U.S. patent application number 11/529668 was filed with the patent office on 2007-01-25 for process for preparation of mixtures of polypeptides using purified hydrobromic acid.
This patent application is currently assigned to Teva Pharmaceutical Industries, Ltd.. Invention is credited to Ben-Zion Dolitzky.
Application Number | 20070021324 11/529668 |
Document ID | / |
Family ID | 36037047 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070021324 |
Kind Code |
A1 |
Dolitzky; Ben-Zion |
January 25, 2007 |
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.
Inventors: |
Dolitzky; Ben-Zion;
(Petach-Tikva, IL) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Assignee: |
Teva Pharmaceutical Industries,
Ltd.
|
Family ID: |
36037047 |
Appl. No.: |
11/529668 |
Filed: |
September 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11223072 |
Sep 9, 2005 |
|
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11529668 |
Sep 27, 2006 |
|
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60608843 |
Sep 9, 2004 |
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Current U.S.
Class: |
530/333 ;
514/1.1; 530/324; 530/402 |
Current CPC
Class: |
C07K 14/4713 20130101;
C07K 14/001 20130101; A61P 25/00 20180101; C07K 14/00 20130101;
A61P 37/06 20180101 |
Class at
Publication: |
514/002 ;
530/402; 530/324 |
International
Class: |
A61K 38/16 20070101
A61K038/16; C07K 14/47 20070101 C07K014/47 |
Claims
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, .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 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,
.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.
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)
Description
[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.
[0044] In a further embodiment, the solution of hydrobromic acid in
acetic acid comprises less than 0.01% of free bromine.
[0045] In yet another embodiment, the solution of hydrobromic acid
in acetic acid comprises less than 0.001% of free bromine.
[0046] In a further embodiment, the solution of hydrobromic acid in
acetic acid is free of free bromine.
[0047] In another embodiment, the solution of hydrobromic acid in
acetic acid comprises less than 1000 ppm of metal ion
impurities.
[0048] In yet another embodiment, the solution of hydrobromic acid
in acetic acid comprises less than 500 ppm of metal ion
impurities.
[0049] In one embodiment, the solution of hydrobromic acid in
acetic acid comprises less than 100 ppm of metal ion
impurities.
[0050] In another embodiment, the solution of hydrobromic acid in
acetic acid comprises less than 30 ppm of metal ion impurities.
[0051] In yet another embodiment, the solution of hydrobromic acid
in acetic acid comprises less than 20 ppm of metal ion
impurities.
[0052] In a further embodiment, the solution of hydrobromic acid in
acetic acid comprises less than 10 ppm of metal ion impurities.
[0053] In another embodiment, the solution of hydrobromic acid in
acetic acid is free of metal ion impurities.
[0054] In yet another embodiment, the mixture of trifluoroacetyl
polypeptides is trifluoroacetyl glatiramer acetate ("TFA GA").
[0055] 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.
[0056] In another embodiment, the solution is pretreated with a
bromine scavenger in order to remove free bromine.
[0057] In one embodiment, the bromine scavenger is phenol.
[0058] In a further embodiment, the solution is produced in a
non-metallic reactor.
[0059] In another embodiment, the solution is prepared in a
glass-lined or Teflon lined reactor.
[0060] In yet another embodiment, the color of the hydrobromic acid
in acetic acid solution is less than 2000 APHA.
[0061] In a further embodiment, the color of the hydrobromic acid
in acetic acid solution is less than 1000 APHA.
[0062] In another embodiment, the color of the hydrobromic acid in
acetic acid solution is less than 700 APHA.
[0063] In yet another embodiment, the color of the hydrobromic acid
in acetic acid solution is less than 500 APHA.
[0064] The subject invention also provides a trifluoroacetyl
product produced by any one of the disclosed processes.
[0065] The subject invention further provides a composition
comprising the trifluoroacetyl product produced by any one of the
disclosed processes, and a carrier.
[0066] 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 more
than 0.1% brominated tyrosine and less than 1000 ppm metal ion
impurities.
[0067] In one embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight from 2000 daltons to
40,000 daltons.
[0068] In another embodiment, the mixture of trifluoroacetyi
polypeptides has an average molecular weight from 4000 daltons to
18,000 daltons.
[0069] In a further embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight from 4000 daltons to
13,000 daltons.
[0070] In another embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight from 13,000 daltons to
19,000 daltons.
[0071] In yet another embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight from 13,500 daltons to
18,500 daltons.
[0072] In a further embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight of 7,000.+-.2,000
daltons.
[0073] In yet a further embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight of 7,000 daltons.
[0074] In another embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight of 14,000 daltons.
[0075] In yet another embodiment, the mixture of trifluoroacetyl
polypeptides has an average molecular weight from 4,700-11,000
daltons.
[0076] In a further embodiment, the mixture of trifluoroacetyl.
polypeptides comprises less than 1000 ppm of metal ion
impurities.
[0077] In yet another embodiment, the mixture of trifluoroacetyl
polypeptides comprises less than 500 ppm of metal ion
impurities.
[0078] In a further embodiment, the mixture of trifluoroacetyl
polypeptides comprises less than 100 ppm of metal ion
impurities.
[0079] In another embodiment, the mixture of trifluoroacetyl
polypeptides comprises less than 30 ppm of metal ion
impurities.
[0080] In a further embodiment, the mixture of trifluoroacetyl
polypeptides comprises less than 20 ppm of metal ion
impurities.
[0081] In another embodiment, the mixture of trifluoroacetyl
polypeptides comprises less than 10 ppm of metal ion.
impurities.
[0082] In yet another embodiment, the mixture of trifluoroacetyl
polypeptides is free of metal ion impurities.
[0083] The subject invention also provides a composition comprising
the mixture of trifluoroacetyl polypeptides and a carrier.
[0084] 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 [0085] a) polymerizing
N-carboxyanhydrides of tyrosine, alanine, .gamma.-benzyl glutamate
and N-trifluoroacetyl lysine to form an aqueous mixture of
protected polypeptides; [0086] 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; [0087] 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 [0088] d) purifying the aqueous mixture of
polypeptides.
[0089] In one embodiment, the average mole fraction in the mixture
is glutamic acid 0.129-0.159; alanine 0.392-0.462; 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.
[0090] The subject invention also provides a process of producing
glatiramer acetate comprising the steps of: [0091] a) polymerizing
N-carboxyanhydrides of tyrosine, alanine, .gamma.-benzyl glutamate
and N-trifluoroacetyl lysine to form protected glatiramer acetate;
[0092] 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; [0093] c)
reacting trifluoroacetyl glatiramer acetate with aqueous piperidine
to form a solution of glatiramer acetate; and [0094] d) purifying
the glatiramer acetate.
[0095] In one embodiment, the product of the step d) is further
subjected to ultrafiltration to remove polypeptide species with
molecular weight less than 5000 daltons.
[0096] 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% nydrobromic 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.
[0097] In another embodiment, the hydrobromic acid in acetic acid
solution is pretreated with a bromine scavenger in order to remove
free bromine.
[0098] In yet another embodiment, the bromine scavenger is
phenol.
[0099] In a further embodiment, the hydrobromic acid in acetic acid
solution is produced in a non-metallic reactor.
[0100] In another embodiment, the hydrobromic acid in acetic. acid
solution is prepared in a glass-lined or Teflon lined reactor.
[0101] In one embodiment, the color of the hydrobromic acid in
acetic acid solution is less than 2000 APHA.
[0102] In another embodiment, the color of the hydrobromic acid in
acetic acid solution is less than 1000 APHA.
[0103] In yet another embodiment, the color of the hydrobromic acid
in acetic acid solution is less than 700 APHA.
[0104] In a further embodiment, the color of the hydrobromic acid
in acetic acid solution is less than 500 APHA.
[0105] The subject invention further provides method of analyzing
the percentage of brominated tyrosine in a sample of alatiramer
acetate comprising the steps of: [0106] a) hydrolyzing glatiramer
acetate to obtain a hydrolyzate; [0107] b) eluting the hydrolyzate
through a chromatographic column; [0108] c) measuring the level of
bromotyrosine in the hydrolyzate; [0109] d) preparing sample
solutions of the amino acid components of glatiramer acetate and of
bromotyrosine; [0110] e) eluting the sample solutions through the
column of step b); and [0111] f) calculating the percentage of
brominated tyrosine in the glatiramer acetate.
[0112] 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; [0113] measuring the percentage of
brominated tyrosine of the batch by a process comprising [0114] a)
hydrolyzing the batch to obtain a hydrolyzate; [0115] b) eluting
the hydrolyzate through a chromatographic column; [0116] c)
measuring the level of bromotyrosine in the hydrolyzate; [0117] d)
preparing sample solutions of the amino acid components of the
batch and of bromotyrosine; [0118] e) eluting the sample solutions
through the column of step b); and [0119] f) calculating the
percentage of brominated tyrosine in the batch; and [0120]
including in the pharmaceutical composition a batch only if its
percentage of brominated tyrosine so measured is less than
0.3%.
[0121] 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%.
[0122] 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%.
[0123] In a further embodiment, the mixture of polypeptides is
glatiramer acetate ("GA").
Terms
[0124] 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.
[0125] 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. 5Bz-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.
[0126] The molar fraction range of each amino acid residue is:
L-Glu 9.129-0.153, L-Ala 0.392-0.462, L-Tyr 0.086-0.100 and L-Lys
0.300-0.374.
[0127] 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: [0128]
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.
[0129] The molecular structures of the identified structure-related
impurities can be derived from the participating monomers i.e.
starting materials.
[0130] 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:
[0131] Residual trifluoroacetyl compounds (expressed as fluoride)
[0132] Residual benzylated glutamyl residues (expressed as benzyl
bromide) [0133] Residual brominated tyrosyl residues (expressed as
bromotyrosine)
[0134] 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.
[0135] The characterization of the impurities is based on their
relative chromatography retention time (RRT) relative to the
L-Tryptophan standard.
[0136] 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
[0137] 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 tyrosine
residue was found to react with bromine to form a
mono-bromotyrosine moiety comprising either 2-bromotyrosine or
3-bromotyrosine.
[0138] 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.
[0139] 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.
[0140] 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
(bromophenols) 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 during
the final purification process.
Metal Impurities
[0141] GA is marketed in two pharmaceutical dosage forms,
lyophilized powder and pre-filled syringes. The syringes, marketed
under the trade name Copaxone.RTM. Injection, generally contained
clear solution. The storage instructions were to keep the syringes
refrigerated. However, red color in aqueous solutions of
Copaxone.RTM. pre-filled solutions was detected. The source of the
color in the solutions was unknown.
[0142] The color appeared when the solutions were kept at room
temperature for 12 to 24 hours.
[0143] 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 ion impurities in the HBr
were chelated by TFA GA and GA. These TFA GA and GA/metal complexes
contributed to the coloration.
[0144] 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-metal 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.
[0145] 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.
[0146] 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
[0147] 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
[0148] Standard solutions containing 2 .mu.g/mL Bromotyrosine were
prepared using distilled water. Amino acid standard stock solution
was prepared using the following amino acids: TABLE-US-00001 L-Glu
About 100 mg L-Ala About 130 mg L-Tyr About 75 mg L-Lys HCl About
200 mg
[0149] 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
[0150] 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 HCl containing 1% of phenol were added
to each of the vials. The vials were heated to 110.degree. C. for
24 hours, under N.sub.2 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.
Chromotography
[0151] The bromotyrosine standard, and each of the hydrolyzates,
were independently eluted through an HPLC column using an eluent of
acetonitrile:water:acetic acid in a ratio of 4:95:1. The column was
equipped with an UV 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
[0152] The percentage of brominated tyrosine moiety in each TFA GA
and GA sample was calculated as follows:
[0153] P=purity of bromotyrosine standard (in percent)
[0154] As=Area of bromotyrosine standard peak
[0155] Ap=Area of bromotyrosine peak in each sample
[0156] Cs=Concentration of bromotyrosine standard (pg/mL)
[0157] Cp=Concentration of glatiramer acetate (or of TFA GA) %
.times. .times. Brominated .times. .times. tyrosine = p * Ap As *
Cs Cp ##EQU1##
[0158] Table 1 shows the effect of free Bromine on the level of
brominated tyrosine moiety in TFA Glatiramer Acetate and in
Glatiramer Acetate TABLE-US-00002 TABLE 1 Effect Of Free Bromine On
The Level Of Brominated Tyrosine Moiety Brominated tyrosine (%)
Bromine (%) TFA Glatiramer Glatiramer Acetate No added Bromine 0.1
0.2 0.5 0.7 1.2 1 1.2 2.2 5 4 No Data
Results
[0159] 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.
[0160] 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
[0161] 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.degree. 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 HBR/Acetic Acid Solution Using Phenol as a Bromine
Scavenger
[0162] 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.
EXAMPLE 4
Levels of Brominated Tyrosine in Various Batches
[0163] The level of Brominated tyrosine moiety in various batches
of glatiramer acetate was measured using the method described in
example 1. TABLE-US-00003 Method of Batch Number Brominated
tyrosine Production of GA moiety concentration OLD METHOD A 0.15 B
0.19 C 0.14 D 0.15 E 0.32 NEW METHOD X None detectable Y None
detectable Z None detectable
Results
[0164] 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.
[0165] 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
[0166] The color of the HBr/acetic acid solution was determined
using standard visual color determination techniques.
[0167] 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
Nov. 16-30, 1996, Vol. 8, No. 16. available at
http://www.hunterlab.com/appnotes/an11.sub.--96br2.pdf.) The APHA
measurement is determined by visual comparison of the solution with
PtCo standards that contain controlled amounts of potassium
chloroplatinate 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, Nov. 16-30, 1996, Vol. 8, No. 16. available at
http://www.hunterlab.com/appnotes/an11.sub.--96br2.pdf.) The APHA
measurement may be made by various instruments well known in the
art.
[0168] APHA color standard "500" and APHA color standard "1000"
were prepared. APHA color standard "500" was prepared by dissolving
1.246 9 Potassium Chloroplatinate, K.sub.2PtCl.sub.6 (equivalent to
50 mg metallic Platinum) and 1.00 g crystallized Cobaltous
Chloride, CoCl.sub.2-6H.sub.20 (equivalent to about 250 mg metallic
Cobalt) in distilled water with 100 ml concentrated HCl and was
diluted to 1000 mL with distilled water.
[0169] APHA color standard "1000" was prepared by dissolving 2.492
g Potassium Chloroplatinate K.sub.2PtCl.sub.6 and 2.00 g
crystallized Cobaltous Chloride CoCl.sub.2-6H.sub.20 in distilled
water with 200 mL concentrated HCl and was diluted to 1000 mL with
distilled water.
[0170] The following batches were produced using non-metal
apparatus as described previously. These samples were color-tested
visually against the color standards by viewing 100 mL Nessler
tubes vertically against a white background. TABLE-US-00004 Batch
Number Color (APHA) M <500 N <500 P 700 Q 350 R <300
[0171] 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.
* * * * *
References