GB2478837A

GB2478837A - Preparation of glatiramer

Info

Publication number: GB2478837A
Application number: GB1104313A
Authority: GB
Inventors: Dharmaraj Ramachandra Rao; Rajendra Narayanrao Kankan; Dilip Ramdas Birari; Santosh Prabhakar Kelkar
Original assignee: Cipla Ltd
Current assignee: Cipla Ltd
Priority date: 2011-03-14
Filing date: 2011-03-14
Publication date: 2011-09-21
Also published as: GB201104313D0

Abstract

The present invention relates to a process for preparing glatiramer base or a pharmaceutically acceptable salt thereof comprising: polymerizing a mixture of N-carboxyanhydride of L-alanine, N- carboxyanhydride of y-benzyl L-glutamate, N- carboxyanhydride of y-benzyl L- tyrosine and N-carboxyanhydride of N-trifluroacetyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a diprotected polypeptide-1 ; adding an acid to the diprotected polypeptide-1 to form monoprotected polypeptide-2, adding a base to the isolated polypeptide-2 to form glatiramer base; and optionally converting the glatiramer base to a pharmaceutically acceptable salt thereof, such as glatiramer acetate (copolymer 1).

Description

PROCESS FOR PREPARING GLATIRAMER

Technical Field of Invention

The present invention provides a novel process for preparing a polypeptide or pharmaceutically acceptable salt thereof. More, specifically the invention provides a process for preparing glatiramer acetate.

Baclcq round of the Invention Glatiramer acetate is the generic name for the drug Copaxone or Copolymer 1. Copaxone is administered by subcutaneous injection at a dose of 20 mg per day. Glatiramer acetate is a random polymer (average molecular mass 6.4 kDa) composed of four amino acids that are found in myelin basic protein. The four naturally occurring amino acids are 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. The average molecular weight of glatiramer acetate s 5,000 -9,000 daltons. Glatiramer acetate is identified by specific antibodies.

Glatiramer is used to reduce episodes of symptoms in patients with relapsing-remitting multiple sclerosis. Glatiramer is in a class of medications called immunomodulators. It works by stopping the body from damaging its own nerve cells (myelin).

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).xCH3COOH (C5H9N04*C3H7N02*C6H 14N202*C9H11 NO3)'xC2H4O2 COPAXONE is a clear, colorless to slightly yellow, sterile, nonpyrogenic solution for subcutaneous injection. Each I ml of solution contains 20 mg of glatiramer acetate and 40 mg of mannitol. The pH range of the solution is approximately 5.5 to 7.0.

A process to prepare Copolymer 1 or glatiramer acetate was first disclosed in US 3849550 wherein polymerization of N-carboxy-anhydrFdes of tyrosne, aFanFne, y-benzyr glutamate and N-trifluroacetyl lysine was carried out at ambient temperature in anhydrous dioxane at room temperature for 24 hours using diethylamine as initiator. The deblocking of y-carboxyl group of glutamic acid was effected with HBr in glacial acetic acid followed by removal of trifluoroacetyl groups from the lysine by 1 M piperidine.

Improvements in the composition of copolymers are disclosed by Konfino et al in US5800808, US5981589, US 6054430; US 6363161, US 6620847, US 6939539, US 6342476 & US 7199098. The process is similar to that disclosed in US 3849550. In the final step, crude copolymer is subjected to dialysis against water until a pH 8 is attained, followed by another dialysis against 0.3% acetic acid to form acetate salt. The salt is further dialyzed against water until a pH =5.5-6 is attained, followed by concentration and lyophilization.

A similar process is described in WO 2006/029411 wherein polymerization of N-carboxyanhydride of L-tyrosine, L-alanine, y-benzyl glutamate and N-trifluroacetyl lysine was carried out with predetermined amount of diethylamine, followed by deprotection of benzyl, removal of trifluoroacetyl group with piperidine base and finally ultra filtration.

The use of excess amount of piperidine base for deprotection has several disadvantages. The piperidine has high boiling point of 106°C. Hence, tangential flow filtration removes low molecular weight glatiramer fragments but it is difficult to remove excess piperidine completely.

The traces of piperidine impart color to the copolymer -1 during lyophilization. Further, the desired amino acid ratio in the glatiranier is not consistent and it varies from batch to batch.

US 7049399 BI describes a process to prepare Copolymer 1. The process comprises the steps of: (a) polymerization of a mixture of the N-carboxyanhydrides of L-alanine, L tyrosine, protected L-glutamate, and protected L-Iysine, to obtain protected copolymer 6 or a salt thereof; (b) deprotection of the protected copolymer 6 or a salt thereof either by palladium catalyzed transfer hydrogenation or palladium catalytic hydrogenation under hydrogen pressure to afford Copolymer 1 or a pharmaceutically acceptable salt thereof in substantially a single step; (c) separation and purification of the Copolymer I or a pharmaceutically acceptable salt thereof.

The process suffers from the several disadvantages, as PdI C deprotects only benzyl group.

This leads to partially protected copolymer 6 and breaking of aniide linkage. Hence, it is difficult to obtain, the copolymer 1 with desired molecular w&ght of 40 KD"TO 5-10 K.

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US20060154862 describes a process to prepare Copolymer 1. The process comprises polymerization of a mixture of the N-carboxyanhydrides of L-alanine, L tyrosine, protected L-glutamate, and protected L-lysine, to obtain protected copolymer 6 or a salt thereof followed by deprotection either by acid hydrolysis or with an aqueous solution of an alkali or alkaline earth metal hydroxide. The disadvantage of the process is use of 140 wt% of alkaline earth metal hydroxide based on the weight of the protected galtiramer. Further, use of high moles of NaOH may not control fragmentation of polymer. Further, the applicant states that the cleavage of N-trifluoroacetyl group of lysine moiety can take place only upon addition of diisopropyl amine and isopropyl amine. The applicant obtained the results wherein, bases like dipropylamine, morpholine, N-methyl-piperazine, dicyclohexylamine, di-sec-butylamine, pyrrolidine and methylamine did not produce a free base form of the polypeptide (page 31, example 30).

Further, diisopropylamine acetate formed during the process, is difficult to remove by TFF filtration. This further gives rise to assay problems in the isolated compound.

US2008/0021 192 describes a process to prepare Copolymer 1, which comprises polymerization of N-carboxyanhydrides of L-alanine, L tyrosine, y-methoxy or benzyl glutamate, and N-Boc-lysine or lysine having cyclic imide derivative; followed by deprotection of glutamate and lysine; dialysis. The deprotection of glutamate and lysine in a single step affects the fragmentation process. Further, the dialysis process is cumbersome as it requires a large volume of water.

The processes disclosed in the prior art to obtain galtiramer acetate are carried out on a small scale. Further, the glatiramer acetate having required average molecular weight is based upon selection of amino acids with desired molecular weight. Further, time needed for obtaining copolymer-1 of desired molecular weight depends on the reaction temperature & size of dipeptide of protected amino acids. The processes disclosed in the prior art involve use of unprotected N-carboxyanhydrides of L-tyrosine in polymerization of amino acids, which results in the brominated tyrosine impurity in subsequent deprotection. Further, presence of free -OH group in tyrosine moiety gives undesired cross linked polymer instead of straight chain polymer.

Hence there was a need to develop a process which is simple, economical and suitable for industrial scale up. The process of the present invention overcome problems associated with

prior art methods for preparing copolymer -1

Object of the invention The object of the present invention is to provide an improved process for the preparation of glatiramer or its pharmaceutically acceptable salts thereof.

Another object of the invention is to provide an improved process for the preparation of glatiramer base.

Another object of the present invention is to provide glatiramer having an average molecular weight of about 5 to 10 KDa and an average molar ratio of four amino acids in the desired range.

Yet another object of the present invention is to provide a process which is simple, economical and suitable for industrial scale-up.

Summary of the invention

According to a first aspect of the invention there is provided a process for preparing a glatiramer base, or a pharmaceutically acceptable salt thereof, comprising: (i) polymerizing a mixture of N-carboxyanhydride of L-alanine, N-carboxyanhydride of y-benzyl L-glutamate, N-carboxyanhydride of y-benzyl L-tyrosine and N-carboxyanhydride of N-trifluroacetyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a diprotected polypeptide-1; (ii) adding an acid to the diprotected polypeptide-1 to form monoprotected polypeptide-2, (iii) adding a base to the isolated polypeptide-2 to form glatiramer base; and optionally (iv) converting the glatiramer base to a pharmaceutically acceptable salt thereof.

In one embodiment the diprotected polypeptide-1 formed in step (I) comprises terminal ends as amine or amide and acid.

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Preferably in step (ii) the acid cleaves y-benzyl group from the glutamate moiety, y-benzyl group from tyrosine moiety and acidolytic peptide bond.

Preferably the initiator is a monoalkyl or dialkyl amine.

Preferably in step (iii) the base cleaves N-trifluoroacetyl group from L-Iysine moiety.

In one embodiment, step (iii) comprises isolating solid polypeptide-2 by quenching in water followed by filtration.

In an embodiment, the process further comprises converting the glatiramer base to a pharmaceutically acceptable salt thereof. The conversion may comprise reacting the base with the corresponding acid to form the salt. Preferably the base is reacted with acetic acid and the pharmaceutically acceptable salt is an acetate salt. However, other appropriate acids could be used to form other pharmaceutically acceptable salts, as would be well known to those of skill in the art.

According to a another aspect of the present invention, there is provided a process for preparing glatiramer comprising preparing a glatiramer base according to the first aspect of the invention; (v) purifying glatiramer base, preferably by tangential flow filtration against water; (vi) treatment of glatiramer base with acetic acid to form acetate salt; (vii) purification of salt, preferably by tangential flow filtration against water.

Preferably the process further comprises the step of: (viii) lyophillization, to obtain glatiramer acetate.

In one embodiment the said glatiramer acetate or copolymer-i has an average molecular weight between 5 to 10 kDa.

In one embodiment the diprotected polypeptide-1 is a straight chain polymer containing protected y-benzyl L-tyrosine moiety.

In one embodiment the monoprotected polypeptide-2 is free of brominated tyrosine impurity.

In one embodiment the acid used in step (ii) for the deprotection is selected from the group comprising of acetic acid, hydrochloric acid, hydrogen bromide, hydrogen fluoride, phosphoric acid, sulfuric acid, trifluoroacetic acid, methane sulfonic acid, trifluoromethane sulfonic acid and mixtures thereof.

In one embodiment the base used in step (iii) for the deprotection is selected from the group comprising of pyrrolidine, piperazine, morpholine, N-methyl-piperazirie, Dicyclohexylamine, Di-sec-butylamine, diisopropylamine, dipropylamine, isopropylamine and aqueous methylamine and mixtures thereof, preferably the base is pyrrolidine in aqueous solution.

In one embodiment in step (v), glatiramer base is substantially free of undesired low molecular weight polypeptide & high molecular weight polypeptide.

In one embodiment the glatiramer base prepared in step (v) has an average molecular weight of from about 5 to about 10 kDa.

In one embodiment, in step (v), the glatiramer base is substantially free of monoprotected polypeptide-2.

In one embodiment, step (v) further comprises acidifying the reaction mass to pH 3 to 4, more preferably 3.5 to 3.7. Preferably the reaction mass is acidified using acetic acid.

In one embodiment, in step (vii) the reaction mass is subjected to tangential flow filtration against water until a pH 4 to 5 is obtained, more preferably pH 4.5.

In one embodiment, in step (vii), glatiramer acetate is substantially free of excess acetic acid and pyrrolidine acetate.

In one embodiment, in step (vii), glatiramer acetate has improved colour characteristics.

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According another aspect of the present invention there is provided a pharmaceutical composition & method comprising glatiramer as described above together with one or more pharmaceutically acceptable excipients.

According to yet another aspect of the present invention there is provided the use of glatiramer as described above in medicine.

According to a still further aspect of the present invention there is provided the use of glatiramer as described above in the treatment of multiple sclerosis.

According to a still further aspect of the present invention there is provided the use of glatiramer as described above in the manufacture of a medicament for the treatment of multiple sclerosis.

According to a still further aspect of the present invention there is provided glatiramer as described above for use in the treatment of multiple sclerosis.

According to a still further aspect of the present invention, there is provided a method of treating multiple sclerosis comprising administering to a patient in need thereof glatiramer as described above.

Detailed description

In an embodiment of the present invention, there is provided an improved process for the preparation of glatiramer acetate, as depicted below in scheme 1:- Scheme 1:-

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0 o:_OO + O__0 0 + Ot_O 0 + H H/

H

H3C (CH2)4 CF3 OCH2Ph (T) (L) (M (G) Diethylamine polymer bound 1 4-dioxane 1JZIIJ HN1CF3 Dipteed Polypeptide-1 CH3H,J H 0 33% l-lBr in AcOH HNXCF3 0 OH Monoprotected Polypeptide-2

HO

1) Pyrrolidine 2) Ultrafiltration

NH

O OH

Glatiramer Base

I

NH

OH GIaramer Base HO 1)AcOH 2) Ultrafiltration NH2 Glatiramer Acetate j[1.CH3COOH

HO

Accordingly, in an embodiment, the present invention provides an improved process for the preparation of glatiramer acetate, which comprises the following steps: (i) polymerizing a mixture of N-carboxyanhydride of L-alanine (NCA of H-Ala-OH/A); N- carboxyanhydride of y-benzyl L-glutamate (NCA of H-Glu ( Obzl) -OHIG); N- carboxyanhydride of y-benzyl L-tyrosine(NCA of H-Tyr ( Bzl)-OH/ T) and N-carboxyanhydride of N-trifluroacetyl L-lysine (NCA of H-Lys (Tfa)-OH/L), in a polar aprotic solvent in presence of an initiator, to form diprotected polypeptide-1; The prior art processes involve polymerization of N-carboxyanhydrides of L-alanine, L-tyrosine, protected L-glutamate, and protected L-lysine. None of these processes involve the use of, N- carboxyanhydrides of protected L-tyrosine. It was surprisingly found that the use of N-carboxyanhydrides of protected L-tyrosine has the following advantages.

a) N-carboxyanhydrides of protected L-tyrosine has greater stability than unprotected L-tyrosine.

b) Use of N-carboxyanhydrides of unprotected L-tyrosine results in formation of brominated tyrosine impurity. Whereas N-carboxyanhydrides of benzylated L-tyrosine does not form such impurity in glatiramer acetate.

c) Use of N-carboxyanhydrides of unprotected L-tyrosine results in formation of cross lined polymers instead of straight chain polymers, due to presence of free -OH group in the amino acid.

The polymerization is carried out in presence of an initiator. The said initiator may be an alkaline initiator such as monoalkyl or dialkylamine. A preferred initiator is selected from the group comprising of diethylamine, diisopropylamine and other cyclic secondary amines.

The polar aprotic solvent is preferably selected from tetrahydrofuran, ethyl acetate, dimethylformamide, dioxane, dimethylsulfoxide. A mixture of polar aprotic solvents may also be used. Most preferably, the polar aprotic solvent is 1,4-dioxane.

The polymerization reaction is preferably carried out at an temperature of from about 10°C to 40°C, more preferably at about 20°C to 30°C.The reaction hours may vary from 1 to 50 hours, more preferably from 20 to 30 hours. Most preferably; the polymerization reaction is carried out at 25° C to 30° C for 24 Hrs.

The solid is isolated by quenching reaction mass in water & acidifying reaction mass with suitable acid, preferably acetic acid. The precipitated solid may be isolated by filtration. The diprotected polypeptide-1 may be further recrystallized from suitable solvent by using crystallization techniques known to those skilled in the art.

(ii) adding an acid to the diprotected polypeptide-1 to form monoprotected polypeptide -2, wherein, the acid cleaves y-benzyl group from the glutamate moiety and y-benzyl group from tyrosine moiety; In the deprotection step (ii), an acid is added to the diprotected polypeptide-1 to form monoprotected polypeptide-2. The acid cleaves y-benzyl group from the glutamate moiety. In addition, the acid cleaves y-benzyl group from tyrosine moiety forming polypeptide -2 as well as causes fragmentation of polypeptide -1.

Further, acid cleaves acidolytic peptide bond, reducing average molecular size of the peptide mixture.

Further, monoprotected polypeptide-2, obtained by the process of present invention is free of brominated tyrosine impurity and thus forms another aspect of the present invention.

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Suitable acids used for the deprotection are selected from the group comprising of acetic acid, hydrochloric acid, hydrogen bromide, hydrogen fluoride, phosphoric acid, sulfuric acid, trifluoroacetic acid, methane sulfonic acid and trifluoromethane sulfonic acid. A mixture of acids may be used. Preferably, a mixture of hydrogen bromide in acetic acid may be used. The acid may be added in the form of an aqueous solution.

Deprotection reaction may be carried out at about 10°C to 40°C for a period of 1 to 40 hours.

(iii) adding a base to the isolated monoprotected polypeptide -2 to form glatiramer base, wherein the base cleaves N-trifluoroacetyl group from L-lysine moiety; In the deprotecting step (iii), a base is added to the polymer-2 to form glatiramer base. The base cleaves N-trifluoroacetyl group from L-lysine moiety. The base used for the deprotection is selected from the group comprising of pyrrolidine, piperazine, morpholine, N-methyl-piperazine, Dicyclohexylamine, Di-sec-butylamine, diisopropylamine, dipropylamine, isopropylamine and aqueous methylamine. Preferably the base used is pyrrolidine. The pyrrolidine being low boiling solvent (87°C) having higher pKa of 11.27, is miscible in water as compared to other bases.

These characteristics proved advantageous while using pyrrolidine as base in the deprotection.

The pyrrolidine is preferably present in an amount from about 0.2 to about 2 molar, more preferably from about 0.25 to 1, molar. Most preferably, the pyrrolidine is present in an aqueous solution in an amount about 0.5 molar, based on the weight of the polymer-2.

The pyrrolidine is miscible in water and hence deprotection reaction can be carried out in water, water miscible solvents or mixture thereof. Preferably, the reaction is carried out in presence of water.

The deprotection reaction is preferably carried out at an temperature of from about 10°C to 40°C, more preferably at about 15°C to 25°CThe reaction hours may very from I to 50 hours, more preferably from 20 to 30 hours. Most preferably; the polymerization reaction is carried out at 25°C for 24 Hrs.

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(iv) purification of glatiramer base by tangential flow filtration against water; The diluted solution of glatiramer base in water, obtained in the step (iii) is subjected to the tangential flow filtration. Undesired low molecular weight polypeptide and high molecular molecular weight polypeptide, are preferably removed by tangential flow filtration. Further piperidine being high boiling liquid, is difficult to remove completely during diafiltration. The traces of piperidine base imparts colour to the galtiramer acetate during lyophilization. Further, the desired amino acid ratio in the galtiramer is not consistent & varies from batch to batch. All these difficulties in step (iv) are overcome by the use of pyrrolidine as deprotecting agent in the step (iii). Further, pyrrolidine being low boiling liquid is removed completely during tangential flow filtration, giving improved colour to the glatiramer acetate during Jyophilization and thus forms another aspect of the present invention.

(v) separation of glatiramer through treatment with acetic acid to form acetate salt & purification of salt by tangential flow filtration against water.

The resulting glatiramer is isolated as an acetate salt, after purification of the crude glatiramer base by a tangential flow filtration against water in step (iv). In a preferred embodiment, the reaction mass is acidified to pH 3 to 4, more preferably pH 3.5 to 3.7, with acid, preferably acetic acid. Preferably the glatiramer base is treated with acetic acid at a suitable temperature in presence of water. The excess acid is preferably removed from the aqueous solution by subjecting to the tangential flow filtration. The reaction mass is preferably subjected to tangential flow filtration against water until a pH 4 to 5 is obtained, more preferably pH 4.5. The tangential flow filtration in aqueous acetic acid not only removes traces of pyrrolidine acetate but also results in the glatiramer acetate salt until the average molecular weight reaches the required value.

(vi) lyophilization to obtain glatiramer acetate.

The aqueous solution of glatiramer acetate is then subjected to lyophilization to remove water. In lyophilization, the aqueous solution of glatiramer acetate is freeze dried under vacuum to remove water (ice) by sublimation without melting, to yield a glatiramer acetate salt product.

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The average molecular weight of the glatiramer acetate is determined by Gel filtration chromatography (GFC). In an embodiment, the glatiramer acetate has a molecular weight pattern in Daltons as shown in Table 1.

Molecular weight in Daltons lnitial(%) 26416 10.0% 16830 20.0% 13103 30.0% 10739 40.0% 8984 50.0% 7577 60.0% 6391 70.0% 5349 80.0% 4383 90.0% 3385 End(%) 1151 In a preferred embodiment of the present invention, the average molecular weight of the glatiramer acetate is in the range of 5 to 10 kDa.

The glatiramer acetate, prepared according to the process of the present invention may be formulated together with one or more pharmaceutically acceptable excipients.

The present invention is described with particular reference to the acetate salt of glatiramer.

However, other pharmaceutically acceptable salts of glatiramer may be prepared in accordance with the present invention as would be known by one of skill in the art.

The invention is explained in more detail in the following working examples. The examples, which illustrate improvement in the method according to the invention, have a purely illustrative character and do not limit the extent of the invention in any respect.

Example I

Process for preparation of diprotected polypeptide-1 N-carboxyanhydride of L-alanine (10 gms, 0.112 mole), N-carboxyanhydride of y-benzyl L-glutamate (5.29 gms, 0.035mo1e), N-carboxyanhydride of N-trifluroacetyl L-lysine (11.64 gms, 0.O79mole) and N-carboxyanhydride of y-benzyl L-tyrosine (3.88 grns, 0.021 mole) were charged in a dry flask containing 2.0 lit 1,4 Dioxane under Inert gas( Argon / Nitrogen) atmosphere in Glove box. The reaction was initiated by adding diethyl amine (2.3 ml, mole). The reaction mass was stirred at 25°C to 30°C for 24 Hrs & then quenched into 6.0 lit. D.l. Water.

The reaction mass was acidified to pH 4 to 5 with acetic acid. The stirring was continued for 30 minutes more. The solid was isolated by filtration, washed with 0.5 lit water and dried, to yield the diprotected polypeptide -1 Yield;-20.0 gms

Example 2

Preparation of monoprotected polypeptide -2 Diprotected polypeptide -1(10 gms) was charged in a flask under stirring followed by addition of 33% HBr in acetic acid (150 ml). The reaction mass was stirred at 24 to 26°C for 13 hours and quenched in Dl water in 2 to 3 hours maintaining temperature at 24 to 26°C and stirred for 1/2 hour. The solid was isolated by filtration, washed with Dl water till neutral pH and suck dried, to yield the monoprotected polypeptide-2.

Example 3

Preparation of glatiramer Monoprotected polypeptide-2 obtained in Example 2 was stirred in 500 ml Dl water, Pyrrolidine (1 ml) was charged and the reaction mass was stirred for 24 hours at 20 to 25°C. The reaction mass was filter over hyulo and washed with 10 ml Dl water.

Example 4

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Preparation and Purification of glatiramer acetate The solution of glatiramer obtained in example 3 was subjected to tangential flow filtration using Dl water. The reaction mass was acidified to pH 3.5 to 3.7 with Acetic acid. The reaction mass was then subjected to tangential flow filtration against water until a pH 4.5, was obtained. The reaction mass was filtered through 0.22 pm membrane filter and washed with Dl water. The reaction mass was lyophilized to obtain solid.

Yield;-4to4.5gms

Claims

IClaims: - 1. A process for preparing glatiramer base or a pharmaceutically acceptable salt thereof comprising: (i) polymerizing a mixture of N-carboxyanhydride of L-alanine, N-carboxyanhydride of y-benzyl L-glutamate, N-carboxyanhydride of y-benzyl L-tyrosine and N-carboxyanhydride of N-trifluroacetyl L-lysine, in a polar aprotic solvent in the presence of an initiator, to form a diprotected polypeptide-1, wherein the diprotected polypeptide-1 comprises terminal ends as amine or amide and acid; (ii) adding an acid to the diprotected polypeptide-1 to form monoprotected polypeptide-2, (iii) adding a base to the isolated polypeptide-2 to form glatiramer base; and optionally (iv) converting the glatiramer base to a pharmaceutically acceptable salt thereof.
2. A process according to claim 1 wherein in step (ii) the acid cleaves y-benzyl group from the glutamate moiety, y-benzyl group from tyrosine moiety and acidolytic peptide bond.
3. A process according to claim I or claim 2 wherein the initiator is a monoalkyl or dialkyl amine.
4. A process according to any one of the preceding claims wherein in step (iii) the base cleaves N-trifluoroacetyl group from L-lysine moiety.
5. A process according to any one of the preceding claims wherein step (iii) comprises isolating solid monoprotected polypeptide-2 by quenching in water followed by filtration.
6 A process according to any one of the preceding claims, optionally further comprising converting the glatiramer base to a pharmaceutically acceptable salt.
7. A process according to claim 6 wherein the conversion comprises reacting the base with acetic acid and the pharmaceutically acceptable salt s an acetate salt.I
8. A process for preparing glatiramer acetate comprising: preparing a glatiramer base according to the process of any one of claims 1 to 5; (v) purifying the glatiramer base; (vi) treatment of the glatiramer base with acetic acid to form acetate salt (vii) purification of acetate salt.
9. The process of claim 8 further comprising the step of: lyophillization, to obtain glatiramer acetate.
10. The process according to claim 8 or claim 9 wherein in step (v) the glatiramer base is purified by tangential flow filtration against water.
11. The process according to any one of claims 8 to 10 wherein in step (vii) the acetate salt is purified by tangential flow filtration against water.
12. The process according to any one of the preceding claims, wherein diprotected polypeptide- 1 is a straight chain polymer containing protected y-benzyl L-tyrosine moiety.
13. The process according to any one of the preceding claims, wherein monoprotected polypeptide-2 is free of brominated tyrosine impurity.
14. The process according to any one of the preceding claims, wherein the acid used in step (ii) for the deprotection is selected from the group comprising of acetic acid, hydrochloric acid, hydrogen bromide, hydrogen fluoride, phosphoric acid, sulfuric acid, trifluoroacetic acid, methane sulfonic acid, trifluoromethane sulfonic acid and mixtures thereof.
15. The process according to any one of the preceding claims, wherein the base used in step (iii) for the deprotection is selected from the group comprising of pyrrolidine, piperazine, morpholine, N-methyl-piperazine, Dicyclohexylamine, Di-sec-butylamine, diisopropylamine, dipropylamine, isopropylamine and aqueous methylamine and mixtures thereof.I
16. The process according to claim 15, wherein the base is pyrrolidine, preferably in aqueous solution.
17. The process according to claim 8, or any one of claims 9 to 16 when dependent on claim 8, wherein in step (v), glatiramer base is substantially free of undesired low molecular weight polypeptide & high molecular weight polypeptide.
18. The process according to claim 8, or any one of claims 9 to 17 when dependent on claim 8, wherein the glatiramer base prepared in step (v) has an average molecular weight from about 5 to about lOkDa.
19. The process according to claim 8, or any one of claims 9 to 18 when dependent on claim 8, wherein in step (v), glatiramer base is substantially free of monoprotected polypeptide-2.
20. The process according to claim 8, or any one of claims 9 to 19 when dependent on claim 8, wherein step (v) further comprises acidifying the reaction mass to pH 3 to 4.
21. The process according to claim 20 wherein the reaction mass is acidified using acetic acid.
22. The process according to claim 8, or any one of claims 9 to 21 when dependent on claim 8, wherein in step (vii) the reaction mass is subjected to tangential flow filtration against water until a pH of 4 to 5 is obtained.
23. The process according to claim 8, or any one of claims 9 to 22 when dependent on claim 8, wherein in step (vii), glatiramer acetate is substantially free of excess acetic acid and pyrrolidine acetate
24. The process according to claim 8, or any one of claims 9 to 23 when dependent on claim 8, wherein in step (vii), glatiramer acetate has improved colour characteristic.
25. The process according to claim 8, or any one of claims 9 to 24 when dependent on claim 8, wherein in step (vii) glatiramer acetate has average molecular weight of about 5 kDa to about 10 kDa.
26. The process according to claim 9, wherein in step (vii) glatiramer acetate has average molecular weight of about 5 kDa to about 10 kDa.
27. A process of preparing a glatiramer base substantially as herein described with particular reference to the examples.
28. A process of preparing glatiramer acetate substantially as herein described with particular reference to the examples.