U.S. patent application number 14/514320 was filed with the patent office on 2015-04-16 for control of copolymer compositions.
The applicant listed for this patent is Momenta Pharmaceuticals, Inc.. Invention is credited to Claire Coleman, John Schaeck, Alicia Thompson.
Application Number | 20150105535 14/514320 |
Document ID | / |
Family ID | 42230467 |
Filed Date | 2015-04-16 |
United States Patent
Application |
20150105535 |
Kind Code |
A1 |
Coleman; Claire ; et
al. |
April 16, 2015 |
CONTROL OF COPOLYMER COMPOSITIONS
Abstract
Methods of making copolymers are described.
Inventors: |
Coleman; Claire; (Pelham,
NH) ; Schaeck; John; (Somerville, MA) ;
Thompson; Alicia; (Danvers, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Momenta Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
42230467 |
Appl. No.: |
14/514320 |
Filed: |
October 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13187243 |
Jul 20, 2011 |
8058235 |
|
|
14514320 |
|
|
|
|
12754344 |
Apr 5, 2010 |
8859489 |
|
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13187243 |
|
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|
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Current U.S.
Class: |
530/330 |
Current CPC
Class: |
C07K 14/001 20130101;
A61P 37/00 20180101; C07K 5/10 20130101 |
Class at
Publication: |
530/330 |
International
Class: |
C07K 5/10 20060101
C07K005/10 |
Claims
1. A method for preparing a composition comprising glatiramer
acetate, the method comprising: polymerizing N-carboxy anhydrides
of L-alanine, benzyl-protected L-glutamic acid, trifluoroacetic
acid protected L-lysine and L-tyrosine to generate a sample
comprising intermediate-1; treating the sample comprising
intermediate-1 to partially depolymerize and deprotect
benzyl-protected L-glutamic acid, thereby generating a sample
comprising intermediate-2, wherein water is present for at least a
portion of the treatment in a range of 4-25% weight/weight (w/w)
against intermediate-1 present at the beginning of treatment;
treating the sample comprising intermediate-2 to deprotect
trifluoroacetic acid protected L-lysine, thereby generating
intermediate-3; further processing the intermediate-3 to generate
glatiramer acetate; and purifying the glatiramer acetate to
generate purified glatiramer acetate.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a continuation and claims priority to
U.S. application Ser. No. 13/187,243 filed Jul. 20, 2011 and claims
priority to Ser. No. 12/754,344, filed on Apr. 5, 2010, and claims
priority to U.S. Provisional Application Ser. No. 61/166,608, filed
on Apr. 3, 2009 and U.S. Provisional Application Ser. No.
61/247,321, filed on Sep. 30, 2009, all of which are hereby
incorporated by reference.
BACKGROUND
[0002] Glatiramer acetate (also known as copolymer-1 and marketed
as the active ingredient in COPAXONE.RTM. by Teva Pharmaceutical
Industries Ltd., Israel) is used in the treatment of the
relapsing-remitting form of multiple sclerosis (RRMS). According to
the COPAXONE.RTM. product label, glatiramer acetate (GA) consists
of the acetate salts of synthetic polypeptides containing four
naturally occurring amino acids: L-glutamic acid, L-alanine,
L-tyrosine, and L-lysine with a reported average molar fraction of
0.141, 0.427, 0.095, and 0.338, respectively. Chemically,
glatiramer acetate is designated L-glutamic acid polymer with
L-alanine, L-lysine and L-tyrosine, acetate (salt). Its structural
formula is:
(Glu, Ala, Lys, Tyr).sub.x.xCH.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.xC.sub.3H.sub.4O.sub.2CAS-147245-92-9
SUMMARY OF THE INVENTION
[0003] The invention is based, at least in part, on the
identification of methods for controlling the level of
L-pyroGlutamic Acid (pyro-Glu) in glatiramer acetate (GA). Pyro-Glu
is present in GA, and the ability to control the level of pyro-Glu
in GA is useful in controlling both product and process quality in
the manufacture of GA.
[0004] Described herein is a method for preparing a composition
comprising glatiramer acetate, comprising: polymerizing N-carboxy
anhydrides of L-alanine, benzyl-protected L-glutamic acid,
trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to
generate a protected copolymer (Intermediate-1); treating the
protected copolymer to partially depolymerize the protected
copolymer and deprotect benzyl protected groups thereby generating
a partially depolymerized, benzyl-deprotected product
(Intermediate-2); treating the partially depolymerized product to
deprotect TFA-protected lysines thereby generating a
TFA-deprotected product (Intermediate-3) and further processing the
Intermediate-3 to create glatiramer acetate, wherein the
improvement comprises: having water present during at least a
portion of the depolymerization step.
[0005] Also described herein is a method for preparing a
composition comprising glatiramer acetate, comprising: polymerizing
N-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic
acid, trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine
to generate a protected copolymer (Intermediate-1); treating the
protected copolymer to partially depolymerize the protected
copolymer and deprotect benzyl protected groups thereby generating
a partially depolymerized, benzyl-deprotected product
(Intermediate-2); and treating the partially depolymerized product
to deprotect TFA-protected lysines thereby generating a
TFA-deprotected product (Intermediate-3); and further processing
the Intermediate-3 to create glatiramer acetate, wherein the
improvement comprises: adjusting the water present during at least
a portion of the depolymerization step so that amount water is
present during at least a portion of the depolymerization step is
within a predetermined range.
[0006] Also described herein is a method for preparing a
composition comprising glatiramer acetate, comprising: polymerizing
N-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic
acid, trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine
to generate a protected copolymer (Intermediate-1); treating the
protected copolymer to partially depolymerize the protected
copolymer and deprotect benzyl protected groups thereby generating
a partially depolymerized, benzyl-deprotected product
(Intermediate-2); treating the partially depolymerized,
benzyl-deprotected product to deprotect TFA-protected lysines
thereby generating acetate TFA-deprotected product
(Intermediate-3); and further processing Intermediate-3 to create
glatiramer acetate, wherein the improvement comprises: controlling
the water present during at least a portion of the depolymerization
step so that amount water is present during at least a portion of
the depolymerization step is within a predetermined range.
[0007] In various embodiments of the forgoing methods: water is
present, adjusted or controlled at the beginning of the
depolymerization step; water is added during the depolymerization
step; the water present during the depolymerization step is present
within a predetermined range (e.g., the predetermined range is
4-25%, 5-25%, 4-20%, 4-16%, 7-15%, 8-14%, 9-13%, 10-12%, 13-19%,
14-18% w/w against Intermediate-1); the depolymerization proceeds
for 16-64 hrs, preferably at least 25 hrs (e.g., 25-55 hrs, at
least 30 hrs, 30-50 hrs, at least 40 hrs, 43-47 hrs); the
depolymerization reaction is carried out at 17-35.degree. C., e.g.,
18-30.degree. C., 18-22.degree. C.; the depolymerization step
comprises contacting the protected copolymer with a solution
comprising phenol, HBr and acetic acid; the concentration of
pyroglu in the purified glatiramer acetate is 2000-7000 ppm (e.g.,
2500-6000 ppm; 2500-5500 ppm; 3000-5000 ppm; 3500-4500 ppm,
2400-6500 ppm); the Mp of the purified glatiramer acetate is
5,000-9,000 Da (e.g., 6,500-7,500 Da); in one embodiment water is
present during the depolymerization step at 11.2% w/w against
Intermediate-1, the depolymerization proceeds for 43-47 hrs at
18-22.degree. C. and the process produces purified glatiramer
acetate in which pyro-Glu is present at 0.24-0.65% w/w (2400-6500
ppm). The improvement further comprises: preparing a pharmaceutical
composition comprising at least a portion of the purified
glatiramer acetate; and in some cases the method further includes
measuring the amount of water in the depolymerization step at least
once.
[0008] Also described is a method for preparing a composition
comprising glatiramer acetate, comprising: polymerizing N-carboxy
anhydrides of L-alanine, benzyl-protected L-glutamic acid,
trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to
generate a protected copolymer; treating the protected copolymer to
partially depolymerize the protected copolymer and deprotect benzyl
protected groups thereby generating a partially depolymerized
product; treating the partially depolymerized product to deprotect
TFA-protected lysines thereby generating a TFA-deprotected product;
and processing the TFA-deprotected product to create glatiramer
acetate, wherein water is present during at least a portion of the
depolymerization step.
[0009] An additional method is a method for preparing a composition
comprising glatiramer acetate, comprising: polymerizing N-carboxy
anhydrides of L-alanine, benzyl-protected L-glutamic acid,
trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine to
generate a protected copolymer; treating the protected copolymer to
partially depolymerize the protected copolymer and deprotect benzyl
protected groups thereby generating a partially depolymerized
product; treating the partially depolymerized product to deprotect
TFA-protected lysines thereby generating glatiramer acetate; and
purifying the glatiramer acetate to create purified glatiramer
acetate, wherein water is present during at least a portion of the
depolymerization step within a predetermined range.
[0010] An additional described method is a method for preparing a
composition comprising glatiramer acetate, comprising: polymerizing
N-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic
acid, trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine
to generate a protected copolymer; treating the protected copolymer
to partially depolymerize the protected copolymer and deprotect
benzyl protected groups thereby generating a partially
depolymerized product; treating the partially depolymerized product
to deprotect TFA-protected lysines thereby generating a
TFA-deprotected product; and processing the a TFA-deprotected
product to create glatiramer acetate, wherein the water present
during at least a portion of the depolymerization step is
controlled to be within a predetermined range.
[0011] In various embodiments of the foregoing methods: water is
present, adjusted or controlled at the beginning of the
depolymerization step; water is added during the depolymerization
step; the water present during the depolymerization step is present
within a predetermined range (e.g., the predetermined range is
4-25%, 5-25%, 13-19%, 14-18% w/w against Intermediate-1); the
depolymerization proceeds for at least 25 hrs (e.g., at least 30
hrs or at least 40 hrs); the depolymerization step comprises
contacting the protected copolymer with a solution comprising
phenol, HBr and acetic acid; the concentration of pyroglu in the
purified glatiramer acetate is 2000-7000 ppm (e.g., 2500-6000 ppm;
2500-5500 ppm; 3000-5000 ppm; 3500-4500 ppm, 2400-6500 ppm
(0.24%-0.65% w/w); the the Mp of the purified glatiramer acetate is
5,000-9,000 Da (e.g., 6,500-7,500 Da); the improvement further
comprises: preparing a pharmaceutical composition comprising at
least a portion of the purified glatiramer acetate; the step of
treating the partially depolymerized product to deprotect
TFA-protected lysines comprises treating the depolymerized product
with piperidine; the protected copolymer is isolated and at least
partially dried prior to treating the protected copolymer to
partially depolymerize the protected copolymer and deprotect benzyl
protected groups; the partially depolymerized product is isolated
and at least partially dried prior to the step of treating the
partially depolymerized product to deprotect TFA-protected lysines;
in some cases the method further includes measuring the amount of
water in the depolymerization step at least once.
[0012] As used herein, a "copolymer", "amino acid copolymer" or
"amino acid copolymer preparation" is a heterogeneous mixture of
polypeptides comprising a defined plurality of different amino
acids (typically between 2-10, e.g., between 3-6, different amino
acids). A copolymer may be prepared from the polymerization of
individual amino acids. The term "amino acid" is not limited to
naturally occurring amino acids, but can include amino acid
derivatives and/or amino acid analogs. For example, in an amino
acid copolymer comprising tyrosine amino acids, one or more of the
amino acids can be a homotyrosine. Further, an amino acid copolymer
having one or more non-peptide or peptidomimetic bonds between two
adjacent residues is included within this definition.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 depicts the structure of pyro-Glu.
[0014] FIG. 2 is a graph depicting the results of studies on the
effect of the presence water in the depolymerization reaction used
in glatiramer acetate production.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Other than a statement about molecular weight and amino acid
composition, which are recited in the FDA-approved label for the
product, the label and other available literature for COPAXONE.RTM.
does not provide detailed information about the physiochemical
characteristics of the product. It has been previously found that
Pyro-Glu (FIG. 1) is a component of Copaxone.RTM. (glatiramer
acetate or GA) that is present within a predetermined range (U.S.
Ser. No. 12/408,058). For example, in many cases the pyro-Glu
content of a GA preparation can be between 2000 ppm and 7000 ppm or
2400-6500 ppm.
[0016] The production of GA entails polymerization of amino acids
to produce a mixture of peptides, referred to as Intermediate-1,
followed by partial depolymerization and deprotection of
Intermediate-1 to yield Intermediate-2. It has now been found that
the level of pyro-Glu in GA can be effectively controlled by
controlling the water present during the depolymerization step of
the GA manufacturing process, for example, by adjusting the water
content at the beginning of and/or during the depolymerization
step, e.g., by adding water to a predetermined level at the
beginning or during the depolymerization step. Moreover, it has now
been found that by properly controlling (e.g., adjusting) the
amount of water present during the depolymerization step and the
duration of the depolymerization step it is possible to produce GA
with a specified pyro-Glu content and a specified peak molecular
weight (Mp). In many cases it is specified to have the pyro-Glu
content of copolymer or GA be 2000 to 7000 ppm, e.g., 2500-5500
ppm, e.g., 3000-5000 ppm, e.g., 3500-4500 ppm, 2400-6500 ppm, and
the water present during the depolymerization reaction or added at
the end of the depolymerization reaction is preferably controlled
or adjusted to achieve this specified pyro-Glu content. In many
cases it is desirable to have the peak molecular weight (Mp) of GA
be 5,000 to 9,000 Da, e.g., 6,000 to 8,000 Da, as measured as
described in U.S. Pat. No. 7,074,580.
Manufacture of Glatiramer Acetate
[0017] Generally, the process for the manufacture of glatiramer
acetate includes the following steps: [0018] Step 1: polymerization
of N-carboxy anhydrides of L-alanine, benzyl-protected L-glutamic
acid, trifluoroacetic acid (TFA) protected L-lysine and L-tyrosine
(collectively referred to as NCAs) to result in a protected
copolymer (Intermediate-1), [0019] Step 2: depolymerization and
benzyl deprotection of Intermediate-1 using hydrobromic acid in
acetic acid (e.g., phenol treated 33% HBr/acetic acid), and [0020]
Step 3: deprotection of the TFA-protected lysines on Intermediate-2
(e.g., by treatment with piperdine) to create Intermediate-3,
followed by processing to generate GA and further purification and
drying of the isolated GA drug substance.
[0021] In Step 1 of GA manufacture, the NCAs are co-polymerized in
a predetermined ratio using diethylamine as an initiator. Upon
consumption of the NCA components, the reaction mixture is quenched
in water. The resulting protected polymer (Intermediate-1) is
isolated and dried. In Step 2 of GA manufacture, Intermediate-1 is
treated with phenol-treated 33% HBr in acetic acid (HBr/AcOH). This
results in the cleavage of the benzyl protecting group on the
glutamic acids as well as cleavage of peptide bonds throughout the
polymer. After a period of time the reaction is quenched with
water, and the product polymer is isolated by filtration and washed
with water. The product polymer, Intermediate-2, has a reduced
molecular weight relative to Intermediate-1. Intermediate-2 is
dried before proceeding to Step 3. In Step 3, Intermediate-2 is
treated with aqueous piperidine to remove the trifluoroacetyl group
on the lysines. The resulting copolymer, Intermediate-3, is
subsequently purified using diafiltration/ultrafiltration and the
resulting acetate salt is dried to produce Glatiramer Acetate drug
substance.
[0022] Methods for the manufacture of GA are described in the
following publications: U.S. Pat. No. 3,849,550; WO 95/031990 and
US 2007-0021324.
Control of pyro-Glu and Depolymerization with Water
[0023] As shown below, GA with a pyro-Glu content of about 4,000
ppm and a peak molecular weight (Mp) about 7,000 Da can prepared by
having water present in the depolymerization reaction at about 16%
w/w against Intermediate-1. While the amount of water present is
expressed here relative to the amount Intermediate 1, the amount of
water present can be expressed in any convenient manner, for
example: w/w against the weight of Intermediate-1 added to the
depolymerization reaction; w/w against the weight of phenol used to
treat the HBr/acetic acid added to depolymerization reaction; w/w
against the total weight of HBr/acetic acid added to
depolymerization reaction; v/v against the total volume of
HBr/acetic acid added to the depolymerization reaction; or w/w
against the total weight of the depolymerization reaction. Thus,
the amount of water present relative to HBr/AcOH on a v/v basis can
be calculated from the amount of water present relative to
Intermediate-1 on a w/w basis as follows:
Vol.sub.(water)/Vol.sub.(HBR/AcOH)=(Wt.sub.(water)/
Wt.sub.(Intermediate-1)).times.(Wt.sub.(Intermediate-1)/Wt.sub.(HBr/AcOH)-
).times.(Wt.sub.(HBr/AcOH)/Vol.sub.(HBr/AcOH)).times.(Vol.sub.(water))
/Wt.sub.(water))=(Wt.sub.(water)/Wt.sub.(Intermediate-1)).times.(Wt.sub.(-
Intermediate-1)/Wt.sub.(HBr/AcOH)).times.(Density.sub.(HBr/AcOH)/Density.s-
ub.(water))
[0024] The water present during the depolymerization reaction can
include water present in the Intermediate-1 added to the
depolymerization reaction (e.g., by using Intermediate-1 that is
not fully dried) and/or water that is added at the beginning or
during the depolymerization reaction. Thus, the amount of water
present during at least a portion of the depolymerization reaction
can be controlled by adding water to the reaction to achieve a
predetermined level of water or by having a certain amount of water
present in the Intermediate-1 added to the reaction or by a
combination of adding water and having water present in the
Intermediate-1. Thus, the amount of water present can be controlled
by simply having a reasonably consistent amount of water present in
the Intermediate-1. Water can be added to the depolymerization
reaction at any time, but is most often present at a predetermined
level, e.g., 4-25%, 5-25%, 10-20%, 4-20%, 4-16%, 7-15%, 8-14%,
9-13%, 10-12%, 13-19%, 14-18%, 15-17%, or 16% w/w against the
weight of Intermediate-1, at the beginning of the depolymerization
reaction. Because the depolymerization reaction can both consume
and produce water, the amount of water present can change slightly
over the course of the depolymerization reaction.
[0025] The amount of water present during the depolymerization step
can impact the pyro-Glu content and molecular weight of the
resulting GA, as shown by the experiments described below. However,
the amount of water present during the depolymerization step can
vary over a reasonable range and still be compatible with the
production of GA having a desirable pyro-Glu content and molecular
weight.
EXAMPLES
Example 1
[0026] The effect of water present during the depolymerization
step, Step 2, on the pyro-Glu content and molecular weight of the
resulting GA was examined as follows. Intermediate-1 was produced
as described above and divided between two depolymerization
reactions (A and B). For Depolymerization reaction A, no water was
added. For Depolymerization reaction B, water was added to 16%
measured w/w against Intermediate-1. Depolymerization was allowed
to proceed at 20.degree. C. Aliquots removed periodically from each
reaction were quenched with water and further processed to produce
GA. The pyro-Glu content (ppm), measured as described below, and
peak molecular weight (Mp) of each of the resulting GA samples were
measured. The results of this analysis are shown in FIG. 2. The
molecular weight (Mp) scale (Da) is on the left axis, the pyro-Glu
concentration scale (ppm) is on the right axis. The time of
Depolymerization reaction A (no added water) is on the upper
horizontal axis, and the time of Depolymerization reaction B (water
present at 16% w/w against intermediate-1) is on the lower
horizontal axis. The scale of the graph is such that the horizontal
line labeled "Midpoint of desired range of GPC-Mp (Da)/Midpoint of
the desired range of pyroGlu (ppm)" indicates both one desirable Mp
molecular weight (7,000 Da) and one desirable pyro-Glu
concentration for GA (4,000 ppm). The lines labeled MW.sub.A and
MW.sub.B depict the Mp molecular weight of the GA produced from
material removed from Depolymerization reaction A and
Depolymerization reaction B, respectively, at various time points.
The lines labeled Py.sub.A and Py.sub.B depict the concentration
pyro-Glu in the GA produced from material removed from
Depolymerization reaction A and Depolymerization reaction B,
respectively, at various time points.
[0027] In the absence of added water, the desired combination of
molecular weight and pyro-Glu concentration is not achieved. As can
be seen in FIG. 2, after about 12 hours (scale on upper horizontal
axis) Depolymerization reaction A (no added water) produces
material that yields GA having a desired pyro-Glu concentration
(about 4,000 ppm), but the molecular weight (Mp) of the GA is about
7,400 Da, above the desired 7,000 Da. As can be also seen in FIG.
2, after about 26 hours (scale on upper horizontal axis)
Depolymerization reaction A (no added water) produces material that
yields GA having a desired Mp (about 7,000 Da), but the pyro-Glu
concentration of the GA is greater than 6,000 ppm, which is above
4,000 ppm (the midpoint of the desired range). In contrast, when
water is added to the depolymerization reaction to 16% (w/w against
Intermediate-1), the desired combination of molecular weight and
pyro-Glu concentration is achieved. As can also be seen from FIG.
2, after about 43 hours Depolymerization reaction B (16% water w/w
against Intermediate-1) produces material that yields GA having a
desired molecular weight (Mp about 7,000 Da) and a desired pyro-Glu
concentration (about 4,000 ppm).
Example 2
[0028] In the study described above pyro-Glu concentration of GA
was measured as follows. N-terminal gyro-Glu residues were cleaved
using Pyrococcus furiosus pyro-glutamate aminopeptidase. Pyro-Glu
in the resulting enzymatic hydrolysate is isolated by reverse phase
liquid chromatography followed by detection at 200 nm using a
reference standard curve prepared with known concentrations of
L-Pyro-glutamate. Neurotensin (a commercially available polypeptide
having 100% pyro-glutamate at the N-terminus) is assayed as a
control to ensure the acceptability of the digestion and adequacy
of the HPLC separation. The chromatographic analysis is performed
using a Waters Atlantis C 18 HPLC column and an isocratic mobile
phase consisting of 100% Water, adjusted to pH 2.1 with phosphoric
acid. Samples and Standards are held at 2-8.degree. C. The peak
corresponding to the pyro-glutamate moiety elutes at a retention
time of approximately 12 minutes. The direct measure of
pyro-glutamate content is on a w/w basis and the results are
expressed as ppm (microgram/gram).
* * * * *