WO2015044876A1

WO2015044876A1 - Molecular weight markers for protected polypeptides

Info

Publication number: WO2015044876A1
Application number: PCT/IB2014/064797
Authority: WO
Inventors: Srinivas Katkam; Sunil Kumar Gandavadi; Munaswamy Sekhar Nariyam; Ambaiah Boini; Ramesh Bochha; Vijay Kumar TIRUNAGARI; Yagna Kiran Kumar Komaravolu; Bala Harsha Vardhan Ganji; Lavanya KOLA
Original assignee: Dr. Reddy's Laboratories Limited
Priority date: 2013-09-25
Filing date: 2014-09-24
Publication date: 2015-04-02
Also published as: WO2015044876A8

Abstract

The present invention provides trifluoroacetyl copolymers useful as standard molecular weight markers in the intermediary stage for obtaining glatiramer acetate having desired average molecular weight. The present invention further provides a process for determining the average molecular weight of glatiramer acetate or other copolymers using molecular weight markers of protected polypeptides.

Description

MOLECULAR WEIGHT MARKERS FOR PROTECTED POLYPEPTIDES

INTRODUCTION

The present application relates to trifluoroacetyl copolymers useful as standard molecular weight markers in the intermediary stage for obtaining glatiramer acetate having desired average molecular weight.

A mixture of polypeptides which do not have the same amino acid sequence referred to as glatiramer acetate (GA) is marketed under the trade name COPAXONE® and comprises the acetate salts of polypeptides containing L-glutamic acid, L-alanine, L- tyrosine and L-lysine at average molar fractions of 0.141 , 0.427, 0.095 and 0.338, respectively. The average molecular weight of COPAXONE® is between 4000 and 1 1 ,000 daltons. Glatiramer acetate is sold in USA as Copaxone®, a registered trademark of Teva Pharmaceutical Industries Ltd. Glatiramer acetate is indicated for reduction of the frequency of relapses in patients with Relapsing-Remitting Multiple Sclerosis (RRMS). Chemically, glatiramer acetate is designated L-glutamic acid polymer with L-alanine, L-lysine, L-tyrosine, acetate (salt). Its structural formula is: (Glu, Ala, Lys, Tyr) _x'_XCH₃COOH

NOg) x'xC₂H₄O₂

Glatiramer acetate (Copolymer 1 ; Cop 1 ) is a mixture of polypeptides composed of alanine, glutamic acid, lysine, and tyrosine in a molar ratio of approximately 4.6:1 .5:3.6:1 .0, respectively, which is synthesized by chemically polymerizing the four amino acids, forming products with average molecular weights ranging from about 4000 to about 1 1 ,000 daltons. The corresponding molar fractions are approximately 0.427 for alanine, 0.141 for glutamic acid, 0.337 for lysine and 0.093 for tyrosine, and may vary by about +/-10%.

To certify a copolymer preparation for use in pharmaceutical products, it is necessary to obtain copolymer preparation with the desired average molecular weight or molecular weight distribution in the preparation. To get the desired average molecular weight of the copolymer in the final stage, it is necessary to control the quality of the protected polypeptides in the intermediary stages. This can be achieved if the process is established such that it leads to the intermediary protected polypeptides substantially with the desired average molecular weight or the molecular weight distribution. Towards achieving this objective, there is a need to provide molecular weight markers useful as standards in order to establish the right process conditions for obtaining the desired protected polypeptides substantially with the desired average molecular weight. The said desired protected polypeptides with the desired average molecular weight can be converted to polypeptides e.g., copolymers with the desired average molecular weights. The molecular weight markers of the said desired protected polypeptides have average molecular weights or molecular weight distribution and physical properties which can be analogous to the desired polypeptides.

Therefore, the objective of the present application is to provide plurality of molecular weight markers of the protected polypeptides, which can be useful as standards for obtaining the desired protected polypeptides substantially with the desired average molecular weights.

SUMMARY

In the first embodiment, the present application provides plurality of molecular weight markers comprising protected amino acids.

In the second embodiment, the present application provides a process for preparation of the plurality of molecular weight markers comprising protected amino acids, which comprises:

a) polymerizing a mixture of protected amino acids to form protected polypeptides, b) partially deprotecting the protected polypeptides obtained in step (a) with a reagent to obtain mixtures of protected polypeptides at different time intervals, c) obtaining plurality of molecular weight markers comprising protected amino acids.

In the third embodiment, the present application provides plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and protected lysine.

In the fourth embodiment, the present application provides a process for preparation of plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and protected lysine, which comprises:

a) polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-protected glutamic acid and protected lysine to obtain a mixture of protected polypeptides, b) partially deprotecting the protected polypeptides comprising tyrosine, alanine, γ- protected glutamic acid and protected lysine obtained in step (a) with a reagent to obtain a mixture of protected polypeptides comprising tyrosine, alanine, glutamic acid and protected lysine at different time intervals,

c) obtaining plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and protected lysine.

In the fifth embodiment, the present application provides plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and Ν^ε trifluoroacetyl lysine.

In the sixth embodiment, the present application provides a process for preparation of plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and Ν^ε trifluoroacetyl lysine, which comprises:

a) polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-protected glutamic acid and Ν^ε trifluoroacetyl lysine to obtain a mixture of protected polypeptides, b) partially deprotecting the protected polypeptides comprising tyrosine, alanine, γ- protected glutamic acid and Ν^ε trifluoroacetyl lysine obtained in step (a) with a reagent to obtain a mixture of protected polypeptides comprising tyrosine, alanine, glutamic acid and Ν^ε trifluoroacetyl lysine at different time intervals, c) obtaining plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and Ν^ε trifluoroacetyl lysine.

In the seventh embodiment, the present application provides the trifluoroacetyl copolymers useful as standard molecular weight markers in the intermediary stage for obtaining glatiramer acetate having desired average molecular weight.

In the eighth embodiment, the present application provides a process for obtaining glatiramer acetate with a desired peak average molecular weight, which comprises:

a) obtaining a mixture of protected polypeptides,

b) partially deprotecting the protected polypeptides obtained in step (a), wherein: i) a gel permeation chromatography column is calibrated with standard molecular weight markers of trifluoroacetyl glatiramer with a predetermined retention time,

ii) testing the reaction sample of deprotection in step (b) periodically until a correlation with the standard retention time is obtained,

iii) terminating the reaction of step (b) after correlation is established,

iv) isolating trifluoroacetyl glatiramer,

c) deprotecting the trifluoracetyl glatiramer, and

d) purifying the resulting polypeptides to obtain glatiramer acetate with a desired average molecular weight.

DEFINITIONS

The following definitions can be used in connection with the words or phrases used in the present application unless the context indicates otherwise.

The term "polypeptide" as used herein refers to any peptide comprising two or more amino acid residues connected by peptide linkage.

The term "amino acid" as used herein refers to an organic compound comprising at least one amino group and at least one acidic group. The amino acid may be a naturally occurring amino acid or be of synthetic origin, or an amino acid derivative or amino acid analog.

The term "protected amino acids" as used herein, refers to amino acids where functional groups in amino acids are derivatized with a suitable protecting group.

The term "protecting group" as used herein, refers to a group used to protect a certain functional group in a chemical synthesis and after the completion of a chemical reaction the said protecting group can be deblocked easily.

The term "protected polypeptide" as used herein refers to polypeptide wherein one or more of the amino acids therein are protected with one or more protecting groups.

The term "trifluoroacetyl copolymers" as used herein refers to trifluoroacetyl glatiramer. The term "marker" as used herein refers to a compound used as reference standard that can be used to characterize a batch sample.

The term "molecular weight marker" as used herein refers to the standards that are used to identify the approximate size or molecular weight of a molecule run on a size exclusion chromatography system.

The term "about" as used in the present application preceding a number and referring to it, means any value which lies within a reasonable range around that number mentioned therein and which can be easily derived and understood by a person skilled in the art.

"As used herein, except where the context requires otherwise, the term

"comprise" and variations of the term, such as "comprising", "comprises" and "comprised of", are not intended to exclude other additives, components, integers or steps."

DETAILED DESCRIPTION

The present application provides protected polypeptides having randomly arranged amino acids, which may be useful as molecular weight markers for determining the peak average molecular weight of polypeptides. Therefore, the protected polypeptides of the present application can be used as molecular weight markers for determining the peak average molecular weight of the desired polypeptides which do not have predetermined or fixed sequence of amino acids.

a) polymerizing a mixture of protected amino acids to obtain protected polypeptides, b) partially deprotecting the protected polypeptides obtained in step (a) with a reagent to obtain mixtures of protected polypeptides at different time intervals, c) obtaining plurality of molecular weight markers comprising protected amino acids.

In step (a) the protected amino acids undergo random polymerization to give protected polypeptides. The resulting protected polypeptides have randomly arranged protected amino acids rather than having protected amino acids arranged in a predetermined or a fixed sequence. The said polymerization may be carried out by processes known in the art. For example, the said polymerization may be carried out by a process similar to that known in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

In step (b), the protected polypeptides obtained in step (a) can be partially deprotected with a reagent to afford protected polypeptides. The said partial deprotection may be carried out by processes that may be suitable for the deprotection of the corresponding protecting group or groups, which can be easily derivable from the art by skilled persons. For example, a suitable reagent for deprotection includes the reagent for similar deprotection disclosed in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

Samples of the reaction mixture from the deprotection reaction in step (b) can be isolated at different time intervals.

Step (c) involves obtaining plurality of molecular weight markers comprising protected amino acids.

The samples isolated at different time intervals in step (b) can be quenched with water and optionally can be subjected to further conventional work up procedures describe herein to obtain samples containing mixtures of protected polypeptides having different molecular weights. Partial deprotection in step (b) optionally results in partially deprotected polypeptides, which still carry protecting groups thereby resulting in protected polypeptides that can be different from the protected polypeptides obtained in step (a). Peak average molecular weights and the corresponding retention time of the isolated molecular weight markers can be obtained for the different samples isolated at different time intervals by using techniques such as GPC (gel permeation chromatography), thereby obtaining plurality of molecular weight markers comprising protected amino acids.

In the third embodiment, the present application provides plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and protected lysine. In the fourth embodiment, the present application provides a process for preparation of plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and protected lysine, which comprises:

a) polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-protected glutamic acid and protected lysine to obtain a mixture of protected polypeptides,

b) partially deprotecting the protected polypeptides comprising tyrosine, alanine, γ- protected glutamic acid and protected lysine obtained in step (a) with a reagent to obtain mixtures of protected polypeptides comprising tyrosine, alanine, glutamic acid and protected lysine at different time intervals,

In step (a) the N-carboxyanhydrides of tyrosine, alanine, γ-protected glutamic acid and protected lysine undergo random polymerization to give protected polypeptides. The resulting protected polypeptides comprising tyrosine, alanine, γ- protected glutamic acid and protected lysine have randomly arranged protected amino acids rather than having protected amino acids arranged in a predetermined or a fixed sequence. The said polymerization may be carried out by processes known in the art. For example, the said polymerization may be carried out by a process known in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

In step (b), the protected polypeptides comprising tyrosine, alanine, γ-protected glutamic acid and protected lysine obtained in step (a) may be partially deprotected with a reagent to afford protected polypeptides comprising tyrosine, alanine, glutamic acid and protected lysine. The said partial deprotection may be carried out by processes that can be suitable for the deprotection of the corresponding protecting group or groups, which can be easily derivable from the art by skilled persons. For example, a suitable reagent for deprotection includes the reagent for similar deprotection disclosed in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

Samples of the reaction mixture from the deprotection reaction in step (b) can be isolated at different time intervals. Step (c) involves obtaining plurality of molecular weight markers comprising tyrosine, alanine, γ-protected glutamic acid and protected lysine.

The samples isolated at different time intervals in step (b) can be quenched with water and optionally can be subjected to further conventional work up procedures described herein to obtain samples containing mixtures of protected polypeptides having different molecular weights. Partial deprotection in step (b) optionally results in partially deprotected polypeptides, which still carry protecting groups thereby resulting in protected polypeptides that can be different from the protected polypeptides obtained in step (a). Peak average molecular weights and the corresponding retention time of the isolated molecular weight markers can be obtained for the different samples isolated at different time intervals by using techniques such as GPC (gel permeation chromatography), thereby obtaining plurality of molecular weight markers comprising protected amino acids.

a) polymerizing N-carboxyanhydrides of tyrosine, alanine, γ-protected glutamic acid and Ν^ε trifluoroacetyl lysine to obtain a mixture of protected polypeptides, b) partially deprotecting the protected polypeptides comprising tyrosine, alanine, γ- protected glutamic acid and Ν^ε trifluoroacetyl lysine obtained in step (a) with a reagent to obtain mixtures of protected polypeptides comprising tyrosine, alanine, glutamic acid and Ν^ε trifluoroacetyl lysine at different time intervals,

c) obtaining plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and Ν^ε trifluoroacetyl lysine.

In step (a) the N-carboxyanhydrides of tyrosine, alanine, γ-protected glutamic acid and Ν^ε trifluoroacetyl lysine undergo random polymerization to give protected polypeptides. The resulting protected polypeptides comprising tyrosine, alanine, γ- protected glutamic acid and Ν^ε trifluoroacetyl lysine have randomly arranged protected amino acids rather than having protected amino acids arranged in a predetermined or a fixed sequence. The said polymerization may be carried out by processes known in the art. For example, the said polymerization may be carried out by a process known in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

In step (b), the protected polypeptides comprising tyrosine, alanine, γ-protected glutamic acid and Ν^ε trifluoroacetyl lysine obtained in step (a) may be partially deprotected with a reagent to afford protected polypeptides comprising tyrosine, alanine, glutamic acid and Ν^ε trifluoroacetyl lysine. The said partial deprotection can be carried out by processes that may be suitable for the deprotection of the corresponding protecting group or groups, which can be easily derivable from the art by skilled persons. For example, a suitable reagent for deprotection includes the reagent for similar deprotection disclosed in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

Step (c) involves obtaining plurality of molecular weight markers comprising tyrosine, glutamic acid, alanine and Ν^ε trifluoroacetyl lysine.

The samples isolated at different time intervals in step (b) can be quenched with water. After stirring for few minutes, the separated solid can be filtered and washed with water. The wet compound can be washed with 10% sodium thiosulfate solution (Na₂S₂O₃ 5H₂O) to obtain samples containing mixtures of protected polypeptides having different molecular weights. Partial deprotection in step (b) optionally results in partially deprotected polypeptides, which still carry protecting groups thereby resulting in protected polypeptides that can be different from the protected polypeptides obtained in step (a). Peak average molecular weights and the corresponding retention time of the isolated molecular weight markers can be obtained for the different samples isolated at different time intervals by using techniques such as GPC (gel permeation chromatography), thereby obtaining plurality of molecular weight markers comprising protected amino acids.

The protected polypeptides that may be useful as molecular markers comprising tyrosine, alanine, glutamic acid and Ν^ε trifluoroacetyl lysine obtained above have defined molar ratios of amino acids. The molar ratios of the amino acids of the protected polypeptides can be similar to mole ratios of amino acids found in the corresponding polypeptide for e.g. copolymer, which may be obtained from the said protected polypeptides. Such a correspondence in molar ratios provides the best molecular weight markers because these markers can have a charge and a molecular shape which is similar to that of the corresponding polypeptide. When structurally dissimilar markers are used, the markers may migrate or elute somewhat differently from the corresponding polypeptide preparations in the chromatographic system, even though those preparations have the same molecular weights as that of markers.

In the seventh embodiment, the present application provides trifluoroacetyl copolymers useful as standard molecular weight markers in the intermediary stage for obtaining glatiramer acetate having desired average molecular weight.

Usefulness of the trifluoroacetyl copolymers as standard molecular weight markers in the intermediary stage in the process of the present application can be understood from the fact that the said trifluoroacetyl copolymers comprise alanine, tyrosine, glutamic acid and Ν^ε trifluoroacetyl lysine are isolated according to the process of the present application and are considered to have desired average molecular weights because the corresponding copolymers e.g., glatiramer acetate obtained from these trifluoroacetyl copolymers in the process of the present application have the desired average molecular weight in the range of about 2000 daltons to about 9000 daltons or preferably between about 4000 to about 8000 daltons or more preferably between about 6000 to about 8000 daltons.

In an aspect of the present application, the trifluoroacetyl copolymers have a predetermined retention time on a molecular sizing column. In an aspect of the present application, the molecular sizing column can be selected from shodex GPC column, KF-603 150 x 6.0mm x 3 m and a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 4 minutes to about 6 minutes on a shodex GPC column, KF- 603 150 x 6.0mm x 3 m.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 4.871 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 4.902 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 4.934 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 5.002 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 5.057 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 30.0 minutes to about 32.0 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 30.732 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 31 .045 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228. In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 31 .225 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 31 .475 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In an aspect of the present application, the molecular weight markers are eluted at a retention time of about 31 .558 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In the eighth embodiment, the present application provides a process for obtaining glatiramer acetate with a desired peak average molecular weight, which comprises: a) obtaining a mixture of protected polypeptides,

iii) terminating the reaction of step (b) after correlation is established, iv) isolating trifluoroacetyl glatiramer,

c) deprotecting the trifluoracetyl glatiramer, and

In a process for obtaining a mixture of protected polypeptides in step (a), the N- carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamic acid and Ν^ε trifluoroacetyl lysine undergo random polymerization to give protected polypeptides. The resulting protected polypeptides comprise tyrosine, alanine, γ-benzyl glutamic acid and Ν^ε trifluoroacetyl lysine which are randomly arranged rather than in a predetermined or a fixed sequence. The said polymerization may be carried out by processes known in the art. For example, the said polymerization may be carried out by a process known in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

Step (b) of the process comprises partially deprotecting the protected polypeptides obtained in step (a), wherein:

i) a gel permeation chromatography column is calibrated with standard molecular weight markers of trifluoroacetyl glatiramer with a predetermined retention time, ii) testing the reaction sample of deprotection in step (b) periodically until a correlation with the standard retention time is obtained,

iii) terminating the reaction of step (b) after correlation is established,

iv) isolating trifluoroacetyl glatiramer,

The protected polypeptides obtained in step (a) are treated with a suitable reagent for deprotection including the reagent for similar deprotection reaction disclosed in the PCT publication WO 201 1/139752, which is incorporated herein by reference in its entirety.

Suitable gel permeation chromatography column as used herein includes Shodex

GPC columns, Styragel columns, sephadex columns, sepharose columns, superpose columns and ultra-hydrogel columns or any other suitable column. Preferably the process of the present application involves the use of shodex GPC column, KF-603 150 x 6.0 mmx 3 m or a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228. These columns are calibrated using standard molecular weight markers of trifluoroacetyl glatiramer with a predetermined retention time.

Suitable gel permeation chromatography include size exclusion chromatography using HPLC, UPLC (ultrahigh pressure liquid chromatography), RP HPLC (reverse phase high performance liquid chromatography) or any other well known in the art.

In one variant, when shodex GPC column, KF-603 150 x 6.0 mm x 3 m is used, the total run time of the reaction sample is about 15 minutes. In another variant, when a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228 is used, the total run time of the reaction sample is about 70 minutes.

The reaction mixture in step (b) is periodically tested by eluting trifluoroacetyl glatiramer (or trifluroroacetyl copolymers) test samples on a suitable GPC column. The said trifluoroacetyl glatiramer test samples can be prepared by taking out samples of deprotection reaction in step (b) and isolating trifluoroacetyl glatiramer by the processes well known in the art e.g., PCT publication WO 201 1/139752. Before analyzing triflouroacetyl glatiramer test samples so prepared by eluting on a gel permeation chromatography column, the said GPC column is calibrated using standard molecular weight markers of trifluoroacetyl glatiramer isolated in the similar way as described herein above. After such calibration with the standard molecular weight markers of trifluoroacetyl glatiramer, the test samples of trifluoroacetyl glatiramer of deprotection reaction in step (b) are eluted on the said GPC column. If the retention time of the reaction sample correlates with the predetermined retention time of standard molecular weight markers of trifluoroacetyl glatiramer, deprotection reaction is terminated and the trifluoroacetyl glatiramer is isolated by the processes as described herein above.

The trifluoroacetyl glatiramer obtained in step (b) is further deprotected and purified to obtain glatiramer acetate with a desired peak average molecular weight of about 2000 to 9000 daltons or preferably about 4000 to about 8000 daltons or more preferably about 6000 to about 8000 daltons.

In an aspect of present application, the predetermined retention time is about 4 minutes to about 6 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3 m.

In an aspect of present application, the predetermined retention time is about 30.0 minutes to about 32.0 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

In an aspect of the present application, calibration of the gel permeation chromatography column using standard molecular weight markers is carried out to establish a linear relationship between retention times of trifluoroacetyl glatiramer and peak average molecular weight of glatiramer acetate.

Markers having different average molecular weights elute at different retention time. Therefore, the retention time is a function of average molecular weight of a marker. There is a linear relationship between the average molecular weight and retention time on a molecular sizing column. As the retention time increases the molecular weight decreases on a molecular sizing column. The standard trifluoroacetyl glatiramer markers obtained in the process of the present application may be treated with 1 M aqueous piperidine for removal of trifluoroacetyl group from the lysine residue, subjected to 1 kd dialysis membrane to remove low molecular weight species and lyophilized. In one variant, the average molecular weight of the resulting glatiramer acetate ranges from about 6000 to about 8000 daltons.

Therefore, the trifluoroacetyl copolymers isolated in the process of the present application may be advantageously used as the standard molecular weight markers in the intermediary stage so as the obtain glatiramer acetate with the desired average molecular weight in the final stage.

The standard molecular weight markers, in turn the process of obtaining glatiramer acetate with the desired average molecular weight using the said standard markers make the process of the present application simple and provide consistence in the quality of glatiramer acetate and hence, is advantageous on a commercial scale.

In the present application, one of skill in the art can readily substitute structurally- related and/or charge-related amino acids without deviating from the spirit of the invention to obtain the corresponding protected polypeptides useful as molecular weight markers based on the need. Thus, the present application further contemplates conservative amino acid substitutions for example for tyrosine, glutamic acid, alanine and lysine in the present application. Such conservative substitutions may be structurally-related amino acid substitutions, including those amino acids which have about the same charge, hydrophobicity and size as tyrosine, glutamic acid, alanine or lysine. For example, lysine is structurally-related to arginine and histidine; glutamic acid is structurally-related to aspartic acid; tyrosine is structurally-related to serine, threonine, phenylalanine and tryptophan; and alanine is structurally-related to valine, leucine and isoleucine. These and other conservative substitutions, such as structurally-related synthetic amino acids, can also contemplated by the present application.

There is a linear relationship between the average molecular weights of the markers and a measurable physical property such as, for example, the molar ellipticity of the markers or the retention time of the markers on a chromatographic column. The protected polypeptides useful as molecular weight markers of the present application have an amino acid composition identical to that of corresponding polypeptides and have molecular weights such that the protected polypeptides useful as molecular weight markers of the present application have molecular weights in a range so that the corresponding polypeptides obtained from the said molecular weight markers can have molecular weights for example in a range of about 1 ,000 Daltons and about 40,000 Daltons or about 2,000 Daltons and about 20,000 Daltons or about 3,000 Daltons and about 15,000 Daltons or about 4,000 Daltons and about 10,000 Daltons or about 5000 daltons and about 9000 daltons or about 6000 daltons and about 8000 daltons or any other desired molecular weights. Molecular weight markers that have chemical and physical characteristics similar to the desired protected polypeptides provide an accurate and robust means of determining the average molecular weights of production batches of the desired polypeptides.

The present application provides derivatives of protected polypeptides e.g., trifluoroacetyl copolymers useful as molecular weight markers for determining the average molecular weight of the polypeptides. It follows from the requirement for an identified average molecular weight that a particular molecular weight marker should not be highly polydispersed and also should have a narrow molecular weight distribution.

Any suitable number of markers may be useful in determining the average molecular weight of the desired protected polypeptides and in turn polypeptides can be isolated. These markers can be used in any molecular size discrimination system using any available molecular weight determination procedure or apparatus. For example, the protected polypeptides useful as molecular weight markers of the present application can be used as standards for the determination of average molecular weight of protected polypeptides or proteins. Such an apparatus useful for the determination of average molecular weight can be any chromatographic technique like GPC (gel permeation chromatography), MALLS (multiple angle laser light scattering) or any other suitable technique. Such chromatographic apparatus can have a molecular weight sizing column which separates protected polypeptides on the basis of their size. Examples of molecular weight sizing columns include Shodex GPC columns, Styragel columns, Sephadex columns, Sepharose columns, superose and ultra-hydrogel columns or any other suitable column.

The plurality of molecular weight markers of the present application generally comprises two or more protected polypeptides. In one variant, plurality molecular weight markers isolated at different time intervals in the process of the present application maybe injected in to a gel permeation chromatography system. Markers having different average molecular weights elute at different retention times. Therefore, the retention time is a function of average molecular weight of a marker. Each time when peak average molecular weight of a sample of a protected polypeptide is to be determined, the plurality of standard molecular weight markers, which have predetermined retention times can be injected in any molecular size discrimination system and correlate the retention time of the sample of protected polypeptide with the retention times of the plurality of standard molecular weight markers. By doing so, we can establish a linear relationship between retention time and the peak average molecular weight of protected polypeptides. With this we can establish whether the desired protected polypeptide is falling within the desired molecular weight range or not thereby establishing the right reaction conditions such as time, temperature, quality and quantity of reagent and so on for the deprotection reaction so as to always result in the protected polypeptide within the desired peak average molecular weight with high yield and quality.

In order to establish the correspondence of the right molecular weight marker to the desired protected polypeptide having the desired average molecular weight range in the deprotection stage i.e., step (b) of the process of the present application, the different isolated molecular weight markers may be subjected to complete deprotection with a reagent capable of completely removing the protecting groups on the amino acids thereby obtaining the corresponding polypeptides. The average molecular weights of these polypeptides can be established by using known molecular size discrimination system and thereby finding out which particular polypeptide has the average molecular weight within the desired range. The corresponding molecular weight marker, from which the polypeptide with the desired average molecular weight range is obtained, can be the standard marker of the protected polypeptide in the previous stage for obtaining the desired protected polypeptide within the desired average molecular weight range.

The protected polypeptide having desired average molecular weight obtained in the process of the present application by correlating with molecular weight markers based on retention time can be converted to the polypeptide for e.g., copolymer-1 , which has the desired average molecular weight.

In one variant, for determining the molecular weight range of desired protected polypeptides, preferred plurality of molecular weight markers elute at retention time of about 4.0 to about 6.0 minutes or about 30.0 to about 32.0 minutes on a molecular sizing column. There is a linear relationship between the molecular weight and retention time of the molecular weight marker on a molecular sizing column. As the retention time increases the molecular weight decreases.

In one variant, the different molecular weight markers isolated at different time intervals in the process of sixth embodiment of the present application may be subjected to complete deprotection using a reagent capable of completely removing the protecting groups on the amino acids such as 1 M piperidine and purified to get polypeptides including copolymer-1 . The average molecular weights of these polypeptides are generally within the range of about 2000 to about 9000 daltons or preferably between 4000 to 8000 daltons or more preferably between 6000 to 8000 daltons. Optionally, the polypeptides have the peak average molecular weight within the range of 6000 to 8000 daltons can be considered as the desired polypeptides and hence, the corresponding molecular weight markers of the protected polypeptides from which such polypeptides are obtained are the desired protected polypeptides. Therefore, the molecular weight markers isolated at different time intervals in the process of the sixth embodiment of the present application can be useful as standards to determine the average molecular weights of the desired protected polypeptides at the intermediary stage. These markers and the desired protected polypeptides can be analyzed using liquid chromatography equipped with UV detector at 278-nm using shorter column, for example Shodex GPC column, KF-603 150 X 6.0 mm X3um or an equivalent at a column temperature of 35°C with a run time of 15 min. Alternatively the markers and desired protected polypeptides can also be analyzed using liquid chromatography equipped with UV detector at 278-nm using longer column, for example styragel column connected in series, consisting of 3 columns with part no's WAT044234, WAT044222, WAT044228 at a column temperature of 40°C with a run time of 70 min. Reaction time, retention times of the illustrative molecular weight markers obtained in the process of sixth embodiment of the present application and peak average molecular weights of the corresponding polypeptides obtained from the said markers are listed in the table below.

The isolated samples of markers obtained in the process of sixth embodiment of the present application may be treated with 1 M aqueous piperidine for removal of trifluoroacetyl group from the lysine residue, subjected to 1 kd dialysis membrane to remove low molecular weight species and Lyophilized. The peak average molecular weight of these polypeptides ranges from 6000-8000 daltons. These markers may be considered as the standard markers of intermediary protected polypeptides for determining the substantial molecular weight of the polypeptides. Though the markers meet the general specification of 5000 - 9000 daltons of the polypeptide i.e. copolymer

1 the protected polypeptide markers with a retention time of 4.871 , 4.902, 4.934, 5.002,

5.057 may be considered as the standard marker of protected polypeptide as the average molecular weight of these polypeptides can be within the range of 6000-8000 daltons which is the desired peak average molecular weight to meet the molecular weight in terms of sameness characterization. The markers M1 , M2, M3, M4 and M5 also meet the general specification of 5000 - 9000 daltons more preferably within the range of 6000-8000 daltons.

To get the desired average molecular weight of the polypeptide, the final product, which is obtained from the corresponding protected polypeptide, it is necessary to control the quality of the protected polypeptides in the intermediary stages itself. This can be achieved if the process is established such that it leads to the desired protected polypeptides substantially with the desired average molecular weight or the molecular weight distribution. Average molecular weight and molecular weight distribution is a key parameter in dictating the API profile in terms of physiochemical properties and biological sameness. To achieve this, monitoring the intermediary stage that is deprotection reaction with respect to average molecular weight through retention time is an important aspect of the process for the preparation of the polypeptide as the final product. Therefore, the molecular weight markers of the present application may be useful as standards in the intermediary stage in order to get the desired molecular weight of the objective polypeptide. In the absence of such molecular weight markers, the quality of the polypeptide can be controlled only by fixing the reaction conditions by conventional trial and error methods and by tedious purification processes.

Not only the reaction conditions, quality of the reagents, engineering parameters involving geometrical similarity like heat transfer area, reactor design, RPM, design of impeller, mass or volume occupancy also play a crucial role in determining the quality of protected polypeptides. Therefore in order to overcome variations in such critical parameters, a robust process can be developed to get the desired average molecular weight by using the molecular weight markers as standards in the intermediary stage. This deprotection reaction also dictates the quality of the polypeptide e.g., copolymer-1 batch that would be acceptable for inclusion in a pharmaceutical composition. If the retention time of protected polypeptide of the production batch is not within the range of the retention time of standard marker(s), the corresponding polypeptide would not be obtained with an acceptable quality in terms of average molecular weight for inclusion in a pharmaceutical composition because average molecular weight and molecular weight distribution is an important parameter in determining the polypeptide profile in terms of physicochemical properties and biological sameness.

Certain specific aspects and embodiments will be further explained by the following examples, being provided only for the purposes of illustration and not to be construed as limiting the scope of the application in any manner.

EXAMPLES

Example 1

Step 1 : Preparation of protected polypeptide.

An N-carboxyanhydride of L-alanine (108.48 g), a N-carboxyanhydride of L- tyrosine (39.52 g), a N-carboxyanhydride of Ν^ε trifluoroacetyl lysine (182.8 g) and a N- carboxyanhydride of γ-benzyl-L-glutamate (79.12g) are charged into a round bottom flask under a nitrogen atmosphere. 1 , 4-Dioxane (7.68 L) is added at 25- 30°C and the mixture is stirred for 15 minutes. Diethyl amine (2.88 ml) is added at 25-30°C and the mixture is stirred at the same temperature for 24 hours. The mixture is poured slowly into water (20.0 L) and the mass is stirred at 25-30°C for 30 minutes. The solid is collected by filtration, washed with water (10 L) and dried under reduced pressure at 25- 35°C to afford 260 g of a protected glatiramer.

Step 2: Preparation of protected polypeptide (trifluoroacetyl glatiramer).

The protected glatiramer from Example-1 (50.0 g) is charged into a round bottom flask at 33°C. A pre-mixed solution of 57% of HI and H₃P0₂ (250 ml) in acetic acid (500 ml) is added and the mixture is stirred at 30-35°C for 22 h. After 17 h every 1 h, 150 ml of the reaction mixture is taken out from the reaction mass and the solid is isolated by adding the reaction mixture to water (1000ml) and the mass is stirred for 15 minutes. The solid is filtered and washed with water (400 ml) to give brown-color compound. The wet compound is washed with 10% sodium thiosulfate solution (Na₂S₂0₃5H₂0) (500 ml) to give white compound. The white compound obtained is slurried with 10% sodium thiosulfate solution (Na₂S₂0₃5H₂0) (500 ml) and stirred for 15 min. It is filtered washed with water (100ml x3) and finally washed with hexane (500 ml) and dried at 25-30°C under reduced pressure to afford 5.4 g of trifluoroacetyl glatiramer for 18 h, 5.4 g of trifluoroacetyl glatiramer for 19 h, 5.4 g of trifluoroacetyl glatiramer for 20 h, 5.1 g of trifluoroacetyl glatiramer for 21 h, and 5.0 g of trifluoroacetyl glatiramer for 22 h

Step 3: Preparation of polypeptides (glatiramer acetate).

Trifluoroacetyl glatiramer at 20 h of above step 2 (2.0 g), piperidine (1 1 ml_), and water (100 mL) are charged into a round bottom flask. The mixture is stirred at 25-35°C for 24 hours, then is subjected to diafiltration using a 1 KDa molecular weight cutoff membrane against ammonium acetate buffer (pH 5.5±0.3) in a stepwise mode of operation, until the pH of the permeate reaches 5.5-6.5. The retentate solution is circulated with 0.3% acetic acid until pH reaches 4.5-4.6 and is diafiltered against water to remove excess acetic acid, until the pH of the retentate reaches 4.8-4.9. The diafiltered sample is then concentrated through a 3 KDa molecular weight cutoff membrane and the concentrated solution is lyophilized to afford 1750 mg of glatiramer acetate.

Peak average molecular weight of polypeptides (glatiramer acetate) by GPC: 7092 Daltons.

Similarly all the samples of trifluoroacetyl glatiramer of step 2 isolated at reaction times from 18 hours to 22 hours are deprotected using aqueous piperidine, subjected to diafiltration as described above and lyophilized to afford Glatiramer acetate. The retention time of the trifluoroacetyl glatiramer and the corresponding Average molecular weight after deprotection are listed below.

3 20 31 .225 7092

4 21 31 .475 6949

5 22 31 .558 6552

EXAMPLE 2

Step 1 : Preparation of protected polypeptide:

A N-carboxyanhydride of L-alanine (108.48 g), a N-carboxyanhydride of L- tyrosine (39.52 g), a N-carboxyanhydride of Ν^ε trifluoroacetyl lysine (182.8 g) and a N- carboxyanhydride of γ-benzyl-L-glutamate (79.12g) are charged into a round bottom flask under a nitrogen atmosphere. 1 , 4-Dioxane (7.68 L) is added at 25- 30°C and the mixture is stirred for 15 minutes. Diethyl amine (2.88 ml) is added at 25-30°C and the mixture is stirred at the same temperature for 24 hours. The mixture is poured slowly into water (20.0 L) and the mass is stirred at 25-30°C for 30 minutes. The solid is collected by filtration, washed with water (10 L) and dried under reduced pressure at 25- 35°C to afford 260 g of a protected glatiramer.

Step 2: Preparation of protected polypeptides (trifluoroacetyl glatiramer) by correlation with standard isolated molecular weight markers of trifluoroacetyl glatiramer:

The protected glatiramer from Example-1 (50.0 g) is charged into a round bottom flask at 33°C. A pre-mixed solution of 57% of HI and H₃P0₂ (250 ml) in acetic acid (500 ml) is added and the mixture is stirred at 30-35°C for 22 h. After 12 h every 1 h, 15 ml of the reaction mixture is taken out from the reaction mass and the solid is isolated by adding the reaction mixture to water (100ml) and the mass is stirred for 15 minutes. The solid is filtered and washed with water (40 ml) to give brown-color compound. The wet compound is washed with 10% sodium thiosulfate solution (Na₂S₂0₃5H₂0) (50 ml) to give white compound. The white compound obtained is slurried with 10% sodium thiosulfate solution (Na₂S₂0₃5H₂0) (50 ml) and stirred for 15 min. It is filtered washed with water (10ml x3) and finally washed with hexane (50 ml) and dried at 25-30°C under reduced pressure to afford trifluoroacetyl glatiramer. S.No Time (h) Retention time of the marker whose Retention

corresponding polypeptide that is obtained after time of complete deprotection has a peak average protected molecular weight of 7000 daltons polypeptide

1 12 31 .345 29.66

2 13 30.40

3 14 30.76

4 15 30.37

5 16 30.85

6 17 31 .40

7 18 31 .31

Step 3: Preparation of polypeptides (glatiramer acetate):

Trifluoroacetyl glatiramer at 12 h of above step 2 (2.0 g), piperidine (1 1 mL), and water (100 mL) are charged into a round bottom flask. The mixture is stirred at 25-35°C for 24 hours, then is subjected to diafiltration using a 1 KDa molecular weight cutoff membrane against ammonium acetate buffer (pH 5.5±0.3) in a stepwise mode of operation, until the pH of the permeate reaches 5.5-6.5. The retentate solution is circulated with 0.3% acetic acid until pH reaches 4.5-4.6 and is diafiltered against water to remove excess acetic acid, until the pH of the retentate reaches 4.8-4.9. The diafiltered sample is then concentrated through a 3 KDa molecular weight cutoff membrane and the concentrated solution is lyophilized to afford glatiramer acetate.

Similarly all the solids obtained from 12h to 18h trifluoroacetyl glatiramer of step 2 are deprotected using aqueous piperidine, subjected to diafiltration as above and lyophilized to afford Glatiramer acetate. The retention time of the trifluoroacetyl glatiramer with respect to the standard retention time and the corresponding Average molecular weight after deprotection are listed below.

whose corresponding time of molecular polypeptide that is obtained protected weight of after complete deprotection polypeptide polypeptides has a peak average molecular after complete weight of 7000 daltons deprotection

12 31 .345 29.66 9675

13 30.40 9240

14 30.76 8840

15 30.37 8341

16 30.85 81 1 1

17 31 .40 8015

18 31 .31 7261

Claims

Claims:

1 . The trifluoroacetyl copolymers useful as standard molecular weight markers in the intermediary stage for obtaining glatiramer acetate having desired average molecular weight.

2. The molecular weight markers of claim 1 with a predetermined retention time on a molecular sizing column.

3. The molecular weight markers of claim 2 wherein the molecular sizing column is selected from a shodex GPC column, KF-603 150 x 6.0mm x 3 m and a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

4. The molecular weight markers of claim 2 which are eluted at a retention time of about 4 minutes to about 6 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3 m.

5. The molecular weight markers of claim 4 which are eluted at a retention time of about 4.871 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3 m.

6. The molecular weight markers of claim 4 which are eluted at a retention time of about 4.902 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3 m.

7. The molecular weight markers of claim 4 which are eluted at a retention time of about 4.934 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

8. The molecular weight markers of claim 4 which are eluted at a retention time of about 5.002 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3pm.

9. The molecular weight markers of claim 4 which are eluted at a retention time of about 5.057 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3 m.

10. The molecular weight markers of claim 2 which are eluted at a retention time of about 30.0 minutes to about 32.0 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

1 1 . The molecular weight markers of claim 10 which are eluted at a retention time of about 30.732 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

12. The molecular weight markers of claim 10 which are eluted at a retention time of about 31.045 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

13. The molecular weight markers of claim 10 which are eluted at a retention time of about 31.225 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

14. The molecular weight markers of claim 10 which are eluted at a retention time of about 31.475 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228

15. The molecular weight markers of claim 10 which are eluted at a retention time of about 31.558 minutes on a styragel column connected in series with part no's

WAT044234, WAT044222 and WAT044228.

16. A process for obtaining glatiramer acetate with a desired peak average molecular weight, which comprises:

a) obtaining a mixture of protected polypeptides, b) partially deprotecting the protected polypeptides obtained in step (a), wherein: i) a gel permeation chromatography column is calibrated with standard molecular weight markers of trifluoroacetyl glatiramer with a predetermined retention time,

iii) terminating the reaction of step (b) after correlation is established,

iv) isolating trifluoroacetyl glatiramer,

c) deprotecting the trifluoracetyl glatiramer, and

17. A process of claim 18, wherein the glatiramer acetate containing a mixture of polypeptides, each of which essentially consist of alanine, tyrosine, glutamic acid and lysine, has a peak average molecular weight of about 6000 to 8000 daltons.

18. A process of claim 18, wherein the predetermined retention time is about 4 minutes to about 6 minutes on a shodex GPC column, KF-603 150 x 6.0mm x 3 m

19. A process of claim 18, wherein the predetermined retention time is about 30.0 minutes to about 32.0 minutes on a styragel column connected in series with part no's WAT044234, WAT044222 and WAT044228.

20. A process of claim 18, wherein calibration of the gel permeation chromatography column using standard molecular weight markers is carried out to establish a linear relationship between retention time of trifluoroacetyl glatiramer and peak average molecular weight of polypeptide.