U.S. patent application number 12/727758 was filed with the patent office on 2010-07-08 for processes for preparing a polypeptide.
Invention is credited to Mariappan Anbazhagan, Johannes Ludescher, Ingolf Macher, Hiren Kumar V. Patel, Mahendra R. Patel, Anup Kumar Ray.
Application Number | 20100174048 12/727758 |
Document ID | / |
Family ID | 36046637 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100174048 |
Kind Code |
A1 |
Ray; Anup Kumar ; et
al. |
July 8, 2010 |
Processes for Preparing a Polypeptide
Abstract
The present invention relates to an improved process for
preparing a polypeptide or pharmaceutically acceptable salt thereof
comprising L-tyrosine, L-alanine, L-glutamate, and L-lysine. The
polypeptide or pharmaceutically acceptable salt thereof is
preferably glatiramer acetate. The process comprises: (a)
polymerizing a mixture of N-carboxyanhydride of L-tyrosine,
N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected
L-glutamate and N-carboxyanhydride of a protected L-lysine, in a
polar aprotic solvent in the presence of an initiator, to form a
protected polypeptide; (b) admixing an acid with the protected
polypeptide formed in Step (a) and a solvent, to form a product;
and (c) admixing a substance selected from the group consisting of
an alkali or alkaline earth metal hydroxide, a carbonate, a
hydrogencarbonate, and mixtures thereof, with the product formed in
Step (b), and a solvent or a mixture of a solvent and water, to
form a deprotected polypeptide or a pharmaceutically acceptable
salt thereof.
Inventors: |
Ray; Anup Kumar; (Staten
Island, NY) ; Patel; Hiren Kumar V.; (Fords, NJ)
; Ludescher; Johannes; (Breitenbach, AU) ;
Anbazhagan; Mariappan; (Monmouth Junction, NJ) ;
Patel; Mahendra R.; (Milltown, NJ) ; Macher;
Ingolf; (Woergl, AU) |
Correspondence
Address: |
SANDOZ INC
506 CARNEFIE CENTER
PRINCETON
NJ
08540
US
|
Family ID: |
36046637 |
Appl. No.: |
12/727758 |
Filed: |
March 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11904422 |
Sep 27, 2007 |
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12727758 |
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11262122 |
Oct 28, 2005 |
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11904422 |
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60723901 |
Oct 5, 2005 |
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60651535 |
Feb 9, 2005 |
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60651372 |
Feb 9, 2005 |
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60623346 |
Oct 29, 2004 |
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Current U.S.
Class: |
530/337 ;
530/300 |
Current CPC
Class: |
C08G 69/10 20130101;
A61P 29/00 20180101; A61K 38/00 20130101; C07K 14/001 20130101;
A61P 37/00 20180101; A61P 37/06 20180101; A61P 25/00 20180101 |
Class at
Publication: |
530/337 ;
530/300 |
International
Class: |
C07K 1/06 20060101
C07K001/06; C07K 2/00 20060101 C07K002/00 |
Claims
1-31. (canceled)
32. A process for preparing a polypeptide comprising L-tyrosine,
L-alanine, L-glutamate and L-lysine, or a pharmaceutically
acceptable salt thereof, wherein said process comprises treating a
protected polypeptide with an aqueous solution of an alkali or
alkaline earth metal hydroxide to form a polypeptide or a
pharmaceutically acceptable salt thereof.
33. A process for preparing a polypeptide comprising L-tyrosine,
L-alanine, L-glutamate and L-lysine, or a pharmaceutically
acceptable salt thereof, wherein said process comprises: (a)
polymerizing a mixture of N-carboxyanhydride of L-tyrosine,
N-carboxyanhydride of L-alanine, N-carboxyanhydride of a protected
L-glutamate and N-carboxyanhydride of a protected L-lysine, in a
polar aprotic solvent in the presence of an initiator, to form a
protected polypeptide; and (b) adding an aqueous solution of an
alkali or alkaline earth metal hydroxide to the protected
polypeptide formed in Step (a) to form a polypeptide or a
pharmaceutically acceptable salt thereof.
34. A process for preparing glatiramer acetate comprising: (a)
polymerizing a mixture of N-carboxyanhydride of L-tyrosine,
N-carboxyanhydride of L-alanine, N-carboxyanhydride of a
.gamma.-benzyl L-glutamate and N-carboxyanhydride of
N.sup..epsilon.-trifluoroacetyl L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
glatiramer; (b) adding an aqueous solution of an alkali or alkaline
earth metal hydroxide to the protected glatiramer formed in Step
(a) to form a glatiramer; and (c) treating the glatiramer with
acetic acid to form glatiramer acetate.
35. The process according to claim 33, wherein Step (b) is
conducted at a temperature of from about -78.degree. C. to about
40.degree. C.
36. The process according to claim 35, wherein Step (b), is
conducted at a temperature of from about -25.degree. C. to about
30.degree. C.
37. The process according to claim 36, wherein Step (b), is
conducted at a temperature of from about -10.degree. C. to about
10.degree. C.
38. The process according to claim 37, wherein Step (b), is
conducted at a temperature of from about 0.degree. C.
39. The process according to claim 32 wherein the pH is from about
13 to about 14.
40. The process according to claim 32 which additionally comprises
a buffer.
41. The process according to claim 40, wherein the buffer is an
acetate buffer and the pH is from about 8 to about 12.
42. The process according to claim 32, wherein the alkali or
alkaline earth metal hydroxide is selected from the group
consisting of calcium hydroxide, lithium hydroxide, magnesium
hydroxide, potassium hydroxide, sodium hydroxide and mixtures
thereof.
43. The process according to claim 42, wherein the alkali or
alkaline earth metal hydroxide is sodium hydroxide.
44. The process according to claim 32, wherein the alkali or
alkaline earth metal hydroxide is present in an amount of from
about 0.1 wt. % to about 400 wt. %, based on the total weight of
the polypeptide or pharmaceutically acceptable salt thereof.
45. The process according to claim 44, wherein the alkali or
alkaline earth metal hydroxide is present in an amount of from
about 10 wt. % to about 300 wt. %, based on the total weight of the
polypeptide or pharmaceutically acceptable salt thereof.
46. The process according to claim 45, wherein the alkali or
alkaline earth metal hydroxide is present in an amount of from
about 140 wt. % to about 260 wt. %, based on the total weight of
the polypeptide or pharmaceutically acceptable salt thereof.
47. A polypeptide or a pharmaceutically acceptable salt thereof
which is prepared by a process comprising treating a protected
polypeptide with an aqueous solution of an alkali or alkaline earth
metal hydroxide to form the polypeptide or pharmaceutically
acceptable salt thereof, wherein said polypeptide comprises
L-tyrosine, L-alanine, L-glutamate and L-lysine.
48. A polypeptide or a pharmaceutically acceptable salt thereof
which is prepared by a process comprising: (a) polymerizing a
mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of
L-alanine, N-carboxyanhydride of a protected L-glutamate and
N-carboxyanhydride of a protected L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
polypeptide; and (b) adding an aqueous solution of an alkali or
alkaline earth metal hydroxide to the protected polypeptide formed
in Step (a) to form a polypeptide or a pharmaceutically acceptable
salt thereof.
49. Glatiramer acetate which is prepared by a process comprising
(a) polymerizing a mixture of N-carboxyanhydride of L-tyrosine,
N-carboxyanhydride of L-alanine, N-carboxyanhydride of a
.gamma.-benzyl L-glutamate and N-carboxyanhydride of
N.sup..epsilon.-trifluoroacetyl L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
glatiramer; (b) adding an aqueous solution of an alkali or alkaline
earth metal hydroxide to the protected glatiramer formed in Step
(a) to form a glatiramer; and (c) treating the glatiramer formed in
Step (b) with acetic acid to form glatiramer acetate.
50-77. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention provides processes for preparing a
polypeptide or pharmaceutically acceptable salt thereof. More
specifically, the invention provides processes for preparing
glatiramer acetate.
BACKGROUND OF THE INVENTION
[0002] COPAXONE.RTM. is the trade name of glatiramer acetate, an
FDA approved drug for the treatment of multiple sclerosis.
COPAXONE.RTM. is also known as Copolymer-1. The COPAXONE.RTM. label
discloses that COPAXONE.RTM. 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 an
average molar fraction of 0.141, 0.427, 0.095 and 0.338,
respectively, and has a weight average molecular weight of 4.7-11.0
kilodaltons (kDa). COPAXONE.RTM. comprises a mixture of
polypeptides having different molecular weights and sequences. The
structural formula of COPAXONE.RTM. 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.s-
ub.2.C.sub.9H.sub.11NO.sub.3).sub.x.xC.sub.2H.sub.4O.sub.2
[0003] COPAXONE.RTM. is a white to off-white, sterile, lyophilized
powder containing 20 mg glatiramer acetate and 40 mg of mannitol.
It is supplied in single use vials for subcutaneous administration
after reconstitution with sterile water.
[0004] Processes for preparing Copolymer-1 or glatiramer acetate
have been described in U.S. Pat. Nos. 3,849,550; 5,800,808;
5,981,589; 6,048,898; 6,054,430; 6,342,476; and 6,362,161. The
process for the synthesis of glatiramer acetate is based on the
polymerization of N-carboxyanhydrides of tyrosine, alanine,
.gamma.-benzyl glutamate and N.sup..epsilon.-trifluoroacetyl lysine
in anhydrous dioxane at room temperature using diethylamine as
initiator, to form a protected polypeptide. The deblocking of the
.gamma.-benzyl groups (first deprotection) is accomplished by
stirring the protected polypeptide in hydrogen bromide/acetic acid
at room temperature. These conditions also facilitate the cleavage
of the copolymer. The next step is the removal of the
N.sup..epsilon.-trifluoroacetyl groups (second deprotection) of the
copolymer by treatment with 1 M piperidine. In the final steps,
glatiramer acetate is obtained by purification of the copolymer
through dialysis, followed by treatment with acetic acid to form
the acetate salt and by another purification by dialysis against
water. Thus, these prior art processes involve the polymerization
of four N-carboxyanhydrides, two deprotection steps, two
purification steps and one acetate salt formation step.
[0005] U.S. Pat. No. 6,620,847 describes a process for preparing
Copolymer-1 which involves treating trifluoroacetyl Copolymer-1
with aqueous piperidine to form a solution of Copolymer-1 and
purifying Copolymer-1.
[0006] U.S. Patent Application Publication No. 2004/0091956
describes a three-step process for preparing glatiramer acetate.
The process involves polymerization of a mixture of the
N-carboxyanhydrides of L-alanine, L-tyrosine, protected L-glutamate
and protected L-lysine, to obtain a protected polypeptide or salt
thereof; and deprotection of the protected polypeptide or salt
thereof by either palladium catalytic transfer hydrogenation or
palladium catalytic hydrogenation under hydrogen pressure.
SUMMARY OF THE INVENTION
[0007] The invention provides a process for preparing a polypeptide
comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a
pharmaceutically acceptable salt thereof, wherein said process
comprises: [0008] (i) polymerizing a mixture of N-carboxyanhydride
of L-tyrosine, N-carboxyanhydride of L-alanine; N-carboxyanhydride
of protected L-glutamate; and N-carboxyanhydride of
N-t-butoxycarbonyl L-lysine, in a polar aprotic solvent in the
presence of an initiator, to form a protected polypeptide, wherein
the protected L-glutamate is selected from the group consisting of
.gamma.-p-methoxybenzyl L-glutamate, .gamma.-benzyl L-glutamate and
mixtures thereof; and [0009] (ii) adding an acid to the protected
polypeptide formed in Step (i) to form a polypeptide or a
pharmaceutically acceptable salt thereof, wherein said acid cleaves
the .gamma.-p-methoxybenzyl group from the glutamate moiety and the
N-t-butoxycarbonyl group from the lysine moiety.
[0010] The invention provides a process for preparing a polypeptide
comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a
pharmaceutically acceptable salt thereof, wherein said process
comprises treating a protected polypeptide with an aqueous solution
of an alkali or alkaline earth metal hydroxide to form a
polypeptide or a pharmaceutically acceptable salt thereof.
[0011] The invention provides a process for preparing a polypeptide
comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a
pharmaceutically acceptable salt thereof, wherein said process
comprises: [0012] (a).sup.2 polymerizing a mixture of
N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine,
N-carboxyanhydride of a protected L-glutamate and
N-carboxyanhydride of a protected L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
polypeptide; [0013] (b).sup.2 admixing an acid with the protected
polypeptide formed in Step (a).sup.2 and a solvent, to form a
product; and [0014] (c).sup.2 admixing a substance selected from
the group consisting of diisopropylamine, isopropylamine, ammonia
and mixtures thereof, with the product formed in Step (b).sup.2,
and water or a mixture of a solvent and water, to form a
deprotected polypeptide or a pharmaceutically acceptable salt
thereof.
[0015] The invention provides a process for preparing a polypeptide
comprising L-tyrosine, L-alanine, L-glutamate and L-lysine, or a
pharmaceutically acceptable salt thereof, wherein said process
comprises: [0016] (a).sup.3 polymerizing a mixture of
N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine,
N-carboxyanhydride of a protected L-glutamate and
N-carboxyanhydride of a protected L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
polypeptide; [0017] (b).sup.3 admixing an acid with the protected
polypeptide formed in Step (a).sup.3 and a solvent, to form a
product; and [0018] (c).sup.3 admixing a substance selected from
the group consisting of an alkali or alkaline earth metal
hydroxide, a carbonate, a hydrogencarbonate and mixtures thereof,
with the product formed in Step (b).sup.3, and a solvent or a
mixture of a solvent and water, to form a deprotected polypeptide
or a pharmaceutically acceptable salt thereof.
DESCRIPTION OF THE INVENTION
[0019] The present invention relates to processes for preparing a
polypeptide or pharmaceutically acceptable salt thereof comprising
L-tyrosine, L-alanine, L-glutamate and L-lysine. The polypeptide or
pharmaceutically acceptable salt thereof is preferably glatiramer
acetate.
I. Acid Hydrolysis Process.
[0020] In one embodiment of the invention, the process comprises:
[0021] (i) polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
protected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl
L-lysine, in a polar aprotic solvent in the presence of an
initiator, to form a protected polypeptide, wherein the protected
L-glutamate is selected from the group consisting of
.gamma.-p-methoxybenzyl L-glutamate, .gamma.-benzyl L-glutamate and
mixtures thereof; and [0022] (ii) adding an acid to the protected
polypeptide formed in Step (i) to form a polypeptide or a
pharmaceutically acceptable salt thereof, wherein said acid cleaves
the .gamma.-p-methoxybenzyl group from the glutamate moiety and the
N-t-butoxycarbonyl group from the lysine moiety.
[0023] In one embodiment of the invention, the process comprises:
[0024] (a) polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
protected L-glutamate and N-carboxyanhydride of N-t-butoxycarbonyl
L-lysine, in a polar aprotic solvent in the presence of an
initiator, to form a protected glatiramer, wherein the protected
L-glutamate is selected from the group consisting of
.gamma.-p-methoxybenzyl L-glutamate, .gamma.-benzyl L-glutamate,
and mixtures thereof; [0025] (b) adding an acid to the protected
glatiramer formed in Step (a) to form a glatiramer, wherein said
acid cleaves the .gamma.-p-methoxybenzyl group from the glutamate
moiety and the N-t-butoxycarbonyl group from the lysine moiety; and
[0026] (c) treating the glatiramer formed in Step (b) with acetic
acid to form glatiramer acetate.
[0027] In the polymerizing step of the processes of the invention,
Step (i), Step (a), Step (a).sup.1, Step (a).sup.1', Step
(a).sup.2, Step (a).sup.2', Step (a).sup.3 and Step (a).sup.3', the
mixture of N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of
L-alanine, N-carboxyanhydride of protected L-glutamate and
N-carboxyanhydride of N-t-butoxycarbonyl L-lysine, are preferably
polymerized at a temperature of from about 10.degree. C. to about
40.degree. C., more preferably about 20.degree. C. to about
30.degree. C. The polymerization reaction preferably takes place
for a period of from about 2 hours to about 80 hours, more
preferably from about 20 hours to about 50 hours. Most preferably,
the polymerization reaction takes place for a period of about 24
hours at a temperature of about 25.degree. C.
[0028] The polar aprotic solvent is preferably selected from
tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide,
dioxane, dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxide
and dichloromethane. Most preferably, the polar aprotic solvent is
1,4-dioxane. A mixture of polar aprotic solvents may also be
used.
[0029] The initiator used in Step (i), Step (a), Step (a).sup.1,
Step (a).sup.1', Step (a).sup.2, Step (a).sup.2', Step (a).sup.3
and Step (a).sup.3', of the processes of the invention may be any
alkylamine initiator, such as a dialkyl or a trialkylamine. Each of
the alkyl groups preferably has 1-6 carbon atoms. A preferred
alkylamine initiator is diethylamine. Preferably, the diethylamine
is present in an amount of from about 0.001 weight percent (wt. %)
to about 2 wt. %, more preferably from about 0.01 wt. % to about
0.02 wt. %, based on the weight of the mixture of
N-carboxyanhydride of L-tyrosine, N-carboxyanhydride of L-alanine,
N-carboxyanhydride of protected L-glutamate and N-carboxyanhydride
of N-t-butoxycarbonyl L-lysine.
[0030] In one embodiment of the invention, water is added to the
polymerization mixture following polymerization. The addition of
water results in precipitation of the protected polypeptide. The
water is preferably removed from the mixture containing water and
protected polypeptide by vacuum filtration and the recovered
protected polypeptide is dried. Methods of drying are known to
those skilled in the art, such as vacuum drying.
[0031] In the deprotecting step, Step (ii), of the process of the
invention, an acid is added to the protected polypeptide which is
formed in Step (i) to form a polypeptide or a pharmaceutically
acceptable salt thereof. The acid cleaves the
.gamma.-p-methoxybenzyl group or .gamma.-benzyl group from the
glutamate moiety and the N-t-butoxycarbonyl group from the lysine
moiety. In addition, the acid cleaves the amide bonds of the
polypeptide or pharmaceutically acceptable salt thereof forming
heterogenous polypeptide fragments.
[0032] In the deprotecting step, Step (b), of the process of the
invention, an acid is added to the protected glatiramer formed in
Step (a) to form a glatiramer. The acid cleaves the
.gamma.-p-methoxybenzyl group or the .gamma.-benzyl group from the
glutamate moiety and the N-t-butoxycarbonyl group from the lysine
moiety. In addition, the acid cleaves the amide bonds of the
glatiramer forming heterogenous glatiramer fragments.
[0033] Suitable acids include, but are not limited to, acetic acid,
hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane
sulfonic acid, trifluoromethane sulfonic acid, phosphoric acid,
trifluroacetic acid and sulfuric acid. A mixture of acids may also
be used. Preferred acids are selected from trifluroacetic acid, a
mixture of acetic acid and hydrochloric acid, a mixture of acetic
acid and hydrogen bromide and a mixture of acetic acid and sulfuric
acid. The acid may be added in the form of an aqueous solution.
[0034] The acid is preferably present in an amount of from about
0.1 wt. % to about 100 wt. %, more preferably from about 1 wt. % to
about 10 wt. %, based on the weight of the protected polypeptide or
protected glatiramer. Most preferably, the acid is present in an
amount of from about 2 wt. % to about 6 wt. %, based on the weight
of the protected polypeptide or protected glatiramer.
[0035] The temperature of the reaction medium during addition of
the acid is preferably from about 10.degree. C. to about 40.degree.
C., more preferably 15.degree. C. to about 30.degree. C. The acid
is preferably added over a period of time from about 1 hour to
about 30 hours, with stirring. Most preferably, the acid is added
to the protected polypeptide or protected glatiramer at a
temperature of about 25.degree. C. for a period of from about 1
hour to about 8 hours, with stirring.
[0036] Excess acid is preferably removed from the reaction mixture
by purging the reaction mixture with nitrogen, lyophilization, or
by means of a rotary evaporator under vacuum to obtain a
deprotected polypeptide in solid form. However, other separation
techniques known to those skilled in the art may also be used.
[0037] The deprotected polypeptide or deprotected glatiramer in the
form of a free base or acid addition salt is preferably dissolved
in water or an aqueous acetic acid solution. Undesired low
molecular weight polypeptide or glatiramer fragments, i.e., less
than about 2 kDa, and high molecular weight polypeptide or
glatiramer fragments, i.e., greater than about 40 kDa, are
preferably removed by such methods as dialysis or diafiltration.
Preferred membranes include Visking partially permeable cellulose
membranes, such as a Size 6 membrane having a molecular weight
cut-off of 12-14 kDa, available from Medicell International Ltd.,
and tangential flow filtration (TFF) membranes, such as a Pellicon
XL PLCCC 10 (50 cm.sup.2) or PLCCC 5 (50 cm.sup.2), available from
Millipore. In a preferred embodiment of the invention, the
deprotected polypeptide or deprotected glatiramer is dissolved in
water and subjected to dialysis, followed by dialysis in aqueous
acetic acid solution.
[0038] The present inventors have determined that the desired
molecular weight polypeptide or a pharmaceutically acceptable salt
thereof may be controlled by dilution, concentration of the acid
added in Step (ii) or Step (b), and/or time.
[0039] In one embodiment of the invention, water is removed from
the deprotected polypeptide. A preferred method of removal is
lyophilization. In lyophilization, the solution is frozen and
placed under vacuum so that the water (ice) vaporizes in the vacuum
(sublimes) without melting and the non-water components
(deprotected polypeptide and residual salt) are left behind in an
undamaged state, i.e., without chemical decomposition. The dried
product of lyophilization contains the deprotected polypeptide and
residual salt.
[0040] In one embodiment of the invention, the deprotected
polypeptide is treated with glacial acetic acid to form glatiramer
acetate salt. The glatiramer acetate salt is collected preferably
by lyophilization to yield a glatiramer acetate salt product.
II. Phase Transfer Process.
[0041] In one embodiment of the invention, the process for
preparing a polypeptide comprising L-tyrosine, L-alanine,
L-glutamate and L-lysine, or a pharmaceutically acceptable salt
thereof, comprises treating a protected polypeptide with an aqueous
solution of an alkali or alkaline earth metal hydroxide to form a
polypeptide or a pharmaceutically acceptable salt thereof.
[0042] In one embodiment of the invention, the process comprises:
[0043] (a).sup.1 polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
a protected L-glutamate and N-carboxyanhydride of a protected
L-lysine, in a polar aprotic solvent in the presence of an
initiator, to form a protected polypeptide; and [0044] (b).sup.1
adding an aqueous solution of an alkali or alkaline earth metal
hydroxide to the protected polypeptide formed in Step (a).sup.1 to
form a polypeptide or a pharmaceutically acceptable salt
thereof.
[0045] In one embodiment of the invention, the process comprises:
[0046] (a).sup.1' polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
a .gamma.-benzyl L-glutamate L-glutamate and N-carboxyanhydride of
N.sup..epsilon.-trifluoroacetyl L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
glatiramer; [0047] (b).sup.1' adding an aqueous solution of an
alkali or alkaline earth metal hydroxide to the protected
glatiramer formed in Step (a).sup.1' to form a glatiramer; and
[0048] (c).sup.1' treating the glatiramer with acetic acid to form
glatiramer acetate.
[0049] In the deprotecting steps, Step (b).sup.1 and Step
(b).sup.1', of the process of the invention, an aqueous solution of
an alkali or alkaline earth metal hydroxide is added to the
protected polypeptide formed in Step (a).sup.1 or to the protected
glatiramer formed in Step (a).sup.1'. In Step (b).sup.1 and Step
(b).sup.1', the protecting groups on the glutamic acid moiety,
i.e., the .gamma.-benzyl group, and the protecting groups on the
lysine moiety, i.e., N.sup..epsilon.-trifluoroacetyl group, are
removed. In addition, the alkali or alkaline earth metal hydroxide
cleaves the amide bonds of the polypeptide or glatiramer forming
heterogenous polypeptide or glatiramer fragments.
[0050] While not wishing to be bound by any particular theory, the
present inventors believe that the removal of the protecting groups
from the protected polypeptide or protected glatiramer results in a
phase transfer of the deprotected polypeptide or deprotected
glatiramer from the organic phase to the aqueous phase.
[0051] In the absence of a buffer in Step (b).sup.1 or Step
(b).sup.1', the pH during Step (b).sup.1 and Step (b).sup.1', is
generally about 13 to about 14 after the addition of the aqueous
solution of an alkali or alkaline earth metal hydroxide. In the
presence of a buffer in Step (b).sup.1 or Step (b).sup.b', the pH
is generally about 8 to about 12. The buffer may be added or formed
in situ. A preferred buffer is an acetate buffer such as sodium
acetate.
[0052] The alkali or alkaline earth metal hydroxide is preferably
selected from calcium hydroxide, lithium hydroxide, magnesium
hydroxide, potassium hydroxide and sodium hydroxide. More
preferably, the alkali or alkaline earth metal hydroxide is sodium
hydroxide. A combination of alkali or alkaline earth metal
hydroxides may also be used.
[0053] The alkali or alkaline earth metal hydroxide is preferably
present in an amount of from about 0.1 wt. % to about 400 wt. %,
more preferably from about 10 wt. % to about 300 wt. %, based on
the weight of the protected polypeptide or protected glatiramer.
Most preferably, the alkali or alkaline earth metal hydroxide is
present in an amount of from about 140 wt. % to about 260 wt. %,
based on the weight of the protected polypeptide or protected
glatiramer.
[0054] An aqueous solution of an alkali or alkaline earth metal
hydroxide is added to the protected polypeptide or protected
glatiramer preferably at a temperature of from about -78.degree. C.
to about 40.degree. C., more preferably -25.degree. C. to about
30.degree. C., for a period of time preferably from about 1 hour to
about 30 hours. Most preferably, an aqueous solution of an alkali
or alkaline earth metal hydroxide is added to the protected
polypeptide or protected glatiramer at a temperature from about
-10.degree. C. to about 10.degree. C., e.g., 0.degree. C., for a
period of from about 1 hour to about 8 hours, with stirring. The
addition of an aqueous solution of an alkali or alkaline earth
metal hydroxide results in a phase separation wherein an organic
phase and an aqueous phase are formed.
[0055] The organic phase substantially contains the polar aprotic
solvent and the protected polypeptide or protected glatiramer. The
aqueous phase substantially contains water, the alkali or alkaline
earth metal hydroxide, and the deprotected polypeptide or
deprotected glatiramer in the form of a free base. The aqueous
phase and the organic phase are preferably separated by using a
centrifuge and decanting the organic phase.
[0056] An additional advantage of the process of the invention is
that the addition of the alkali or alkaline earth metal hydroxide
causes deprotection of the protected polypeptide to form a
deprotected polypeptide, or deprotection of the protected
glatiramer to form a deprotected glatiramer. In addition, the
alkali or alkaline earth metal hydroxide causes cleavage of the
amide bonds in the deprotected polypeptide or deprotected
glatiramer to form polypeptide or glatiramer fragments.
[0057] The aqueous phase is preferably treated with an organic or
mineral acid to achieve a pH of about 7 to about 8. Such organic or
mineral acids to adjust the pH are well-known to those skilled in
the art and include, but are not limited to, acetic acid, formic
acid, oxalic acid and hydrochloric acid. Dilute hydrochloric acid
is preferred. The amount of organic or mineral acid used is
preferably an equivalent amount based on the amount of the alkali
or alkaline earth metal hydroxide added during Step (b).sup.1 or
Step (b).sup.1' which is sufficient to produce a pH of about 7 to
about 8.
[0058] Undesired low molecular weight polypeptide or glatiramer
fragments, i.e., less than about 2 kDa, and high molecular weight
polypeptide or glatiramer fragments, i.e., greater than about 40
kDa, are preferably removed by such methods as dialysis or
diafiltration. Preferred membranes include Visking partially
permeable cellulose membranes such as a Size 6 membrane having a
molecular weight cut-off of 12-14 kDa, available from Medicell
International Ltd., and tangential flow filtration (TFF) membranes,
such as a Pellicon XL PLCCC 10 (50 cm.sup.2) or PLCCC 5 (50
cm.sup.2), available from Millipore. In a preferred embodiment of
the invention, the deprotected polypeptide or deprotected
glatiramer is subjected to dialysis in water, followed by dialysis
in aqueous acetic acid solution.
III. Process Using an Amine or Ammonia.
[0059] In one embodiment of the invention, the process comprises:
[0060] (a).sup.2 polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
a protected L-glutamate and N-carboxyanhydride of a protected
L-lysine, in a polar aprotic solvent in the presence of an
initiator, to form a protected polypeptide; [0061] (b).sup.2
admixing an acid with the protected polypeptide formed in Step
(a).sup.2 and a solvent, to form a product, preferably, the acid is
admixed with a solution or suspension comprising the protected
polypeptide and solvent; and [0062] (c).sup.2 admixing a substance
selected from the group consisting of diisopropylamine,
isopropylamine, ammonia, and mixtures thereof, with the product
formed in Step (b).sup.2, and water or a mixture of a solvent and
water, to form a deprotected polypeptide or a pharmaceutically
acceptable salt thereof.
[0063] In one embodiment of the invention, the process comprises:
[0064] (a).sup.2' polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
a protected L-glutamate and N-carboxyanhydride of a protected
L-lysine, in a polar aprotic solvent in the presence of an
initiator, to form a protected glatiramer, wherein said protected
L-glutamate is selected from the group consisting of
.gamma.-p-methoxybenzyl L-glutamate, .gamma.-benzyl L-glutamate and
mixtures thereof; [0065] (b).sup.2' admixing an acid with the
protected glatiramer formed in Step (a).sup.2' and a solvent, to
form a product; [0066] (c).sup.2' admixing a substance selected
from the group consisting of diisopropylamine, isopropylamine,
ammonia and mixtures thereof, with the product formed in Step
(b).sup.2', and water or a mixture of a solvent and water, to form
a deprotected glatiramer; and [0067] (d).sup.2' treating the
deprotected glatiramer formed in Step (c).sup.2' with acetic acid
to form glatiramer acetate.
[0068] In the first deprotecting step, Step (b).sup.2 and Step
(b).sup.2' of the process of the invention, an acid is admixed with
the protected polypeptide formed in Step (a).sup.2 or protected
glatiramer formed in Step (a).sup.2', and a solvent to form a
product. Preferably, the acid is added to a mixture, i.e., a
solution or suspension, comprising the protected polypeptide or
protected glatiramer and a solvent. In Step (b).sup.2 and Step
(b).sup.2' the protecting groups on the glutamic acid moiety, i.e.,
.gamma.-p-methoxybenzyl group and/or .gamma.-benzyl group, are
removed. While not wishing to be bound by any particular theory,
the present inventors believe that the acid also cleaves amide
bonds of the protected polypeptide or protected glatiramer forming
heterogenous polypeptide or glatiramer fragments.
[0069] Suitable acids include, but are not limited to, acetic acid,
hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane
sulfonic acid, trifluoromethane sulfonic acid, phosphoric acid,
trifluroacetic acid and sulfuric acid. A mixture of acids may also
be used. Preferred acids are selected from trifluroacetic acid, a
mixture of acetic acid and hydrochloric acid, a mixture of acetic
acid and hydrogen bromide and a mixture of acetic acid and sulfuric
acid. The acid may be added in the form of an aqueous solution.
[0070] The acid is preferably present in an amount of from about
0.1 wt. % to about 100 wt. %, more preferably from about 1 wt. % to
about 10 wt. %, based on the weight of the protected polypeptide or
protected glatiramer. Most preferably, the acid is present in an
amount of from about 2 wt. % to about 6 wt. %, based on the weight
of the protected polypeptide or protected glatiramer.
[0071] The temperature of the reaction medium during addition of
the acid is preferably from about 10.degree. C. to about 40.degree.
C., more preferably 15.degree. C. to about 30.degree. C. The acid
is preferably added over a period of time from about 1 hour to
about 30 hours, with stirring. Most preferably, the acid is added
to the protected polypeptide or protected glatiramer at a
temperature of about 25.degree. C. for a period of from about 1
hour to about 8 hours, with stirring.
[0072] The solvent used in the first deprotection step, Step
(b).sup.2 and Step (b).sup.2', is selected from polar protic
solvents and polar aprotic solvents. Preferably, the solvent used
in Step (b).sup.2 and Step (b).sup.2' is selected from acetic acid,
tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide,
1,4-dioxane, dimethoxyethane, 1,2-dichloroethylene,
dimethylsulfoxide and dichloromethane. A mixture of solvents may
also be used. Most preferably, the solvent used in Step (b).sup.2
and Step (b).sup.2' is tetrahydrofuran or acetic acid.
[0073] The amount of solvent used in Step (b).sup.2 and Step
(b).sup.2' is preferably from about 1-fold (wt.) to about
1,000-fold (wt.), more preferably, from about 10-fold (wt.) to
about 500-fold (wt.), based on the amount of protected polypeptide
or protected glatiramer which is used in Step (b).sup.2 or Step
(b).sup.2'.
[0074] In the second deprotecting step, Step (c).sup.2 and Step
(c).sup.2' of the process of the invention, a substance selected
from the group consisting of diisopropylamine, isopropylamine,
ammonia and mixtures thereof, is admixed with the product formed in
Step (b).sup.2 or Step (b).sup.2', and water or a mixture of a
solvent and water, to form a deprotected polypeptide or deprotected
glatiramer.
[0075] Preferably, the substance selected from the group consisting
of diisopropylamine, isopropylamine, ammonia and mixtures thereof,
is added to a mixture, i.e., a solution or suspension, comprising
the product formed in Step (b).sup.2 or Step (b).sup.2', and water
or a mixture of a solvent and water. The ammonia is in the form of
NH.sub.3 (aqueous) or NH.sub.3 (gas). Preferably an aqueous
solution of ammonia is used having a pH of about 7 to about 14. The
addition of a substance selected from the group consisting of
diisopropylamine, isopropylamine, ammonia and mixtures thereof, to
the product formed in Step (b).sup.2 or Step (b).sup.2' preferably
removes the protecting group, such as
N.sup..epsilon.-trifluoroacetyl group, of the lysine moiety.
Preferably the deprotected polypeptide is a deprotected glatiramer
in the form of a free base.
[0076] The substance selected from the group consisting of
diisopropylamine, isopropylamine, ammonia and mixtures thereof, is
preferably present in an amount of from about 1-fold (wt.) to about
1,000-fold (wt), more preferably from about 10-fold (wt.) to about
500-fold (wt), based on the total weight of the product of Step
(b).sup.2 or Step (b).sup.2' which is used in Step (c).sup.2 or
Step (c).sup.2'. Most preferably, the substance selected from the
group consisting of diisopropylamine, isopropylamine, ammonia and
mixtures thereof, is present in an amount of from about 50-fold
(wt.) to about 150-fold (wt).
[0077] The substance selected from the group consisting of
diisopropylamine, isopropylamine, ammonia and mixtures thereof, is
preferably admixed with a solution or suspension comprising the
product formed in Step (b).sup.2 or Step (b).sup.2', and water or a
mixture of a solvent and water, at a temperature of from about
10.degree. C. to about 60.degree. C., more preferably 15.degree. C.
to about 40.degree. C., for a period of time preferably from about
1 minute to about 60 hours, to form a deprotected polypeptide or
deprotected glatiramer. Suitable solvents include, but are not
limited to, tetrahydrofuran, ethyl acetate, dimethyl furan,
dimethylformamide, 1,4-dioxane, dimethoxyethane,
1,2-dichloroethylene, dimethylsulfoxide and dichloromethane. A
preferred solvent is tetrahydrofuran.
[0078] More preferably, the substance selected from the group
consisting of diisopropylamine, isopropylamine, ammonia and
mixtures thereof, is admixed with a solution or suspension
comprising the product formed in Step (b).sup.2 or Step (b).sup.2',
and water or a mixture of a solvent and water, at a temperature of
from about 20.degree. C. to about 30.degree. C., for a period of
from about 1 hour to about 30 hours, to form a deprotected
polypeptide or deprotected glatiramer. Unreacted diisopropylamine
or ammonia, or any solvent or water, is preferably removed by
evaporation or vacuum distillation.
[0079] Undesired low molecular weight polypeptide or glatiramer
fragments, i.e., less than about 2 kDa, and high molecular weight
polypeptide or glatiramer fragments, i.e., greater than about 40
kDa, are preferably removed by such methods as dialysis or
diafiltration. Preferred membranes include Visking partially
permeable cellulose membranes, such as a Size 6 membrane having a
molecular weight cut-off of 12-14 kDa, available from Medicell
International Ltd., and TFF membranes, such as a Pellicon XL PLCCC
10 (50 cm.sup.2) or PLCCC 5 (50 cm.sup.2), available from
Millipore. In a preferred embodiment of the invention, the
deprotected polypeptide or deprotected glatiramer is subjected to
dialysis in water, followed by dialysis in aqueous acetic acid
solution.
IV. Process Using an Alkali or Alkaline Earth Metal Hydroxide,
Carbonate or Hydrogencarbonate.
[0080] In one embodiment of the invention, the process comprises:
[0081] (a).sup.3 polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
a protected L-glutamate and N-carboxyanhydride of a protected
L-lysine, in a polar aprotic solvent in the presence of an
initiator, to form a protected polypeptide; [0082] (b).sup.3
admixing an acid with the protected polypeptide formed in Step
(a).sup.3 and a solvent, to form a product, preferably, the acid is
admixed with a solution or suspension comprising the protected
polypeptide and solvent; and [0083] (c).sup.3 admixing a substance
selected from the group consisting of an alkali or alkaline earth
metal hydroxide, a carbonate, a hydrogencarbonate and mixtures
thereof, with the product formed in Step (b).sup.3, and a solvent
or a mixture of a solvent and water, to form a deprotected
polypeptide or a pharmaceutically acceptable salt thereof.
[0084] In one embodiment of the invention, the process comprises:
[0085] (a).sup.3' polymerizing a mixture of N-carboxyanhydride of
L-tyrosine, N-carboxyanhydride of L-alanine, N-carboxyanhydride of
a protected L-glutamate and N-carboxyanhydride of
N.sup..epsilon.-trifluoroacetyl L-lysine, in a polar aprotic
solvent in the presence of an initiator, to form a protected
glatiramer, wherein said protected L-glutamate is selected from the
group consisting of .gamma.-p-methoxybenzyl L-glutamate,
.gamma.-benzyl L-glutamate and mixtures thereof; [0086] (b).sup.3'
admixing an acid with a mixture comprising the protected glatiramer
formed in Step (a).sup.3 and a solvent, to form a product; [0087]
(c).sup.3' admixing a substance selected from the group consisting
of an alkali or alkaline earth metal hydroxide, a carbonate, a
hydrogencarbonate and mixtures thereof, with a mixture comprising
the product formed in Step (b).sup.3', and a solvent or a mixture
of a solvent and water, to form a deprotected glatiramer; and
[0088] (d).sup.3' treating the deprotected glatiramer formed in
Step (c).sup.3' with acetic acid to form glatiramer acetate.
[0089] In the first deprotecting step, Step (b).sup.3 and Step
(b).sup.3' of the process of the invention, an acid is admixed with
the protected polypeptide formed in Step (a).sup.3 or protected
glatiramer formed in Step (a).sup.3', and a solvent to form a
product. Preferably, the acid is added to a mixture, i.e., a
solution or suspension, comprising the protected polypeptide or
protected glatiramer and a solvent. In Step (b).sup.3 and Step
(b).sup.3' the protecting groups on the glutamic acid moiety, i.e.,
.gamma.-p-methoxybenzyl group and/or .gamma.-benzyl group, are
removed. While not wishing to be bound by any particular theory,
the present inventors believe that the acid also cleaves amide
bonds of the protected polypeptide or protected glatiramer forming
heterogenous polypeptide fragments.
[0090] Suitable acids include, but are not limited to, acetic acid,
hydrochloric acid, hydrogen bromide, hydrogen fluoride, methane
sulfonic acid, trifluoromethane sulfonic acid, phosphoric acid,
trifluroacetic acid and sulfuric acid. A mixture of acids may also
be used. Preferred acids are selected from trifluroacetic acid, a
mixture of acetic acid and hydrochloric acid, a mixture of acetic
acid and hydrogen bromide and a mixture of acetic acid and sulfuric
acid. The acid may be added in the form of an aqueous solution.
[0091] The acid is preferably present in an amount of from about
0.1 wt. % to about 100 wt. %, more preferably from about 1 wt. % to
about 10 wt. %, based on the weight of the protected polypeptide or
protected glatiramer. Most preferably, the acid is present in an
amount of from about 2 wt. % to about 6 wt. %, based on the weight
of the protected polypeptide or protected glatiramer.
[0092] The temperature of the reaction medium during addition of
the acid is preferably from about 10.degree. C. to about 40.degree.
C., more preferably 15.degree. C. to about 30.degree. C. The acid
is preferably added over a period of time from about 1 hour to
about 30 hours, with stirring. Most preferably, the acid is added
to the protected polypeptide or protected glatiramer at a
temperature of about 25.degree. C. for a period of from about 1
hour to about 8 hours, with stirring.
[0093] The solvent used in the first deprotection step, Step
(b).sup.3 and Step (b).sup.3', is selected from polar protic
solvents and polar aprotic solvents. Preferably, the solvent used
in Step (b).sup.3 and Step (b).sup.3' is selected from acetic acid,
tetrahydrofuran, ethyl acetate, dimethyl furan, dimethylformamide,
1,4-dioxane, dimethoxyethane, 1,2-dichloroethylene,
dimethylsulfoxide and dichloromethane. A mixture of solvents may
also be used. Most preferably, the solvent used in Step (b).sup.2
and Step (b).sup.2' is tetrahydrofuran or acetic acid.
[0094] The amount of solvent used in Step (b).sup.3 and Step
(b).sup.3' is preferably from about 1-fold (wt.) to about
1,000-fold (wt.), more preferably, from about 10-fold (wt.) to
about 500-fold (wt.), based on the amount of protected polypeptide
or protected glatiramer which is used in Step (b).sup.3 or Step
(b).sup.3'.
[0095] In the second deprotecting step, Step (c).sup.3 and Step
(c).sup.3', of the process of the invention, a substance selected
from the group consisting of an alkali or alkaline earth metal
hydroxide, a carbonate, a hydrogencarbonate and mixtures thereof,
is admixed with the product formed in Step (b).sup.3 or Step
(b).sup.3', and water or a mixture of a solvent and water, to form
a deprotected polypeptide or deprotected glatiramer.
[0096] Preferably, the substance selected from the group consisting
of an alkali or alkaline earth metal hydroxide, a carbonate, a
hydrogencarbonate and mixtures thereof, is added to a mixture,
i.e., a solution or suspension, comprising the product formed in
Step (b).sup.3 or Step (b).sup.3', and water or a mixture of a
solvent and water. The addition of a substance selected from the
group consisting of an alkali or alkaline earth metal hydroxide, a
carbonate, a hydrogencarbonate and mixtures thereof, to the product
formed in Step (b).sup.3 or Step (b).sup.3' preferably removes the
N.sup..epsilon.-trifluoroacetyl group of the lysine moiety.
Preferably the deprotected polypeptide is a deprotected glatiramer
in the form of a free base.
[0097] The substance selected from the group consisting of an
alkali or alkaline earth metal hydroxide, a carbonate and a
hydrogencarbonate, and includes calcium hydroxide, lithium
hydroxide, magnesium hydroxide, potassium hydroxide, barium
hydroxide, sodium hydroxide, calcium carbonate, lithium carbonate,
magnesium carbonate, potassium carbonate, sodium carbonate, calcium
hydrogencarbonate, lithium hydrogencarbonate, magnesium
hydrogencarbonate, potassium hydrogencarbonate and sodium
hydrogencarbonate. More preferably, the substance used in Step
(c).sup.3 or Step (c).sup.3' is selected from the group consisting
of sodium hydroxide, lithium hydroxide and potassium hydroxide.
[0098] The substance selected from the group consisting of an
alkali or alkaline earth metal hydroxide, a carbonate, a
hydrogencarbonate and mixtures thereof, is preferably present in an
amount of from about 1-fold (wt.) to about 1,000-fold (wt), more
preferably from about 10-fold (wt.) to about 500-fold (wt), based
on the total weight of the product of Step (b).sup.3 or Step
(b).sup.3' which is used in Step (c).sup.3 or Step (c).sup.3'. Most
preferably, the substance selected from the group consisting of an
alkali or alkaline earth metal hydroxide, a carbonate, a
hydrogencarbonate and mixtures thereof, is present in an amount of
from about 50-fold (wt.) to about 150-fold (wt).
[0099] The substance selected from the group consisting of an
alkali or alkaline earth metal hydroxide, a carbonate, a
hydrogencarbonate and mixtures thereof, is preferably admixed with
a solution or suspension comprising the product formed in Step
(b).sup.3 or Step (b).sup.3', and water or a mixture of a solvent
and water, at a temperature of from about 10.degree. C. to about
60.degree. C., more preferably 15.degree. C. to about 40.degree.
C., for a period of time preferably from about 1 minute to about 60
hours, to form a deprotected polypeptide or deprotected glatiramer.
Suitable solvents include, but are not limited to, tetrahydrofuran,
ethyl acetate, dimethyl furan, dimethylformamide, 1,4-dioxane,
dimethoxyethane, 1,2-dichloroethylene, dimethylsulfoxide and
dichloromethane. A preferred solvent is tetrahydrofuran.
[0100] More preferably, the substance selected from the group
consisting of an alkali or alkaline earth metal hydroxide, a
carbonate, a hydrogencarbonate and mixtures thereof, is admixed
with a solution or suspension comprising the product formed in Step
(b).sup.3 or Step (b).sup.3', and water or a mixture of a solvent
and water, at a temperature of from about 20.degree. C. to about
30.degree. C., for a period of from about 1 hour to about 30 hours,
to form a deprotected polypeptide or deprotected glatiramer.
Unreacted alkali or alkaline earth metal hydroxide, carbonate,
hydrogencarbonate or any solvent or water, is preferably removed by
evaporation or vacuum distillation.
[0101] The amount of solvent or a mixture of a solvent and water
which is used in Step (c).sup.3 or Step (c).sup.3' is preferably
from about 1-fold (wt.) to about 1,000-fold (wt.), more preferably,
from about 10-fold (wt.) to about 500-fold (wt.), based on the
total weight of the product of Step (b).sup.3 or Step (b).sup.3'
which is used in Step (c).sup.3 or Step (c).sup.3'.
[0102] After the second deprotection step, Step (c).sup.3 and Step
(c).sup.3', any layers are separated preferably in separatory
funnel, or the solvent is preferably removed by evaporation or
vacuum distillation. The deprotected polypeptide or deprotected
glatiramer is preferably obtained as a solution in water.
[0103] Undesired low molecular weight polypeptide or glatiramer
fragments, i.e., less than about 2 kDa, and high molecular weight
polypeptide or glatiramer fragments, i.e., greater than about 40
kDa, are preferably removed by such methods as dialysis or
diafiltration. Preferred membranes include Visking partially
permeable cellulose membranes, such as a Size 6 membrane having a
molecular weight cut-off of 12-14 kDa, available from Medicell
International Ltd., and TFF membranes, such as a Pellicon XL PLCCC
10 (50 cm.sup.2) or PLCCC 5 (50 cm.sup.2), available from
Millipore. In a preferred embodiment of the invention, the
deprotected polypeptide or deprotected glatiramer is subjected to
dialysis in water, followed by dialysis in aqueous acetic acid
solution.
[0104] The weight average molecular weight of the polypeptide or a
pharmaceutically acceptable salt thereof which is prepared in
accordance with the processes of the invention is preferably from
about 2 kDa to about 30 kDa, more preferably from about 4.7 Da to
about 11 kDa, and most preferably from about 7 kDa to about 10 kDa,
as determined by gas permeation chromatography (GPC). Preferably,
the polypeptide or pharmaceutically acceptable salt thereof is
substantially free of polypeptide fragments having a molecular
weight of greater than about 40 kDa. Preferably, the polypeptide or
pharmaceutically acceptable salt thereof is substantially free of
polypeptide fragments having a molecular weight of less than about
2 kDa. As used herein, "substantially free" means less than about
5% by weight, more preferably less than about 2.5% by weight of the
polypeptide prepared according to the process of the invention.
[0105] The polypeptide or pharmaceutically acceptable salt thereof,
preferably glatiramer acetate, prepared according to the process of
the invention may be formulated by conventional methods known in
the art.
[0106] In one embodiment of the invention, the polypeptide or
pharmaceutically acceptable salt thereof, which is prepared by the
process of the invention, is dissolved or suspended in an
acceptable pharmaceutical liquid vehicle, such as water, and the
solution or suspension is injected into the body.
[0107] In one embodiment of the invention, the glatiramer acetate
salt is dissolved in a mixture containing water and mannitol, and
the solution is injected into the body.
[0108] Typically, the polypeptide or a pharmaceutically acceptable
salt thereof is administered daily to patients suffering from
multiple sclerosis, e.g., at a dosage of 20 mg of glatiramer
acetate.
[0109] The following non-limiting examples illustrate further
aspects of the invention.
[0110] Examples 1-7 relate to an acid hydrolysis process for
preparing glatiramer acetate.
EXAMPLE 1
Preparation of a Protected Polypeptide.
[0111] N-Carboxyanhydride of L-alanine (860 mg, 7.5 mmol),
N-carboxyanhydride of .gamma.-benzyl L-glutamate (600 mg, 2.3
mmol), N-carboxyanhydride of N-t-butoxycarbonyl L-lysine (1410 mg,
5.2 mmol) and N-carboxyanhydride of L-tyrosine (300 mg, 1.4 mmol)
are placed in a three-neck flask. Distilled anhydrous dioxane (57
mL) is added. Diethylamine (3.4 .mu.L) is added. The resulting
mixture is stirred mechanically for 24 hours at a temperature of
approximately 22-25.degree. C. The mixture is slowly poured into
100 mL deionized water and filtered under vacuum. The solid is kept
under vacuum for 12 hours.
EXAMPLE 2
Preparation of a Protected Polypeptide.
[0112] N-Carboxyanhydride of L-alanine (430 mg, 3.75 mmol),
N-carboxyanhydride of .gamma.-benzyl L-glutamate (300 mg, 1.15
mmol), N-carboxyanhydride of N-t-butoxycarbonyl L-lysine (705 mg,
2.6 mmol) and N-carboxyanhydride of L-tyrosine (150 mg, 0.7 mmol)
are placed in a three-neck flask. Distilled anhydrous dioxane (28.5
mL) is added. Diethylamine (1.7 .mu.L) is added. The resulting
mixture is stirred mechanically for 24 hours at a temperature of
approximately 22-25.degree. C. The mixture is slowly poured into
100 mL deionized water and filtered under vacuum. The solid is kept
under vacuum for 12 hours.
EXAMPLE 3
[0113] Deprotecting the Protected Polypeptide Prepared in Example 1
with HBr/Acetic Acid.
[0114] The protected polypeptide prepared in Example 1 (200 mg) is
suspended in 7 mL of 33% hydrogen bromide in acetic acid. The
starting material slowly dissolves forming a red brown solution.
The mixture is stirred for 17 hours at a temperature of
approximately 22.degree. C. The solution of HBr/acetic acid is
evaporated to dryness using a rotary evaporator under reduced
pressure. To this residue, 100 mL of water is added to dissolve the
solid. The solution is placed in a Visking partially permeable
cellulose membrane which is in the form of a tube, Size 6, having a
molecular weight cut-off of 12-14 kDa. Size 6 tube has a diameter
of 27/32 inches, 21.5 mm and a width of 32-34 mm. The tube is
available from Medicell International Ltd. The tube containing the
solution is stirred in a beaker of water. Polypeptide fragments
having a molecular weight less than about 2 kDa are removed by
osmosis from the dialysis tube. The tube is removed from the water
and stirred in a beaker containing 0.3% acetic acid in water. The
resulting product is removed from the tube and lyophilized to
obtain glatiramer acetate as a pure white solid.
EXAMPLE 4
[0115] Deprotecting the Protected Polypeptide Prepared in Example 1
with HCl/Acetic Acid.
[0116] The protected polypeptide prepared in Example 1 (200 mg) is
suspended in 20 mL of a mixture prepared of 9.4 mL concentrated
hydrochloric acid adjusted to 20 mL with glacial acetic acid. The
starting material slowly dissolves forming a slightly turbid
solution. The mixture is stirred for 17 hours at a temperature of
approximately 22.degree. C. The solution of HCl/acetic acid is
evaporated to dryness using a rotary evaporator under reduced
pressure. To this residue, 100 mL of water is added to dissolve the
solid. The solution is placed in a Visking partially permeable
cellulose membrane which is in the form of a tube, Size 6, having a
molecular weight cut-off of 12-14 kDa. Size 6 tube has a diameter
of 27/32 inches, 21.5 mm and a width of 32-34 mm. The tube is
available from Medicell International Ltd. The tube containing the
solution is stirred in a beaker of water. Polypeptide fragments
having a molecular weight less than about 2 kDa are removed by
osmosis from the dialysis tube. The tube is removed from the water
and stirred in a beaker containing 0.3% acetic acid in water. The
resulting product is removed from the tube and lyophilized to
obtain glatiramer acetate as a pure white solid.
EXAMPLE 5
[0117] Deprotecting the Protected Polypeptide Prepared in Example 2
with HCl/Acetic Acid.
[0118] The protected polypeptide prepared in Example 2 (200 mg) is
suspended in 20 mL of a mixture prepared of 9.4 mL concentrated
hydrochloric acid adjusted to 20 mL with glacial acetic acid. The
starting material slowly dissolves forming a slightly turbid
solution. The mixture is stirred for 17 hours at a temperature of
approximately 22.degree. C. The solution of HCl/acetic acid is
evaporated to dryness using a rotary evaporator under reduced
pressure. To this residue, 100 mL of water is added to dissolve the
solid. The solution is placed in a Visking partially permeable
cellulose membrane which is in the form of a tube, Size 6, having a
molecular weight cut-off of 12-14 kDa. Size 6 tube has a diameter
of 27/32 inches, 21.5 mm and a width of 32-34 mm. The tube is
available from Medicell International Ltd. The tube containing the
solution is stirred in a beaker of water. Polypeptide fragments
having a molecular weight less than about 2 kDa are removed by
osmosis from the dialysis tube. The tube is removed from the water
and stirred in a beaker containing 0.3% acetic acid in water. The
resulting product is removed from the tube and lyophilized to
obtain glatiramer acetate as a pure white solid.
EXAMPLE 6
Relative Molecular Weight Determination by GPC Using a UV
Detector.
GPC-UV Detector Conditions:
TABLE-US-00001 [0119] Eluent: Phosphate buffer 0.05 M, pH 7.4, 5.6
g Na.sub.2HPO.sub.4, 116 g NaCl/4 L water Column: PSS Suprema, 10
.mu.m, 100 A, 8 .times. 300 mm. Temperature: 23.degree. C. Pump:
TSP AS 3000 autosampler inj. - Vol.: 50 .mu.L Concentration: about
2.0 mg/mL Detector: TSP UV2000 at 276 nm GPC- PSS WinGPC Vers. 7.2
Software: Samples: COPAXONE .RTM. (Lot # 5308036) wherein the
mannitol has been removed by dialysis COPAXONE .RTM. (Lot #
8040341) wherein the mannitol has been removed by dialysis Sample A
is glatiramer acetate prepared in Example 4 according to the
process of the invention Sample B is glatiramer acetate prepared in
Example 5 according to the process of the invention Sample The
weighted samples are dispersed in the eluent and Preparation:
allowed to stand for full hydration at room temperature for about
12 hours. The sample solution is filtered through a 1.0 filter unit
(Schleicher & Schuell)
[0120] The test results for GPC are summarized in Table I.
TABLE-US-00002 TABLE I Sample M.sub.w [kD] COPAXONE .RTM. (Lot #
5308036) 7.923 COPAXONE .RTM. (Lot # 8040341) 9.524 Sample A 8.551
Sample B 7.689
[0121] The test results in Table I clearly show that the weight
average molecular weight of glatiramer acetate prepared by the
process of the invention, Samples A and B, is 8.551 kDa and 7.689
kDa, respectively, and the weight average molecular weight of
COPAXONE.RTM. is 7.923 kDa and 9.524 kDa, depending on the lot
number.
EXAMPLE 7
Relative Molecular Weight Determination by Gel Permeation
Chromatography (GPC) Using a Refractive Index Detector.
GPC-RI Detector Conditions:
TABLE-US-00003 [0122] Eluent: Phosphate buffer 0.05 M, pH 7.4, 5.6
g Na.sub.2HPO.sub.4, 116 g NaCl/4 L water Column: PSS Suprema, 10
.mu.m, 100 A, 8 .times. 300 mm Temperature: 23.degree. C. Pump: TSP
AS 3000 autosampler inj. - Vol.: 50 .mu.L Concentration: about 2.0
mg/mL Detector: Shodex RI 71 GPC-Software: PSS WinGPC Vers. 7.2
Samples: COPAXONE .RTM. (Lot # 5308036) wherein the mannitol has
been removed by dialysis COPAXONE .RTM. (Lot # 8040341) wherein the
mannitol has been removed by dialysis Sample A is glatiramer
acetate prepared in Example 4 according to the process of the
invention Sample B is glatiramer acetate prepared in Example 5
according to the process of the invention Sample The weighted
samples are dispersed in the Preparation: eluent and allowed to
stand for full hydration at room temperature for about 12 hours.
The sample solution is filtered through a 1.0 filter unit
(Schleicher & Schuell)
[0123] The test results for GPC with RI detector are summarized in
Table II.
TABLE-US-00004 TABLE II Sample M.sub.w [kD] COPAXONE .RTM. (Lot #
5308036) 8.663 COPAXONE .RTM. (Lot # 8040341) 9.641 Sample A 9.581
Sample B 8.224
[0124] The test results in Table II clearly show that the weight
average molecular weight of glatiramer acetate prepared by the
process of the invention, Samples A and B, is 9.581 kDa and 8.224
kDa, respectively, and the weight average molecular weight of
COPAXONE.RTM. is 8.663 kDa and 9.641 kDa, depending on the lot
number.
[0125] Examples 8-17 relate to a phase transfer process for
preparing glatiramer acetate.
[0126] The present inventors have established a scale of 1 (low) to
5 (high) to quantify the influence of the alkaline compound (sodium
hydroxide) on glatiramer formation. The test results are summarized
in Table III.
TABLE-US-00005 TABLE III Recovery of Desired NaOH Conc. Molecular
Low Product with in 15 mL Weight Product Molecular Desired THF + 75
mg from Aqueous Weight Molecular Glatiramer Free Phase Phase
Polypeptide Weight Base Separation SM = 75 mg Formation Cut-off 0.1
N/5 mL No 1 1 1 0.25 N/5 mL 1 5 1 5 0.25 N/10 mL 1 5 1 5 0.5 N/5 mL
3 3 2 4 0.5 N/10 mL 4 3 3 3 0.5 N/10 mL + 5 3 2 4 10 mL water 1.0
N/5 mL 5 3 5 1
[0127] The results in Table III clearly show that 0.1-1.0 N sodium
hydroxide solutions may be used to prepare the polypeptide or a
pharmaceutically acceptable salt thereof products of the invention.
The results in Table III also show that the use of a sodium
hydroxide concentration of at least 0.25 N is preferred to
facilitate phase separation of the organic phase and aqueous
phase.
EXAMPLE 8
Polymerization Step (a).
[0128] N-Carboxyanhydride of tyrosine (30 mg, 0.010 mm),
N-carboxyanhydride of alanine (62 mg, 0.054 mm), N-carboxyanhydride
of .gamma.-benzyl glutamate (42 mg, 0.016 mm) and
N-carboxyanhydride of E-N-trifluoroacetyllysine (100 mg, 0.037 mm),
are placed in a single-neck flask with a magnetic stirrer. To this
mixture is added 20 mL of dry tetrahydrofuran. A clear solution is
obtained. Diethylamine (10 .mu.L) is added. The resulting mixture
is stirred for 48 hours. The tetrahydrofuran is removed by
evaporation. Water (150 mL) is added to the residue and the
stirring is continued. A white solid is obtained. The solid is
filtered and dried in a dessicator under vacuum.
[0129] The yield is determined to be 154 mg.
EXAMPLE 9
Deprotecting Step (b).
[0130] Protected glatiramer acetate, 75 mg, prepared in Example 8
is transferred to a single-neck flask provided with a magnetic
stirrer. Tetrahydrofuran (15 mL) is added to the flask and stirred.
A clear solution is obtained. Aqueous solution of sodium hydroxide
(8 mL) 0.5 N, is added. The addition of the sodium hydroxide
solution results in the mixture becoming hazy. The mixture is
stirred for 1 hour at 24-26.degree. C. The formation of two phases
is observed. The bottom layer is separated and acidified using
dilute aqueous 1 N HCl solution to pH=6.0 with stirring. The crude
glatiramer free base solution is filtered using a nylon filter (0.2
micron Nylon Acrodisk).
EXAMPLE 10
[0131] Protected glatiramer acetate, 75 mg, prepared in Example 8
is transferred to a single-neck flask provided with a magnetic
stirrer. Tetrahydrofuran (15 mL) is added to the flask and stirred.
A clear solution is obtained. Aqueous solution of sodium hydroxide
(10 mL) 0.25 N is added. The addition of the sodium hydroxide
solution results in the mixture becoming hazy. The mixture is
stirred for 16 hours at 25-26.degree. C. The reaction mixture is
centrifuged for 15 minutes. The formation of two phases is
observed. The bottom layer is separated and acidified using dilute
aqueous HCl solution to pH=7-7.5 with stirring. Stirring is
continued for an additional 30 minutes and the pH is determined to
be pH=8.0. The crude glatiramer free base solution is filtered.
EXAMPLE 11
Polymerization Step.
[0132] N-Carboxyanhydride of tyrosine (30 mg, 0.010 mm),
N-carboxyanhydride of alanine (62 mg, 0.054 mm), N-carboxyanhydride
of .gamma.-benzyl glutamate (42 mg, 0.016 mm) and
N-carboxyanhydride of E-N-trifluoroacetyllysine (100 mg, 0.037 mm),
are placed in a single-neck flask with a magnetic stirrer. To this
mixture is added 20 mL of dry dioxane. A clear solution is
obtained. Diethylamine (10 .mu.L) is added. The resulting mixture
is stirred for 48 hours. To this, water (150 mL) is added slowly
with stirring. A white solid is obtained. The solid is filtered and
dried in a dessicator under vacuum.
[0133] The yield is determined to be 170 mg.
EXAMPLE 12
Deprotecting Step.
[0134] Protected glatiramer acetate, 75 mg, prepared in Example 11
is transferred to a single-neck flask provided with a magnetic
stirrer. Dioxane (15 mL) is added to the flask and stirred. A clear
solution is obtained. Aqueous solution of sodium hydroxide (10 mL)
0.5 N, is added. The addition of the sodium hydroxide solution
results in the mixture becoming hazy. The mixture is stirred for 16
hours at 25-26.degree. C. The reaction mixture is centrifuged for
15 minutes. The formation of two phases is observed. The bottom
layer is separated and acidified using dilute aqueous HCl solution
to pH=7-7.5 with stirring. Stirring is continued for an additional
30 minutes and the pH is determined to be pH=8.0. The crude
glatiramer free base solution is filtered.
EXAMPLE 13
Deprotecting Step Conducted at Lower Temperature.
[0135] Protected glatiramer acetate, 75 mg, prepared in Example 12
is transferred to a single-neck flask provided with a magnetic
stirrer. Tetrahydrofuran (15 mL) is added to the flask and the
temperature of the solution is reduced to 0.degree. C. Aqueous
solution of sodium hydroxide (10 mL) 0.5 N, is added to the
solution while maintaining a temperature of 0.degree. C. The
addition of the sodium hydroxide results in the solution becoming
hazy. The solution is stirred for 3 hours at 0.degree. C. The
formation of two phases is observed. The bottom layer is separated
and acidified using dilute aqueous HCl solution to pH=7-7.5 with
stirring at 0.degree. C. Stirring is continued for an additional 30
minutes and the pH is determined to be approximately pH=8.0. The
crude glatiramer free base solution is filtered.
EXAMPLE 14
[0136] Deprotecting Step with Acetate Buffer.
[0137] Protected glatiramer acetate, 75 mg, prepared in Example 12
is transferred to a single-neck flask provided with a magnetic
stirrer. Tetrahydrofuran (15 mL) is added to the flask and the
temperature of the solution is reduced to 0.degree. C. Aqueous
solution of sodium hydroxide (10 mL) 0.5 N, and acetic acid (2 mL)
is added to the solution while maintaining a temperature of
0.degree. C. and a pH=12. The addition of the sodium hydroxide and
acetic acid results in the solution becoming hazy. The solution is
stirred for 3 hours at 0.degree. C. The formation of two phases is
observed. The bottom layer is separated and acidified using dilute
aqueous HCl solution to pH=7-7.5 with stirring at 0.degree. C.
Stirring is continued for an additional 30 minutes and the pH is
determined to be approximately pH=8.0. The crude glatiramer free
base solution is filtered.
EXAMPLE 15
[0138] Protected glatiramer acetate, 75 mg, prepared in Example 8
is transferred to a single-neck flask provided with a magnetic
stirrer. Dioxane (15 mL) is added to the flask and stirred. A clear
solution is obtained. Aqueous solution of sodium hydroxide (10 mL)
0.25 N is added. The addition of the sodium hydroxide solution
results in the mixture becoming hazy. The mixture is stirred for 16
hours at 25-26.degree. C. The reaction mixture is centrifuged for
15 minutes. The formation of two phases is observed. The bottom
layer is separated and acidified using dilute aqueous HCl solution
to pH=7-7.5 with stirring. Stirring is continued for an additional
30 minutes and the pH is determined to be pH=8.0. The crude
glatiramer free base solution is filtered.
EXAMPLE 16
Diafiltration (Tangential Flow Filtration).
[0139] The crude glatiramer free base solution prepared in Example
9 is diluted to 120 with water. The dilute solution is first
filtered through a 10 K diafiltration membrane, Pellicon XL, PLCCC
10 (50 cm.sup.2), available from Millipore, and then, filtered
through a 10 K diafiltration membrane, Pellicon XL, PLCCC 5 (50
cm.sup.2), available from Millipore. The concentrated solution
obtained is lyophilized. A white powder is obtained.
EXAMPLE 17
Chromatographic Method of Purification of Glatirimer Acetate.
[0140] The crude glatiramer free base solution prepared in Example
9 is subjected to chromatographic separation. A column for gel
filtration, FRACTOGEL TSK HW55 (600.times.26 mm) is prepared in a
Superformance 26 Merck cartridge according to the manufacturer's
instructions. The column is equilibrated with 0.2M ammonium acetate
buffer pH 5.0, 30 mL of glatiramer free base solution samples (20
mg/mL, in 0.2 M ammonium acetate pH 5.0) are loaded on the column
and fractions are collected every 10 minutes. A fraction having an
average molecular weight of 7-8 KDa is isolated.
[0141] Examples 18-25 relate to a process for preparing glatiramer
acetate using an alkali or alkaline earth metal hydroxide,
carbonate, or a hydrogen carbonate.
EXAMPLE 18
Preparation of a Protected Polypeptide.
[0142] N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM),
N-carboxyanhydride of L-alanine (620 mg, 5.4 mM),
N-carboxyanhydride of .gamma.-benzyl L-glutamate (430 mg, 1.6 mM)
and N-carboxyanhydride of N.sup..epsilon.-trifluoroacetyl L-lysine
(1.01 g, 3.73 mM), are placed in a single-neck flask (100 mL) with
a magnetic stirrer. To this mixture is added 40 mL of
tetrahydrofuran. Diethylamine (10 .mu.L) is added. The resulting
mixture is stirred for 24 hours at a temperature of approximately
25.degree. C. The mixture is slowly poured into 100 mL water while
stirring. A solid is precipitated. The solid is filtered after 2
hours of stirring and washed with water. The solid is resuspended
in 100 mL water and filtered. The solid is kept under vacuum for
approximately 12 hours.
EXAMPLE 19
Preparation of a Protected Polypeptide.
[0143] N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM),
N-carboxyanhydride of L-alanine (620 mg, 5.4 mM),
N-carboxyanhydride of .gamma.-benzyl L-glutamate (430 mg, 1.6 mM)
and N-carboxyanhydride of N.sup..epsilon.-trifluoroacetyl L-lysine
(1.01 g, 3.73 mM), are placed in a single-neck flask (100 mL) with
a magnetic stirrer. To this mixture is added 40 mL of dioxane.
Diethylamine (10 .mu.L) is added. The resulting mixture is stirred
for 48 hours at a temperature of approximately 25.degree. C. The
mixture is slowly poured into 100 mL water while stirring. A solid
is precipitated. The solid is filtered and washed with water. The
solid is resuspended in 100 mL water and filtered. The solid is
kept under vacuum for approximately 12 hours.
EXAMPLE 20
[0144] Cleavage of .gamma.-Benzyl Moiety from the Polypeptide
Prepared in Example 2.
[0145] The protected polypeptide prepared in Example 19, 100 mg, is
suspended in tetrahydrofuran (20 mL) and cooled in an ice water
bath. Concentrated sulfuric acid, 4 mL, is added. The resulting
clear solution is stirred for 20 hours at a temperature of
approximately 25.degree. C. The solvent, tetrahydrofuran, is
removed by evaporation at 25.degree. C. to form a viscous liquid.
Water, 50 mL, is added to the viscous liquid with stirring. A white
precipitate forms which is filtered under vacuum and dried over
phosphorous pentoxide under vacuum at 25.degree. C. for
approximately 12 hours in the dark. A white solid is obtained. The
solid is filtered and dried in a dessicator under vacuum. The yield
is determined to be 75 mg.
EXAMPLE 21
[0146] Cleavage of N.sup..epsilon.-trifluoroacetyl Moiety from the
Polypeptide Prepared in Example 20.
[0147] The protected polypeptide prepared in Example 20, 75 mg, is
dispersed in 12 mL of tetrahydrofuran, 4 mL of 0.5 M aqueous sodium
hydroxide is added with stirring. The mixture is stirred for 3
hours at ambient temperature (approximately 22.degree. C.). The
lower aqueous layer is separated and acidified with acetic acid to
pH=6.0.
EXAMPLE 22
[0148] Cleavage of .gamma.-Benzyl Moiety from the Polypeptide
Prepared in Example 19.
[0149] The protected polypeptide prepared in Example 19, 1 g, is
suspended in 50 mL of a mixture prepared of 47 mL concentrated HCl
adjusted to 100 mL with glacial acetic acid. The starting material
slowly dissolves forming a slightly turbid solution. The mixture is
stirred for 18 hours at a temperature of approximately 22.degree.
C. The solution is poured into 1,000 mL of stirred water. A white
precipitate is formed. The suspension is stirred for another 3
hours and then filtered. The product is washed with water and dried
under vacuum at 50.degree. C. for approximately 17 hours.
EXAMPLE 23
[0150] Cleavage of N.sup..epsilon.-trifluoroacetyl Moiety from the
Polypeptide Prepared in Example 22.
[0151] The protected polypeptide prepared in Example 22, 300 mg, is
dispersed in 45 mL of tetrahydrofurane, 25 mL of 0.5 M aqueous
sodium hydroxide is added with stirring. The mixture is stirred for
3 hours at ambient temperature (approximately 22.degree. C.). A
clear, two-phase liquid system is formed. The lower aqueous layer
is separated and acidified with acetic acid to pH=6.0. The clear,
colorless solution is filled into dialysis bags and dialyzed at
ambient temperature once against 0.3% aqueous acetic acid, and then
against water until a pH of 5.5 is reached. This solution is
filtered and lyophilized to yield glatiramer acetate as a white
solid.
EXAMPLE 24
Diafiltration (Tangential Flow Filtration).
[0152] The glatiramer acetate solution prepared in Example 21 is
adjusted to 120 mL with water to provide a 0.5-0.6 mg/mL
concentration of the glatiramer acetate. The dilute solution is
first filtered through a 30 K diafiltration membrane, Pellicon XL,
PLCCC 10 (50 cm.sup.2), available from Millipore, and then,
filtered through a 3 K diafiltration membrane, Pellicon XL, PLCCC 5
(50 cm.sup.2), available from Millipore. The concentrated solution
obtained is lyophilized to provide glatiramer acetate in solid
form.
EXAMPLE 25
Chromatographic Method of Purification of Glatiramer Acetate.
[0153] The glatiramer as extract of pH=6 prepared in Example 23 is
concentrated in vacuo to dryness and subjected to chromatographic
separation. A column for gel filtration, FRACTOGEL TSK HW55
(600.times.26 mm) is prepared in a Superformance 26 Merck cartridge
according to the manufacturer's instructions. The column is
equilibrated with 0.2 M ammonium acetate buffer pH 5.0, 30 mL of
glatiramer free base solution samples (20 mg/mL, in 0.2 M ammonium
acetate pH 5.0) are loaded on the column and fractions are
collected. A fraction having an average molecular weight of 7-10
kDa is isolated.
[0154] Examples 26-33 relate to a process for preparing glatiramer
acetate using an amine or ammonia.
EXAMPLE 26
Preparation of a Protected Polypeptide.
[0155] N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM),
N-carboxyanhydride of L-alanine (620 mg, 5.4 mM),
N-carboxyanhydride of .gamma.-benzyl L-glutamate (430 mg, 1.6 mM)
and N-carboxyanhydride of N.sup..epsilon.-trifluoroacetyl L-lysine
(1.01 g, 3.73 mM), are placed in a single-neck flask (100 mL) with
a magnetic stirrer. To this mixture is added 40 mL of
tetrahydrofuran. Diethylamine (10 .mu.L) is added. The resulting
mixture is stirred for 24 hours at a temperature of approximately
25.degree. C. The mixture is slowly poured into 100 mL water while
stirring. A solid is precipitated. The solid is filtered after 2
hours of stirring and washed with water. The solid is resuspended
in 100 mL water and filtered. The solid is kept under vacuum for
approximately 12 hours.
EXAMPLE 27
Preparation of a Protected Polypeptide.
[0156] N-Carboxyanhydride of L-tyrosine (207.19 mg, 1.0 mM),
N-carboxyanhydride of L-alanine (620 mg, 5.4 mM),
N-carboxyanhydride of .gamma.-benzyl L-glutamate (430 mg, 1.6 mM)
and N-carboxyanhydride of N.sup..epsilon.-trifluoroacetyl L-lysine
(1.01 g, 3.73 mM), are placed in a single-neck flask (100 mL) with
a magnetic stirrer. To this mixture is added 40 mL of dioxane.
Diethylamine (10 .mu.L) is added. The resulting mixture is stirred
for 48 hours at a temperature of approximately 25.degree. C. The
mixture is slowly poured into 100 mL water while stirring. A solid
is precipitated. The solid is filtered and washed with water. The
solid is resuspended in 100 mL water and filtered. The solid is
kept under vacuum for approximately 12 hours.
EXAMPLE 28
[0157] Cleavage of .gamma.-Benzyl Moiety from the Polypeptide
Prepared in Example 27.
[0158] The protected polypeptide prepared in Example 27, 100 mg, is
suspended in tetrahydrofuran (20 mL) and cooled in an ice water
bath. Concentrated sulfuric acid, 4 mL, is added. The resulting
clear solution is stirred for 20 hours at a temperature of
approximately 25.degree. C. The solvent, tetrahydrofuran, is
removed by evaporation at 25.degree. C. to form a viscous liquid.
Water, 50 mL, is added to the viscous liquid with stirring. A white
precipitate forms which is filtered under vacuum and dried over
phosphorous pentoxide under vacuum at 25.degree. C. for
approximately 12 hours in the dark. A white solid is obtained. The
solid is filtered and dried in a dessicator under vacuum. The yield
is determined to be 75 mg.
EXAMPLE 29
[0159] Cleavage of .gamma.-Benzyl Moiety from the Polypeptide
Prepared in Example 27.
[0160] The protected polypeptide prepared in Example 27, 1 g, is
suspended in 50 mL of a mixture prepared of 47 mL concentrated HCl
adjusted to 100 mL with glacial acetic acid. The starting material
slowly dissolves forming a slightly turbid solution. The mixture is
stirred for 18 hours at a temperature of approximately 22.degree.
C. The solution is poured into 1,000 mL of stirred water. A white
precipitate is formed. The suspension is stirred for another 3
hours and then filtered. The product is washed with water and dried
under vacuum at 50.degree. C. for approximately 17 hours.
EXAMPLE 30
[0161] Evaluation of Amines to be Used to Cleave the
N.sup..epsilon.-trifluoroacetyl Moiety from the Polypeptide
Prepared in Example 28.
[0162] The polypeptide prepared in Example 28, 75 mg, is suspended
in 15 mL of water. An amine, 7 mL, is added to the suspension to
provide an amine concentration of 3 M. A list of amines is provided
in Table IV. Since a deprotected polypeptide is soluble in water,
the reaction is monitored by the clarity of the solution. The
results for each of the amines are summarized in Table IV.
TABLE-US-00006 TABLE IV Amine Result Morpholine No clear solution
after 48 hours. N-methyl-piperazine No clear solution after 48
hours. Dicyclohexylamine No clear solution after 48 hours.
Di-sec-butylamine No clear solution after 48 hours. Pyrrolidine No
clear solution after 48 hours. Diisopropylamine Clear solution
after about 1 hour. Dipropylamine No clear solution after 48 hours.
Isopropylamine Clear solution after about 1.5 hours. Methylamine
(aqueous) No clear solution after 48 hours.
[0163] The results in Table IV clearly show that a free base form
of the polypeptide prepared in Example 28 is formed only upon the
addition of diisopropylamine or isopropylamine. The results in
Table I also show that dipropylamine, morpholine,
N-methyl-piperazine, dicyclohexylamine, di-sec-butylamine,
pyrrolidine, and methylamine failed to produce a free base form of
the polypeptide.
[0164] Thus, applicants unexpectedly determined that in the second
deprotection step of the process of the invention, Step (b),
diisopropylamine and isopropylamine were the only amines that
successfully removed the N.sup..epsilon.-trifluoroacetyl group of
the lysine moiety.
EXAMPLE 31
[0165] Cleavage of the N.sup..epsilon.-trifluoroacetyl Moiety from
the Polypeptide Prepared in Example 28.
[0166] The polypeptide prepared in Example 28, 75 mg, is suspended
in 15 mL of water. Diisopropylamine, 7 mL, is added to the
suspension to provide an amine concentration of 3 M. A milky-white
solution becomes clear in approximately 1 hour and the clear
solution is stirred at 25.degree. C. for 20 hours. The reaction
mixture is evaporated at approximately 25.degree. C. to form crude
glatiramer free base in the form of a viscous liquid. Fifty percent
(50%) acetic acid (15 mL) is added to the mixture and stirred for
30 minutes to form a glatiramer acetate solution.
EXAMPLE 32
Diafiltration (Tangential Flow Filtration).
[0167] The glatiramer acetate solution prepared in Example 31 is
diluted to 120 mL with water. The dilute solution is first filtered
through a 30 K diafiltration membrane, Pellicon XL, PLCCC 10 (50
cm.sup.2), available from Millipore, and then, filtered through a 3
K diafiltration membrane, Pellicon XL, PLCCC 5 (50 cm.sup.2),
available from Millipore. The concentrated solution obtained is
lyophilized to provide glatiramer acetate in solid form.
EXAMPLE 33
Chromatographic Method of Purification of Glatiramer Acetate.
[0168] The glatiramer acetate solution prepared in Example 31 is
concentrated in vacuo to dryness and subjected to chromatographic
separation. A column for gel filtration, FRACTOGEL TSK HW55
(600.times.26 mm) is prepared in a Superformance 26 Merck cartridge
according to the manufacturer's instructions. The column is
equilibrated with 0.2 M ammonium acetate buffer pH 5.0, 30 mL of
glatiramer free base solution samples (20 mg/mL, in 0.2 M ammonium
acetate pH 5.0) are loaded on the column and fractions are
collected. A fraction having an average molecular weight of 7-10
kDa is isolated.
[0169] While the invention has been described with particular
reference to certain embodiments thereof, it will be understood
that changes and modifications may be made by those of ordinary
skill within the scope and spirit of the following claims:
* * * * *