Process for preparation of mixtures of polypeptides using purified hydrobromic acid
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. A process for obtaining a pharmaceutical composition containing a mixture of polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and lysine, and wherein the mixture has a desired average molecular weight, which comprises
i) treating a solution of hydrobromic acid in acetic acid with a bromine scavenger so as to prepare a treated hydrobromic acid in acetic acid solution, and
ii) a) polymerizing N-carboxyanhydrides of tyrosine, alanine, y-benzyl glutamate and N-trifluoroacetyl lysine to form a mixture of protected polypeptides;
b) deprotecting the protected polypeptides with the treated hydrobromic acid in acetic acid solution prepared in part i), to form a mixture of trifluoroacetyl polypeptides;
c) reacting the mixture of trifluoroacetyl polypeptides with aqueous piperidine to form a solution of aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine; and
d) purifying the mixture of polypeptides.
2. The process of claim 1 , wherein in the mixture the molar fraction of alanine is 0.427, of glutamic acid is 0.141, of lysine is 0.337 and of tyrosine is 0.093.
3. A process of producing glatiramer acetate comprising the steps of:
i) treating a solution of hydrobromic acid in acetic acid with a bromine scavenger so as to prepare a treated hydrobromic acid in acetic acid solution, and
ii) a) polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate and N-trifluoroacetyl lysine to form protected polypeptides;
b) deprotecting protected polypeptides with the treated hydrobromic acid in acetic acid solution prepared in part i) to form trifluoroacetyl glatiramer acetate;
c) reacting trifluoroacetyl glatiramer acetate with aqueous piperidine to form a solution of glatiramer acetate; and
d) purifying the glatiramer acetate.
4. The process of claim 3 , further comprising subjecting the product of the step d) to ultrafiltration.
5. The process of claim 4 , wherein the hydrobromic acid in acetic acid solution is 10%-36% hydrobromic acid in acetic acid.
6. The process of claim 5 , wherein the hydrobromic acid in acetic acid solution is 33% hydrobromic acid in acetic acid.
7. The process of claim 3 , wherein the bromine scavenger is phenol.
8. The process of claim 3 , wherein in part
i) the hydrobromic acid in acetic acid solution is produced in a non-metallic reactor.
9. The process of claim 3 , wherein in part
i) the hydrobromic acid in acetic acid solution is prepared in a glass-lined or polytetrafluoroethylene-lined reactor.
10. The process of claim 3 , wherein the color of the hydrobromic acid in acetic acid solution is less than 2000 APHA.
11. The process of claim 10 , wherein the color of the hydrobromic acid in acetic acid solution is less than 1000 APHA.
12. The process of claim 11 , wherein the color of the hydrobromic acid in acetic acid solution is less than 700 APHA.
13. The process of claim 12 , wherein the color of the hydrobromic acid in acetic acid solution is less than 500 APHA.
14. The process of claim 3 , wherein the hydrobromic acid in acetic acid solution is 10%-36% hydrobromic acid in acetic acid.
15. The process of claim 3 , where in the hydrobromic acid in acetic acid solution is 33% hydrobromic acid in acetic acid.
16. In a process for obtaining a pharmaceutical composition containing a mixture of polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of alanine, glutamic acid, tyrosine and lysine, and wherein the mixture has a desired average molecular weight, which process comprises:
a) polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate and N-trifluoroacetyl lysine to form a mixture of protected polypeptides;
b) deprotecting the protected polypeptides with a solution of hydrobromic acid in acetic acid, to form a mixture of trifluoroacetyl polypeptides;
c) reacting the mixture of trifluoroacetyl polypeptides with aqueous piperidine to form a solution of aqueous mixture of polypeptides, each of which consists essentially of alanine, glutamic acid, tyrosine and lysine; and
d) purifying the mixture of polypeptides,
the improvement comprising:
pretreating the solution of hydrobromic acid in acetic acid with a bromine scavenger prior to use of the solution in step b) for deprotecting the protected polypeptides.
17. In a process of producing glatiramer acetate comprising the steps of:
a) polymerizing N-carboxyanhydrides of tyrosine, alanine, y-benzyl glutamate and N-trifluoroacetyl lysine to form a mixture of protected polypeptides;
b) deprotecting the protected polypeptides with a solution of hydrobromic acid in acetic acid, to form trifluoroacetyl glatiramer acetate;
c) reacting the trifluoroacetyl glatiramer acetate with aqueous piperidine to form a solution of glatiramer aceate; and
d) purifying the glatiramer acetate,
the improvement comprising:
pretreating the solution of hydrobromic acid in acetic acid with a bromine scavenger prior to use of the solution in step b) for deprotecting the protected polypeptides.
18. The process of claim 16 , wherein the hydrobromic acid in acetic acid solution is from 10% to 36% hydrobromic acid in acetic acid.
19. The process of claim 16 , wherein the hydrobromic acid in acetic acid solution is 33% hydrobromic acid in acetic acid.
20. The process of claim 17 , wherein the hydrobromic acid in acetic acid solution is from 10% to 36% hydrobromic acid in acetic acid.
21. The process of claim 17 , wherein the hydrobromic acid in acetic acid solution is 33% hydrobromic acid in acetic acid.