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  Process for preparation of mixtures of polypeptides using purified hydrobromic acidUSPTO Application #: 20070021324Title: 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. (end of abstract) Agent: Cooper & Dunham, LLP - New York, NY, US Inventor: Ben-Zion Dolitzky USPTO Applicaton #: 20070021324 - Class: 514002000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai The Patent Description & Claims data below is from USPTO Patent Application 20070021324. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims benefit of U.S. Provisional Application No. 60/608,843, filed Sep. 9, 2004, the contents of which are hereby incorporated herein by reference. [0002] Throughout this application various publications are referenced by their full citations. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. BACKGROUND OF THE INVENTION [0003] A mixture of polypeptides which do not all have the same amino acid sequence referred to as glatiramer acetate (GA) is marketed under the tradename Copaxone.RTM. and comprises the acetate salts of polypeptides containing L-glutamic acid, L-alanine, L-tyrosine and L-lysine at average molar fractions of 0.141, 0.427, 0.095 and 0.338, respectively. The average molecular weight of Copaxone.RTM.. is between 4,700 and 11,000 daltons. ("Copaxone", Physician's Desk Reference, (2000), Medical Economics Co., Inc., (Montvale, N.J.), 3115.) Chemically, glatiramer acetate is designated L-glutamic acid polymer with L-alanine, L-lysine, L-tyrosine, acetate (salt). Its structural formula is: (Glu, Ala, Lys, Tyr).sub.x..chi.CH.sub.3COOH (C.sub.5H.sub.9NO.sub.4.C.sub.3H.sub.7NO.sub.2.C.sub.6H.sub.14N.sub.2O.su- b.2.C.sub.9H.sub.11NO.sub.3).sub.x..chi.C.sub.2H.sub.4O.sub.2 CAS--147245-92-9 ("Copaxone", Physician's Desk Reference, (2000), Medical Economics Co., Inc., (Montvale, N.J.), 3115.) [0004] Glatiramer acetate is approved for reduction of the frequency of relapses in patients with relapsing-remitting multiple sclerosis. Multiple sclerosis has been classified as an autoimmune disease. Glatiramer acetate has also been disclosed for use in the treatment of other autoimmune diseases (Publication No. US 2002/0055466 A1 for R. Aharoni et al.), inflammatory non-autoimmune diseases (Publication No. US 2005/0014694 A1 for V. Wee Yong et al.; and U.S. Patent Application No. 2002/0077278 Al, published Jun. 20, 2002 (Young et al.)) and to promote nerve regeneration and/or to prevent or inhibit secondary degeneration which may follow primary nervous system injury (Publication No. US 2003/0004099 A1 for M. Eisenbach-Schwartz et al.; and U.S. Patent Application No. 2002/0037848 Al, published Mar. 28, 2002 (Eisenbach-Schwartz)). Furthermore, glatiramer acetate has been disclosed as a treatment for immune mediated diseases (e.g., U.S. Pat. No. 6,514,938 B1, issued Feb. 4, 2003 (Gad et al.); PCT International Publication No. WO 01/60392, published Aug. 23, 2001 (Gilbert et al.); and PCT International Publication No. WO 00/27417, published May 19, 2000 (Aharoni et al.) as well as diseases associated with demyelination (PCT International Publication No. WO -1/97846, published Dec. 27, 2001 (Moses et al.). [0005] The manufacturing process as detailed in the above patents involves reacting protected polypeptides with 33% hydrobromic acid in acetic acid. (U.S. Pat. No. 5,800,808, issued Sep. 1, 1998 to Konfino, et al.) This deprotection reaction removes the gamma benzyl protecting group from the 5-carboxylate of the glutamate residue and cleaves the polymer to smaller polypeptides to form a trifluoroacetyl polypeptide. (U.S. Pat. No. 5,800,808, issued Sep. 1, 1998 to Konfino, et al.) The time needed to obtain GA of the proper average molecular weight of between 7,000.+-.2,000 daltons depends on the reaction temperature and the molecular weight profile of the protected glatiramer acetate. (U.S. Pat. No. 5,800,808, issued Sep. 1, 1998 to Konfino, et al.) The deprotection occurs at a temperature of between 20.degree. C. and 28.degree. C. (U.S. Pat. No. 5,800,808, issued Sep. 1, 1998 to Konfino, et al.). A test reaction is performed on every batch at different time periods to determine the reaction time needed at a given temperature to achieve trifluoroacetyl polypeptides of a proper molecular weight profile. (U.S. Pat. No. 5,981,589, issued Nov. 9, 1999 to Konfino, et al.) The amount of time needed for the reaction ranges, for example, between 10 and 50 hours. (U.S. Pat. No. 5,800,808, issued Sept. 1, 1998 to Konfino, et al.). In addition, U.S. Pat. Nos. 5,981,589, 6,048,898, 6,054,430, 6,342,476, 6,362,161, and 6,620,847, also relate to compositions and methods for manufacture of mixtures of polypeptides, including GA. [0006] This invention provides an improved manufacturing process. SUMMARY OF THE INVENTION [0007] The subject invention provides 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, .gamma.-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. [0008] The subject invention also provides 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, .gamma.-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 1000 ppm of metal ion impurities. [0009] The subject invention further provides 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, .gamma.-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. [0010] The subject invention also provides a composition comprising the trifluoroacetyl product produced by any one of the subject invention processes, and a carrier. [0011] The subject invention further provides 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. The subject invention also provides a composition comprising the mixture of trifluoroacetyl polypeptides and a carrier. [0012] The subject invention also provides 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 [0013] a) polymerizing N-carboxyanhydrides of tyrosine, alanine, .gamma.-benzyl glutamate and N-trifluoroacetyl lysine to form a mixture of protected polypeptides; [0014] b) deprotecting the protected polypeptides with a solution of hydrobromic acid in acetic acid, the solution comprises less than 0.5% of free bromine and less than 1000 ppm of metal ion impurities, to form a mixture of trifluoroacetyl polypeptides; [0015] c) reacting the a 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 [0016] d) purifying the mixture of polypeptides. [0017] The subjection invention further provides process of producing glatiramer acetate comprising the steps of: [0018] a) polymerizing N-carboxyanhydrides of tyrosine, alanine, .gamma.-benzyl glutamate and N-trifluoroacetyl lysine to form protected glatiramer acetate; [0019] b) deprotecting protected glatiramer acetate with a solution of hydrobromic acid in acetic acid, the solution comprises less than 0.5% of free bromine and less than 1000 ppm of metal ion impurities, to form trifluoroacetyl glatiramer acetate; [0020] c) reacting trifluoroacetyl glatiramer acetate with aqueous piperidine to form a solution of glatiramer acetate; and [0021] d) purifying the glatiramer acetate. [0022] The subject invention yet further provides a method of analyzing the percentage of brominated tyrosine in a sample of glatiramer acetate comprising the steps of: [0023] a) hydrolyzing glatiramer acetate to obtain a hydrolyzate; [0024] b) eluting the hydrolyzate through a chromatographic column; [0025] c) measuring the level of bromotyrosine in the hydrolyzate; [0026] d) preparing sample solutions of the amino acid components of glatiramer acetate and of bromotyrosine; [0027] e) eluting the sample solutions through the column of step b); and [0028] e) calculating the percentage of brominated tyrosine in the glatiramer acetate. [0029] The subject invention also provides a process for preparing a pharmaceutical composition containing a mixture of polypeptides which do not all have the same amino acid sequence, where each polypeptide consists essentially of glutamic acid, alanine, tyrosine and lysine, wherein the mixture has a predetermined percentage of brominated tyrosine acceptable for inclusion in a pharmaceutical composition, which comprises obtaining a batch of a mixture of polypeptides having nonuniform amino acid sequences, where each polypeptide consists essentially of glutamic acid, alanine, tyrosine and lysine; [0030] measuring the percentage of brominated tyrosine of the batch by a process comprising [0031] a) hydrolyzing the batch to obtain a hydrolyzate; [0032] b) eluting the hydrolyzate through a chromatographic column; [0033] c) measuring the level of bromotyrosine in the hydrolyzate; [0034] d) preparing sample solutions of the amino acid components of the batch and of bromotyrosine; [0035] e) eluting the sample solutions through the column of step b); and [0036] f) calculating the percentage of brominated tyrosine in the batch; and [0037] inluding in the pharmaceutical composition a batch only if its percentage of brominated tyrosine so measured is less than 0.3%. DETAILED DESCRIPTION OF THE INVENTION [0038] The subject invention provides 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, .gamma.-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. [0039] In one embodiment, the improvement further comprises use of a solution of hydrobromic acid in acetic acid that comprises less than 1000 ppm of metal ion impurities. [0040] The subject invention further provides 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, .gamma.-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 1000 ppm of metal ion impurities. [0041] The subject invention yet further provides 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, .gamma.-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. [0042] In one embodiment, the solution of hydrobromic acid in acetic acid comprises less than 0.1% of free bromine. [0043] In another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 0.05% of free bromine. Continue reading... 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