FOR
MIXTURES OF POLYPEPTIDSS USING PURIFIED HYPUODRQMie-fteiP
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
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® and comprises the acetate salts of polypeptides containing L-glutaroic 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® is between 4,700 and 11,000 daltons. ("Copaxone", Physician's Desk Reference, (2000), Medical Economics Co., Inc., (Montvale, HJ), 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) x'xCH3COOH (CsHgNO* • C3H7N02 • C6Hi4N202 • C9HnN03) x • XC2H4O2 • CAS - 147245-92-9
("Copaxone", Physician's Desk Reference, (2000), Medical Economics Co., Inc., (Montvale, NJ), 3115.)
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 Al for R. Aharoni et al.), inflammatory non-autoimmune diseases (Publication No. US 2005/00X4694 Al for V. Wee Yong et al.; and U.S. Patent Application No. 2002/0077278 Al, published June 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 Al for M. Eisenbach-Schwartz et al.; and U.S. Patent Application No. 2002/0037848 Al, published March 28, 2002 (Eisenbach-Schwartz)). Furthermore, glatiramer acetate has been disclosed as a treatment for immune mediated diseases {e.g., U.S. Patent Ho. 6,514,938 Bl, issued February 4, 20O3 (Gad et al.); PCT International Publication No. WO 01/60392, published August 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 December 27, 2O01 (Moses et al.).
The manufacturing process as detailed in the above patents involves reacting protected polypeptides with 33% hydrobromic acid in acetic acid. (U.S. Patent No. 5,800,808, issued September I, 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. Patent No. 5,800,808, issued September 1, 1998 to Konfino, et al.) The time needed to obtain GA of the proper average
molecular weight of between 7,00012,000 daltons depends on the reaction temperature and the molecular weight profile of the protected glatiramer acetate. (U.S. Patent No. 5,800,808, issued September 1, 1998 to Konfino, et al.) The deprotection occurs at a temperature of between 20°C and 28°C (U.S. Patent No. 5,800,808, issued September 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 polypeptidea of a proper molecular weight
profile. (U.S. Patent No. 5>981,589, issued November 9,
1999 to Konfino, et al.) The amount of time needed for the
reaction ranges, for example, between 10 and 50 hours.
(U.S. Patent No. 5,800,808, issued September 1, 1998 to
Konfino, et al.). In addition, U.S. Patent Hos. 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.
This invention provides an improved manufacturing process.

Summary Of The Invention
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, y-benzyl glutamate, tyrosine and trifluoroacetyl lysine is deprotected with1 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.
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, y-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.
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, y-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.
The subject invention also provides a. composition comprising the trifluoroacetyl product produced by any one of the subject invention processes, and a carrier.
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, wberein 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.
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
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,
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;
c) reacting the a mixture of trifluoroacetyl
polypeptides with aqueous pipcridine to form a
solution of aqueous mixture of polypeptides,
each of which consists essentially of alanine,
glutaraic acid, tyrosine and lysine; and
d) purifying the mixture of polypeptides.
The subjection invention further provides 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 protected
glatiramer acetate;
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;
c) reacting trifluoroacetyl glatiramer acetate
with aqueous piperidine to form a solution of
glatiramer acetate; and
d) purifying the glatiramer acetate.
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:
a) hydrolyzing glatiramer acetate to obtain a
hydrolyzate;
b) elating the hydrolyzate through a
chromatographic column;
c) measuring the level of bronotyrosine 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
.e) calculating the percentage of brominated tyrosine in the glatiramer acetate.
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;
measuring the percentage of brominated tyrosine of
the batch by a process comprising
a) hydrolyzing the batch to obtain a hydrolyzate;
b) eluting the hydrolyzate through a
chromatographic column;
c) measuring the level of bronotyrosine in the
hydrolyzate;
d) preparing sample solutions of the amino acid
components of the batch and of bromotyrosine;
e) eluting the sample solutions through the column
of step b); and
f) calculating the percentage of brominated
tyrosine in the batch; and
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
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, y-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.
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.
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, y-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.
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, y-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 I
In one embodiment, the solution of hydrobrotnic acid in acetic acid comprises less than 0.1% of free bromine.
In another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 0.05% of free bromine.
In a further embodiment, the solution of hydrobromic acid in acetic acid comprises less than 0.01% of free bromine. .
In yet another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 0.001% of free bromine.
In a further embodiment, the solution of hydrobromic acid in acetic acid is free of free bromine.
In another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 1000 ppm of metal ion
impurities.
In yet another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 500 ppm of metal ion impurities.
In one embodiment, the solution of hydrobromic acid in acetic acid comprises less than 100 ppm of metal ion impurities.
In another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 30 ppm of metal ion impurities.
In yet another embodiment, the solution of hydrobromic acid in acetic acid comprises less than 20 ppm of metal ion impurities.
In a further embodiment, the solution of hydrobromic acid in acetic acid comprises less than 10 ppm of metal ion
impurities.
In another embodiment, the solution of hydrobromic- acid in acetic acid is free of metal ion impurities.
In yet another embodiment, the mixture of trifluoroacetyl polypeptides is trifluoroacetyl glatiramer acetate ("TFA GA") .
In an embodiment, the hydrobromic acid in acetic acid solution is from 10% to 36% hydrobromic acid in acetic acid. In another embodiment, the hydrobromic acid in acetic acid is from 16% to 33% hydrobromic acid in acetic

acid; 18% to 33% hydrobromic acid in acetic acid; 20% to 37% hydrobromic acid in acetic acid; 20% to 33% hydrobromic acid in acetic acid; 22% to 33% hydrobromic acid in acetic acid; 24% to 33% hydrobromic acid in acetic acid; 25% to 35% hydrobromic acid in acetic acid; 26% to 33% hydrobromic acid in acetic acid; 28% to 33% hydrobromic acid in acetic acid; 30% to 34% hydrobromic acid is acetic acid; 30% to 33% hydrobromic acid in acetic acid; or 32% to 33% hydrobromic acid in acetic acid. In a further embodiment, the solution is 33% hydrobromic acid in acetic acid. In another embodiment, the solution is 16% hydrobromic acid in acetic acid.
In another embodiment, the solution is pretreated with a bromine scavenger in order to remove free bromine.
In one embodiment, the bromine scavenger is phenol.
In a further embodiment, the solution is produced in a non-metallic reactor.
In another embodiment, the solution is prepared in a glass-lined or Teflon lined reactor.
In yet another embodiment, the color of the hydrobromic acid in acetic acid solution is less than 2000 APHA.
In a further embodiment, the color of the hydrobromic acid in acetic acid solution is less than 1000 APHA.
In- another embodiment, the color of the hydrobromic acid in acetic acid solution is less than 700 APHA. In yet another embodiment, the color of the hydrobromic acid in acetic acid solution is less than 500 APHA.
The subject invention also provides a trifluoroacetyl product produced by any one of the disclosed processes.
The subject invention further provides a composition comprising the trifluoroacetyl product produced by any one of the disclosed processes, and a carrier.
The subject invention yet 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 wore than 0,1% brominated tyrosine and less than 1000 ppm metal ion impurities.
In one embodiment, the mixture of trifluoroacetyl polypeptides has an average molecular weight from 2000 da1tons to 40,000 daltons.
In another embodiment, the mixture of trifluoroacetyl polypeptides has an average molecular weight from 4000 daltons to 18,000 daltons.
In a further embodiment, the mixture of trifluoroacetyl polypeptides has an average molecular weight from 4000 daltons to 13,000 daltons.
In another embodiment, the mixture of trifluoroacetyl polypeptides has an average molecular weight from 13,000 daltons to 19,000 daltons. In yet another embodiment, the mixture of trif luoroacetyl polypeptides has an average molecular weight from 13,500 daltons to 18,500 daltons.
In a further embodiment, the mixture of trif luoroacetyl polypeptides has an average molecular weight of 7,000 ±2,000 daltons.
in yet a further embodiment, the mixture of trifluoroacetyl polypeptides has an average molecular weight of 7,000 daltons.
In another embodiment, the mixture of trif luoroacetyl polypeptides has an average molecular weight of 14,000 daltons.
In yet another embodiment, the mixture of trifluoroacetyl polypeptides has an average molecular weight from 4,700 -11,000 daltons.
In a further embodiment, the mixture of trifluoroacetyl polypeptides comprises less than 1000 ppm o.f metal ion impurities.
In yet another embodiment, the mixture of trifluoroacetyl polypeptides comprises less than 500 ppm of metal ion impurities.
In a further embodiment, the mixture of trifluoroacetyl polypeptides comprises less than 100 ppm of metal ion impurities.
In another embodiment, the mixture of trifluoroacetyl polypeptides comprises less than 30 ppm of metal ion impurities.
In a further embodiment, the mixture of trifluoroacetyl polypeptides comprises less than 20 ppm of metal ion impurities.
In another embodiment, the mixture of trifluoroacetyl polypeptides comprises, less than 10 ppm of metal ion-impurities.
In yet another embodiment, the mixture of trifluoroacetyl polypeptides is free of metal ion impurities.
The subject invention also provides a composition comprising the mixture of trifluoroacetyl polypeptides and a carrier.
The subject invention further provides 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
a) polymerizing N-carboxyanhydrides of tyrosine,
alanine, y-benzyl glutamate and N-trifluoroacetyl
lysine to form an aqueous mixture of protected
polypeptides;
b) deprotecting the protected polypeptides 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, to form an aqueous mixture of trifluoroacetyl polypeptides;
c) reacting the an aqueous 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 aqueous mixture of polypeptides.
In one embodiment, the average mole fraction in the mixture is glutamic acid 0.129-0.159; alanine 0.392-O.462; tyrosine 0.086-0.100; and lysine 0.300-0.374. In
a specific embodiment, the average mole fraction in the mixture of glutamic acid is 0.141, of alanine is 0.427, of tyrosine is 0.093, and of lysine is 0.337.-
The subject invention also provides 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 protected glatiramer acetate;
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;
c) reacting trifluoroacetyl glatiramer acetate with
aqueous piperidine to form a solution of glatiramer
acetate; and
d) purifying the glatiramer acetate.
In one embodiment, the product of the step d) is further subjected to ultraflltration to remove polypeptide species with molecular weight less than 5000 daltons.
In an embodiment, the hydrobromic acid in acetic acid solution is from 10% to 36% hydrobromic acid in acetic acid. In another embodiment, the hydrobromic acid in acetic acid is from 16% to 33% hydrobroroic acid in acetic acid; 18% to 33% hydrobromic acid in acetic acid; 20% to 37% hydrobronic acid in acetic acid; 20% to 33% hydrobromic acid in acetic acid; 22% to 33% hydrobromic acid in acetic acid; 24% to 33% hydrobromic acid in acetic acid; 25% to 35% hydrobromic acid in acetic acid; 26% to 33% hydrobromic acid in acetic acid; 28% to 33% hydrobromic acid in acetic acid; 30% to 34% hydrobromic acid is acetic acid; 30% to 33% hydrobromic acid in acetic acid; or 32% to 33% hydrobromic acid in acetic acid. In a further embodiment, the solution is 33% hydrobromic acid in acetic acid. In another embodiment, the solution is 16% hydrobromic acid in acetic acid.
In another embodiment, the hydrobromic acid in acetic acid solution is pretreated with a bromine scavenger in order to remove free bromine.
In yet another embodiment, the bromine scavenger is phenol.
In a further embodiment, the hydrobromic acid in acetic acid solution is produced in a non-metallic reactor.
In another embodiment, the hydrobromic acid in acetic, acid solution is prepared in a glass-lined or Teflon lined reactor.
In one embodiment, the color of the hydrobromic acid in acetic acid solution is less than 2000 APHA.
In another embodiment, the color of the hydrobromic acid in acetic acid solution is less than 1000 APHA.
In yet another eiribodiMent, the color of the hydrobromic acid in acetic acid solution is less than 700 APHA.
In a further embodiment, the color of the'. hydrobromic acid in acetic acid solution is less than 500 APHA.
The subject invention further provides 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.
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 glutanic 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 nonuniforra amino acid sequences, where each polypeptide consists essentially of glutamic acid, alanine, tyrosine and lysine;
measuring the percentage of brominated tyrosine of the
batch by a process comprising
a) hydrplyzing the batch 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 the batch and of bromotyrosine;
e) eluting the sample solutions through the column of
step b); and
f) calculating the percentage of brominated tyrosine in
the batch; and
including in the pharmaceutical composition a batch only if its percentage of brominated tyrosine so measured is less than 0.3%.
In one embodiment, the batch is acceptable for inclusion in the pharmaceutical composition only if its percentage of brominated tyrosine so measured is less than 0.2%.
In another embodiment:, the batch is acceptable "for inclusion in the pharmaceutical composition only if its percentage of brominated tyrosine so measured is less than 0.1%.
In a further embodiment, the mixture of polypeptides is glatiramer acetate ("GA").
TERMS
The term "average molecular weight" as used in this application means the molecular weight of the species of polypeptides present in the mixture in the highest relative proportion (i.e. the peak maximum) when the mixture is subjected to separation by molecular -weight on an HPLC gel permeation column. This value can be obtained in several ways, e.g. from the retention time on a calibrated column; or from a correlation between the
location of the peak and the location of the cochromatographed copolymer markers of defined sequence and molecular weight. Other methods of determining an average molecular weight such as by light scattering may be employed and will correspond substantially to the value obtained from the peak maximum.
A polypeptide mixture according to this invention as exemplified is the acetate salt of synthetic polypeptides prepared by chemically reacting four activated amino acid derivatives (two of them L-Glutamic acid and L-lysine protected): L-Glutamic acid (L-Glu), L-alanine (L-Ala) , L-tyrosine (L-Tyr) and L-lysine (L-Lys) (two of them protected i.e. SBz-Glutamate derivative and 6N-TFA-Lysine derivative) in a specified ratio. The term '"mixture" as used in this document generally refers to in the ^mixture of polypeptides of the invention" comprising L-glutamic acid, L-alanine, L-tyrosine, and L-lysine, and both terms are meant to include residual impurities from the manufacturing process.
The molar fraction range of each amino acid residue is : L-Glu 0.129-0.153, L-Ala 0.392-0 ..462, L-Tyr 0.086-0.100 and L-Lys 0.300-0.374.
Because no reaction goes to completion 100% and although practically all impurities are eliminated, small amounts can remain. Such impurities may be of .the following three types:
• Structure-related substances, which are protected
amino acid residues such as 5-BZ-L-glutamyl and/or
N6-TFA-L-Lysyl residues, originating from
incomplete removal of the protecting groups. In addition, the polypeptide mixture of the invention molecules may contain brominated L-tyrosyl residues, formed during production due to the presence of free bromine in the HBr/acetic acid reagent.
The molecular structures of the identified structure-related impurities can be derived from the participating monomers i.e. starting materials.
These identified impurities are quantified (after chemical conversion) by comparison to the specific Reference Standards, which are either derivatives or part of the impurities themselves:
- Residual trifluoroacetyl compounds (expressed as
fluoride)
- Residual benzylated glutamyl residues (expressed
as benzyl bromide)
- Residual brominated tyrosyl residues (expressed
as bromotyrosine)
Unidentified related substances (determined by RP-HPLC): these are small molecular size polypeptides of the same origin with similar structures. These substances probably have similar response factors and the concentration (%) of each impurity can be calculated as % peak area relative to the the polypeptide mixture of the invention peak area.
The characterization of the impurities is based on
their relative chromatography retention time (RRT) relative to the L-Tryptophan standard.
Residual solvents and inorganic impurities covered in the specification such as the residual solvent 1,4 dioxane, residual piperidine and heavy metals.
DISCUSSION
Free Bromine
In the manufacturing process for mixtures of polypeptides, such as GA, 33% hydrobromic acid in acetic acid is used to deprotect protected GA. For example, during the development of the production process for GA it was found that some of the tyrosine residues in trifluoroacetyl GA (TFA GA) and in GA were brominated. This impurity was isolated and identified using an analytical procedure that is described in detail in the examples. The tyros ine residue was found to react with bromine to form a mono-bromotyrosine moiety comprising either 2-brombtyrosine or 3-bromotyrosine.
After much investigation the inventors discovered that the brominated tyrosine impurity was introduced "into the GA through free bromine in HBr/acetic acid. The free bromine was present in 33% HBr/acetic acid bought from .a supplier and used in the production process.
Measures were taken in order to decrease the level of free bromine in 33% HBr/acetic acid. For example, pre-treatment ..of HBr/acetic acid with a bromine scavenger was effective in removing some of the free bromine from the HBr/acetic acid solution.
One of the bromine scavengers used in the HBr purification process was phenol. In addition to phenol, other reducing agents, such as sodium bisulfite, may be used. Phenol was chosen as a bromine scavenger because it and its reaction product with bromine (bromophenpls) are both essentially non-reactive with protected polypeptides, such as protected GA, TFA polypeptides, such as TFA GA and polypeptides, such as GA, and they are easy to remove from the solution of GA during the purification process. Similarly, any bromine scavenging agent may be used provided that it, and its reaction product with bromine, are not reactive with protected polypeptides, such as protected GA, TFA polypeptides, such as TFA GA and polypeptides, such as GA, and it is easily removable daring the final purification process.
Metal Impurities
GA is marketed in two pharmaceutical dosage forms, lyophilized powder and pre-filled syringes. The syringes, marketed under the trade name Copaxone® Injection, generally contained clear solution. The storage instructions were to keep the syringes refrigerated. However, red color in aqueous solutions of Copaxone® pre-filled solutions was detected. The source of the color in the solutions was unknown.
The color appeared when the solutions were kept at room temperature for 12 to 24 hours.
It was determined that production of HBr in metal apparatus led to trace metallic ion impurities in the HBr. When HBr was later mixed with protected GA, the metallic
-Vision impurities in the HBr were chelated by TFA GA and GA. These TFA GA and GA/metal complexes contributed to the coloration.
As a result, another measure taken to ensure purity, e.g., in the GA product, was the use of a non-metal reactor for the production of 33% HBr/acetic acid solution. The reactor used for the production of HBr/acetic acid solution was glass lined in order to prevent the formation of impurities which could later affect the purity of, e.g., the GA. In order to prevent contact of HBr solution with metal, parts of the piping used were Teflon-lined. Similarly, other types of non-reactive, acid resistant non-metal apparatus can be used to prevent the formation of trace Metal ions in the HBr/acetic acid solution. The use of a non-wetal apparatus for the production of HBr/acetic acid solution was successful in eliminating the red color from the GA. When the non-metal apparatus was used for the production of the HBr/acetic acid solution, the result was that the solution was essentially free of metal ions and the red GA was not formed.
In addition, the color of every batch of HBr/acetic acid
is measured to determine level of impurities before being
used to deprotect protected GA. It was found that levels
of metal ion impurity in HBr solution could be-determined
by visual analysis. HBr solution with a color below 2000
APHA was shown to produce glatiramer acetate without red
color. . •
The invention will be -exemplified but not limited by the following examples. EXPERIMENTAL DETAILS
EXAMPLE 1 - INFLUENCE OF BROMINE CONCENTRATION IN HBR/ACETIC ACID ON BROMOMINATED TYROSINE MOIETY IN TFA GA AND IN GA
In order to determine the effect of free bromine in hydrobromic acid/ acetic acid on the level of brominated tyrosine moiety impurity in TFA-GA and GA, hydrobromic acid in acetic acid was contaminated with various amounts of bromine. In the experiment, HBr which was not pretreated with bromine scavenger was used in the manufacturing process. Various levels of bromine impurity (measured as percentage of HBr/Acetic acid solution) were added. The level of brominated tyrosine moiety impurity in TFA GA and in GA was Measured by hydrolyzing TFA GA and GA to its amino acid components, and then using HPLC to determine the amount of bromotyrosine in relation to the TFA GA and GA.
PROCEDURE
Preparation of standard solutions
Standard solutions containing 2 pg/mL Bromotyrosine were' prepared using distilled water. Amino acid standard stock solution was prepared using the following amino acids:

(Table Removed)
The amino acids were dissolved in water. A few drops of 5 N NaOH were added and water was added to a final volume of 25 mL.
Hydrolysis
10 mg of glatiramer acetate and 10 mg of TFA GA were each independently weighed into 5 mL hydrolysis vials. A negative control vial was prepared by adding 0.5 mL of the amino acid standard stock solution to a 5 mL hydrolysis vial. 0.5 mL of water and 0.5 mL of concentrated HC1 containing 1% of phenol were added to each of the vials. The vials were heated to 110°C for 24 hours, under N£ atmosphere. The samples were then cooled to room temperature. Each of the hydrolyzates were transferred to 5 mL ;volumetric flasks and filled to volume with distilled water.
ChrCTOOtoqraphy
The t>romotyrosine standard, and each of the hydrolyzates, were independently eluted through an HPI*C column using an eluent of acetonitrile : water : acetic acid in a ratio of 4 : 95 : 1. The column was equipped with an DV detector and data recording system. The amino acid standard is used as a negative control to determine which peak in the glatiramer acetate hydrolyzate corresponds to bromotyrosine.
Data Analysis
The percentage of brominated tyrosine moiety in each TFA
GA and GA sample was calculated as follows:
P = purity of bromotyrosine standard (in percent)
As = Area of bromotyrosine standard peak
Ap *= Area of bromotyrosine peak in each sample
Cs = Concentration of bromotyrosine standard (ug/mL)
Cp = Concentration of glatiramer acetate (or of TFA GA)
Table 1 shows the effect of free Bromine on the level of brominated tyrosine moiety in TFA Glatiramer Acetate and 5 in Glatiramer Acetate
Table 1. Effect Of Free Bromine On The Level Of Brominated Tyrosine Moiety

(Table Removed)
Results
From the above example it can be seen that contamination of HBr with bromine leads to higher levels of brominated tyrosine moiety in TFA GA and in GA, relative to the standard reaction in which no bromine was added. When no bromine was added, since the HBr was not treated with a bromine scavenger, some free bromine was still available and brominated tyrosine moiety contamination of GA and TFA GA was still evident.
In order to produce GA with brominated tyrosine moiety impurity at a level of less than 0.2%, the level of free Bromine in HBr must be lowered by the addition of a bromine scavenger.
EXAMPLE 2 - PRODUCTION OF 33% HBR IN ACETIC ACID SOLUTION
The glass-lined reactor is rinsed with acetic acid, then emptied. 1013 kg of acetic acid is added into the reactor. The acetic acid is maintained at a temperature of 10-20°C. 522 kg of HBr gas is introduced into the reactor while mixing the solution. After the gas is introduced, the solution is mixed for an additional 30 minutes. The solution is tested to determine if HBr content is 33.
EXAMPLE 3 - PURIFICATION OF HER/ ACETIC ACID SOLUTION USING PHENOL AS A BROMINE SCAVENGER
A solution of 33% HBr in acetic acid was poured into a glass-lined reactor. Phenol was weighed and added to the HBr solution in a weight ratio of 1 to 100. The solution was then stirred for 12 to 24 hours. The purified HBr solution is then added to protected glatiramer acetate. The reaction of the HBr with protected GA forms TFA GA. The TFA GA is reacted with piperidine to form GA. Brominated tyrosine moiety in various batches of glatiramer acetate was measured using the method described in example 1.

(Table Removed)
Results
The HBr produced using the new method, as described in
example 2 and treated with phenol as in example 3, was free of Bromine and metallic impurities. .Therefore the glatiramer acetate which was produced was substantially free of brominated tyrosine moiety.
The HBr which was bought from external suppliers (old method) had impurities, and therefore the glatiramer acetate produced using it also had brominated tyrosine moiety impurities, even though phenol was used as a tyrosine scavenger.
EXAMPLE 5 - COLOR DETERMINATION
The color of the HBr/acetic acid solution was determined using standard visual color determination techniques.
The American Public Health Association (APHA) color index
is a single number yellowness index where each APHA unit
is based on a dilution of the 500 ppm stock solution of
platinum-cobalt {PtCo). (HunterLab, APHA Background,
Applications Note, Insight on Color November 16-30, 1996,.
Vol. 8, No. 16. available at
http: //www.hunterlab.com/appnotes/anil 96br2.pdf.) The
APHA measurement is determined by visual comparison of the
solution with PtCo standards that contain controlled
amounts of potassium cbloroplatinate and cobaltous
chloride. Each number unit is the equivalent of 1 mg of
platinum per liter of solution (ppm). The standards and
corresponding measurements are designated according to
their ppm measurement, i.e. the No. 20 APHA standard
contains 20 ppm of platinum. American Chemical Society,
General Directions and Procedures: Measurement of Physical
Properties available at
http://pubs.acs.org/reagent demo/sec b002.html.), distille water has an APHA value of 0, and the stock solution has an APHA value of 500 ppm. (HunterLab, APHA Background, Applications Not, Insight on Color, November 16-30, 1996, Vol. 8, No. 16. available at http://www.hunterlab.com/appnotes/an11 96br2.pdf.) The APHA measurement may be made by various instruments well known in the art.
APHA color standard "500" and APHA color standard "1.000" were prepared. APHA color standard "500" was prepared by
dissolving 1.246 g Potassium Chloroplatinate, K2PtCl6 (equivalent to 50 mg metallic Platinum) and 1.00 g crystallized Cobaltous Chloride, CoCl2-6HzO (equivalent to about 250 mg metallic Cobalt) in distilled water with 100 ml concentrated HC1 and was diluted to 1000 mL with distilled water.
APHA color standard "1000" was prepared by dissolving
2.492 g Potassium Chloroplatinate K2PtCl6 and 2.00 g
crystallized Cobaltous Chloride CoClz-6H20 in distilled
water with 200 mL concentrated HC1 and was diluted to 1000
mL with distilled water. .
The following batches were produced using non-metal apparatus as described previously. These samples were color-tested visually against the color standards by viewing lOOmL Nessler tubes vertically against a white background.

(Table Removed)
The color of these batches of HBr/acetic acid indicated that they were essentially free of bromine and metal ion impurities. Because the color was less than 2000 APHA, these batches were considered essentially free of metal ion impurities.

We Claim:
1. In a process for obtaining trifluoroacetyl glatiramer
acetate, wherein during the process a batch of a mixture
of polypeptides, each of which consists essentially of
alanine, -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,
use of a solution of hydrobromic acid in acetic acid, which solution comprises less than 1000 ppm of metal ion impurities, or
a step of pretreatment of the solution of hydrobromic acid with a bromine scavenger in order to remove free bromine.
2. A process of producing a mixture of trifluoroacetyl glatiramer acetate, wherein the mixture has a desired average molecular weight comprising deprotecting a mixture of polypeptides each consisting essentially of alanine, -kenzyl 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.
3. A process of producing glatiramer acetate comprising the steps of:
a) polymerizing N-carboxyanhydrides of tyrosine, alanine,  benzyl glutamate and N-trif luoroacetyl lysine to form a mixture of protected glatiramer acetate;
b) deprotecting the 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;
c) reacting the trifluoroacetyl glatiramer acetate with aqueous piperidine to form a solution of glatiramer acetate; and
d) purifying the glatiramer acetate.

4. The process of any of claims 1-3, wherein the color of the hydrobromic acid in acetic acid solution is less than 2,000 APHA, less than 1000 APHA, less than 700 APHA, or less than 50 0 APHA.
5. The process of any one of claims 1-4, wherein the solution of hydrobromic acid in acetic acid comprises less than 500 ppm metal ion impurities, less than 100 ppm metal ion impurities, less than 3 0 ppm metal ion impurities, less than 20 ppm metal ion impurities, less than 10 ppm metal ion impurities, or is free of metal ion impurities.
6. The process of any one of claims 1-5, wherein the hydrobromic acid in acetic acid solution is produced in a non-metallic reactor.
7. The process of any one of claims 1-5, wherein the hydrobromic acid in acetic acid solution is prepared in a glass-lined or Teflon-lined reactor.
8. A process for preparing a pharmaceutical composition containing glatiramer acetate, wherein the glatiramer acetate has a predetermined percentage of brominated
tyrosine acceptable for inclusion in a pharmaceutical composition, which comprises
obtaining a batch of glatiramer acetatee;
measuring the percentage of brominated tyrosine of the batch by a process comprising
a) hydrolyzing the batch 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 the batch and of bromotyrosine;
e) eluting the sample solutions through the column of step b); and
f) calculating the percentage of brominated tyrosine in the batch; and
including in the pharmaceutical composition a batch only if its percentage of brominated tyrosine so measured is less than 0.3% by weight.
9. The process of claim 8, wherein the batch is acceptable for inclusion in the pharmaceutical composition only if its percentage of brominated tyrosine so measured is less than 0.2%.
10. The process of claim 8, wherein the batch is acceptable for inclusion in the pharmaceutical composition only if
its percentage of brominated tyrosine so measured is less than 0.1%.
11. A composition comprising trifluoroacetyl glatiramer acetate, wherein the trifluoroacetyl glatiramer acetate hasno more than 0.1% brominated tyrosine by weight and less than 1,000 ppm metal ion impurities.
12. A composition comprising glatiramer acetate, wherein the mixture has no more than 0.1% brominated tyrosine by weight and less than 1,000 ppm of metal ion impurities.
13. The composition of any one of claims 11-12, having an average molecular weight of 4,700 daltons to 11,000 daltons.
14. The composition of any of claims 11-13, wherein the composition comprises less than 500 ppm of metal ion impurities.
15. The composition of any of claims 11-13, wherein the composition comprises less than 100 ppm of metal ion impurities.
16. The composition of any of claims 11-13, wherein the composition comprises less than 30 ppm of metal ion impurities.
17. The composition of any of claims 11-13, wherein the composition comprises less than 2 0 ppm of metal ion impurities.
18. The composition of any of claims 11-13, wherein the composition comprises less than 10 ppm of metal ion impurities.
19. The composition of any of claims 11-13, wherein the
composition is free of metal ion impurities.
20. The composition of any of claims 11-19, wherein the color
of the composition is less than 2,000 APHA.
21. The composition of any of claims 11-19, wherein the color
of the composition is less than 1,000 APHA.
22. The composition of any of claims 11-19, wherein the color
of the composition is less than 700 APHA.
23. The composition of any of claims 11-19, wherein the color
of the composition is less than 500 APHA.
24. Trifluoroacetyl glatiramer acetate produced by the
process of any one of claims 1, 2, or 4-7.
25. Glatiramer acetate produced by the process of any one of claims 3-7.