Process for preparation of mixtures of polypeptides using purified hydrobromic acid

Abstract

The subject invention provides an improved process for obtaining a mixture of polypeptides having nonuniform amino acid sequences, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and lysine where the resulting mixture of polypeptides comprises less than 0.3% brominated tyrosine and less than 1000 ppm metal ion impurities.

1 . In a process for obtaining a mixture of trifluoroacetyl polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and trifluoroacetyl lysine, wherein the mixture has a desired average molecular weight and wherein during the process a batch of a mixture of polypeptides, each of which consists essentially of alanine, γ-benzyl glutamate, tyrosine and trifluoroacetyl lysine is deprotected with a solution of hydrobromic acid in acetic acid, the improvement comprising use of a solution of hydrobromic acid in acetic acid, which solution comprises less than 0.5% of free bromine or less than 1000 ppm of metal ion impurities.  
     
     
         2 . The process of  claim 1 , wherein if the improvement comprises use of a solution of hydrobromic acid in acetic acid that comprises less than 0.5% of free bromine, then the improvement further comprises use of a solution of hydrobromic acid in acetic acid that comprises less than 1000 ppm of metal ion impurities.  
     
     
         3 . (canceled)  
     
     
         4 . A process of producing a mixture of trifluoroacetyl polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and trifluoroacetyl lysine, wherein the mixture has a desired average molecular weight comprising deprotecting a mixture of polypeptides each consisting essentially of alanine, γ-benzyl glutamate, tyrosine and trifluoroacetyl lysine with a solution of hydrobromic acid in acetic acid, which solution comprises less than 0.5% of free bromine and less than 1000 ppm of metal ion impurities.  
     
     
         5 . The process of  claim 1 , wherein the solution of hydrobromic acid in acetic acid comprises less than 0.1% of free bromine.  
     
     
         6 - 9 . (canceled)  
     
     
         10 . The process of  claim 1 , wherein the solution of hydrobromic acid in acetic acid comprises less than 1000 ppm of metal ion impurities.  
     
     
         11 - 14 . (canceled)  
     
     
         15 . The process of  claim 1 , wherein the mixture of trifluoroacetyl polypeptides is TFA GA.  
     
     
         16 . The process of  claim 1 , wherein the solution is 10-36% hydrobromic acid in acetic acid.  
     
     
         17 . (canceled)  
     
     
         18 . The process of  claim 1 , wherein the solution is pretreated with a bromine scavenger in order to remove free bromine.  
     
     
         19 . The process of  claim 18 , wherein the bromine scavenger is phenol.  
     
     
         20 . The process of  claim 1 , wherein the solution is produced in a non-metallic reactor.  
     
     
         21 . The process of  claim 1 , wherein the solution is prepared in a glass-lined or Teflon lined reactor.  
     
     
         22 . The process of  claim 1 , wherein the color of the hydrobromic acid in acetic acid solution is less than 2000 APHA.  
     
     
         23 - 25 . (canceled)  
     
     
         26 . A trifluoroacetyl product produced by the process of  claim 1 .  
     
     
         27 . A composition comprising the trifluoroacetyl product produced by the process of claims  1 , and a carrier.  
     
     
         28 . A mixture of trifluoroacetyl polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and trifluoroacetyl lysine, wherein the mixture has a desired average molecular weight, no more than 0.1% brominated tyrosine and less than 1000 ppm metal ion impurities.  
     
     
         29 . The mixture of  claim 28 , having an average molecular weight from 2000 daltons to 40,000 daltons.  
     
     
         30 - 33 . (canceled)  
     
     
         34 . The mixture of  claim 29 , wherein the mixture comprises less than 1000 ppm of metal ion impurities.  
     
     
         35 - 38 . (canceled)  
     
     
         39 . A composition comprising the mixture of  claim 28  and a carrier.  
     
     
         40 - 53 . (canceled)  
     
     
         54 . A method of analyzing the percentage of brominated tyrosine in a sample of glatiramer acetate comprising the steps of: 
 a) hydrolyzing glatiramer acetate to obtain a hydrolyzate;    b) eluting the hydrolyzate through a chromatographic column;    c) measuring the level of bromotyrosine in the hydrolyzate;    d) preparing sample solutions of the amino acid components of glatiramer acetate and of bromotyrosine;    e) eluting the sample solutions through the column of step b); and    f) calculating the percentage of brominated tyrosine in the glatiramer acetate.    
     
     
         55 - 58 . (canceled)