present in an amount of from about 0.1 wt. % to about 5 wt. %, based on the total weight of solvent or mixture of a solvent and water, which is used in Step (c) 3 .  
   
   
       74 . The process according to  claim 63 , wherein the solvent or mixture of a solvent and water, which is used in Step (c) 3 , is present in an amount from about 1-fold (wt.) to about 1000-fold (wt.), based on the total weight of the product of Step (b) 3 , which is used in Step (c) 3 .  
   
   
       75 . The process according to  claim 74 , wherein the solvent or mixture of a solvent and water, which is used in Step (c) 3 , is present in an amount from about 10-fold (wt.) to about 500-fold (wt.), based on the total weight of the product of Step (b) 3 , which is used in Step (c) 3 .  
   
   
       76 . The process according to  claim 63 , wherein the substance selected from the group consisting of an alkali or alkaline earth metal hydroxide, a carbonate, a hydrogencarbonate and mixtures thereof, is selected from the group consisting of calcium hydroxide, lithium hydroxide, magnesium hydroxide, potassium hydroxide, barium hydroxide, sodium hydroxide, calcium carbonate, lithium carbonate, magnesium carbonate, potassium carbonate, sodium carbonate, calcium hydrogencarbonate, lithium hydrogencarbonate, magnesium hydrogencarbonate, potassium hydrogencarbonate and sodium hydrogencarbonate.  
   
   
       77 . The process according to  claim 76 ,wherein the substance is selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide.