WO2011071799A2

WO2011071799A2 - Purification of bivalirudin

Info

Publication number: WO2011071799A2
Application number: PCT/US2010/059045
Authority: WO
Inventors: Pratap Reddy Padi; Santhana Krishnan Srinivasan; Venugopal Rao Dama; Basanthi Devi; Chakravarthula Kalyan Narasimham Nalla; Ramesh Bochha; Parameswara Reddy Koche; Yagna Kiran Kumar Komaravolu
Original assignee: Dr. Reddy's Laboratories Ltd.; Dr. Reddy's Laboratories, Inc.
Priority date: 2009-12-11
Filing date: 2010-12-06
Publication date: 2011-06-16
Also published as: WO2011071799A3

Abstract

Provided are processes for the purification of bivalirudin. Also provided is substantially pure bivalirudin.

Description

DULI\ o.t-^tut)

PURIFICATION OF BIVALIRUDIN

INTRODUCTION

Aspects of the present application relate to processes for the purifying bivalirudin. Further aspects of the application relate to substantially pure

bivalirudin.

Hirudin, a 65-amino acid polypeptide, is a potent thrombin inhibitor, naturally occurring in the salivary glands of medicinal leeches. Bivalirudin, also known as hirulog-8, is a synthetic peptide based on hirudin and is a 20-amino acid polypeptide. It has a chemical name D-phenylalanyl-L-prolyl-L-arginyl-L-prolyl- glycyl-glycyl-glycyl-glycyl-L-asparagyl-glycyl-L-aspartyl-L-phenylalanyl-L-glutamyl- L-glutamyl-L-isoleucyl-L-prolyl-L-glutamyl-L-glutamyl-L-tyrosyl-L-leucine

trifluoroacetate (salt) hydrate. Bivalirudin directly inhibits thrombin, a key component in blood clot formation and extension. It is the active ingredient in a lyophilized product for injection, sold as ANGIOMAX^®.

U.S. Patent No. 5,196,404 describes a method for the preparation of bivalirudin using BOC-L-leucine-O-divinylbenzene resin. The process involves a sequential approach of adding Boc-protected amino acids on divinylbenzene resin. The peptide sequence obtained was fully deprotected and uncoupled from the resin using an anhydrous mixture of HF, p-cresol, and ethyl methyl sulfate, followed by lyophilization to dryness. The crude product was purified by reverse- phase HPLC employing an Applied Biosystems 151 A liquid chromatographic system and a Vydac C-m column (2.2x25 cm). The column was equilibrated in 0.1 % TFA/water and eluted with a linear gradient of increasing acetonitrile concentration from 0 to 80% over 45 minutes in the 0.1 % TFA at a flow rate of 4.0 mL/minute.

T. Okayama et al., "Anticoagulant Peptides; Synthesis, Stability, and Antithrombin Activity of Hirudin C-Terminal-Related Peptides and Their Disulfated Analog," Chemical & Pharmaceutical Bulletin, Vol. 44, No. 7, pages 1344-1350 (1996) describe the synthesis of various hirulog derivatives using conventional solution-phase methods. The process involves the use of Fmoc-protected amino acid p-alkoxybenzyl alcohol resin as the starting resin. The synthesis was performed using DCC or water-soluble carbodiimide and HOBt as active ester coupling agents, and TFA in 1.5% water and 1.5% anisole as the cleavage solution. The crude peptide was purified by reverse-phase HPLC on a Vydac de cartridge column (47x300 mm) using a linear gradient of 0-50% acetonitrile over 50 minutes at a flow rate of 80 mL/minute of 0.1 % TFA.

T. Steinmetzer et al., "Design and evaluation of novel bivalent thrombin inhibitors based on aminophenylalanines," European Journal of Biochemistry, Vol. 265, Issue 2, pages 598-605 (1999) disclose a process for the preparation of hirulog analogues (BZA-1 hirulog). However, the preparation of bivalirudin is not disclosed. The article also discloses a process for the purification of hirulog analogues by preparative HPLC performed on a Shimadzu LC-8 A, Vydac C8, 5 μιτι reversed phase column (250x5 mm) and with solvents: A, 0.1 % aqueous TFA; B, 0.1 % TFA in acetonitrile, gradient 10% B to 55% B in 120 minutes and with a flow rate of 10 mL/min.

International Application Publication No. WO 2006/041945 A2 describes a counterion exchange process for purifying cyclic or non-cyclic peptides, which involves loading a peptide onto a RP-HPLC column, washing the column with an aqueous solution of a pharmaceutically acceptable counterion salt; and eluting the peptide from the column with a solvent mixture of an organic solvent and an acid of the pharmaceutically acceptable counterion, wherein the aqueous solution has a pH of at least about 6. However, the document does not disclose a specific process for purifying bivalirudin.

U.S. Patent Application Publication No. 2007/0093423 A1 describes a process for preparing bivalirudin peptide sequence on a hyperacid labile resin, which allows cleavage of the peptide from the resin in presence of mild acidic conditions, and involves the use of amino acids suitably protected with Boc or Fmoc. The application also discloses a process for the purification of bivalirudin by preparative HPLC using a Ci₈ RP-HPLC column, to obtain fractions containing bivalirudin at a purity >97.5%, containing not more than 0.5% Asp⁹-bivalirudin and not more than 0.5% of any other impurity.

International Application Publication No. WO 2008/109079 A2 describes a process for the preparation of high purity bivalirudin, wherein crude semi- protected bivalirudin dissolved in aqueous acetonitrile is loaded on a preparative Ci8 RP-HPLC column and purified to obtain fractions containing >95% pure product, which is reacted with a deprotecting agent and the resulting solution of bivalirudin is loaded on a HPLC preparative column loaded with RP C-18 resin, 15 μητι, and purified using a linear gradient of water (0.1 % TFA)/acetonitrile (10% to 15% acetonitrile in 5 minutes and to 38% in 40 minutes) and purified to obtain fractions containing bivalirudin at a purity >97.5%. The counterion was exchanged to TFA and pure fractions were collected and lyophilized to obtain a final dry peptide that is >99.0% pure (HPLC), containing not more than 0.5% [ASP⁹]- bivalirudin, not more than 0.5% [+Gly]-bivalirudin and not more than 0.5% of any other impurity.

U.S. Patent Application Publication No. 2008/0051558 A1 describes a process for purifying bivalirudin, wherein crude bivalirudin in acetic acid solution is passed through Ci₈ or C₈ column, wherein the liquid phase is 0.01 -0.5M acetic acid, phosphoric acid or trifluoroacetic acid (TFA)-acetonitrile ( 0-90:90-10, v/v); the flow rate is 50-1 ,500 ml/minute and the detection wavelength is 250-280 nm. The desired fractions are collected, salt is removed, and the solution lyophilized to obtain the purified bivalirudin.

The known processes for the preparation of bivalirudin may result in impurities due to side-chain modification, sequence modification, undesired impurities formation during the intermediate steps, etc. The above-mentioned processes for the purification of bivalirudin using reversed phase high- performance liquid chromatography (RP-HPLC) may result in improved purity, however, they have drawbacks in that the conditions as disclosed do not result in the separation of some of the process-related peptide impurities, and the stability of the column deteriorates and does not produce reproducible and consistent results.

As the selection of column, packing media, and the mobile phase/solvent/buffer system for eluting the column is important, there still exists need for a more robust, user convenient and up-scalable process for the purification of bivalirudin.

SUMMARY

Aspects of the present application relates to processes for the purification of bivalirudin. Further aspects of the application also relate to substantially pure bivalirudin. An aspect of the present application provides process for the purification of bivalirudin, embodiments comprising:

a) loading crude or semi-purified bivalirudin onto a column;

b) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;

c) collecting fractions of desired bivalirudin purity and pooling;

d) loading the pooled fractions onto the column;

e) washing the column with about 0.1 % trifluoroacetic acid;

f) eluting the product from the column with a composition of water and acetonitrile; and

g) isolating purified bivalirudin.

In another aspect, the present application provides process for the purification of bivalirudin, embodiments comprising:

a) loading bivalirudin onto an ion chromatography column;

b) eluting bivalirudin from the column with a gradient of about 0.05% formic acid and a mixture of about 0.05% formic acid with about 0.5 M ammonium formate;

c) collecting fractions of desired bivalirudin purity;

d) loading the bivalirudin fractions of desired bivalirudin purity onto a RP-HPLC column;

e) eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and ammonium acetate buffer; optionally

f) mixing the pooled fractions obtained in step e) with ammonium acetate buffer, loading onto a RP-HPLC column (5 pm, 100 A) and eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and orthophosphoric acid buffer having pH about 2.9 to about 3.1 ; and g) isolating purified bivalirudin.

I. Purification by Ion Chromatography

a) loading crude bivalirudin onto an ion chromatography column; b) eluting bivalirudin from the column with a gradient of about 0.05% formic acid and a mixture of about 0.05% formic acid with about 0.5 M ammonium formate;

c) collecting fractions of desired bivalirudin purity;

II. Purification by Reverse Phase Chromatography

a) loading the bivalirudin fractions onto a RP-HPLC column;

b) eluting bivalirudin fractions from the RP-HPLC column with

acetonitrile and ammonium acetate buffer;

c) collecting the fraction of desired bivalirudin purity;

and/or

d) loading the bivalirudin fractions onto a RP-HPLC column;

e) eluting bivalirudin fractions from the RP-HPLC column with

acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;

f) collecting the fraction of desired bivalirudin purity; and

g) isolating purified bivalirudin.

In yet another aspect, the present application provides substantially pure bivalirudin having purity greater than about 98.5% and each of the impurities [Asp⁹]-bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGly]-bivalirudin, [D-Asn⁹]- bivalirudin, and [D-phe¹²]-bivalirudin being present at less than about 1%.

In a further aspect, the present application provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [D-Asn⁹]-bivalirudin.

In another aspect, the present application provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [-Gly]-bivalirudin.

DETAILED DESCRIPTION

Aspects of the present application relate to processes for the purification of bivalirudin. Further aspects of the application relate to substantially pure bivalirudin.

An aspect of the present application provides process for the purification of bivalirudin, embodiments comprising:

a) loading crude or semi-purified bivalirudin onto a column; b) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;

c) collecting fractions of desired bivalirudin purity and pooling;

d) loading the pooled fractions onto the column;

e) washing the column with about 0.1 % trifluoroacetic acid;

g) isolating purified bivalirudin.

All of the steps of the above process are individually described herein below.

Step a) involves loading the sample of crude or semi-purified bivalirudin onto a column.

A purification process of the present invention may be carried out by eluting bivalirudin though a preparative HPLC column, wherein the column is packed with high pure C-18 reverse phase media (5 pm, 100 A) under a dynamic axial compression mode with operating pressures up to about 200 bar.

Crude bivalirudin may be prepared by any known process, including a process disclosed in U.S. Patent Application Publication No. 2009/0062511 A1.

Crude bivalirudin may optionally be purified prior to loading onto the column by Ion exchange chromatography or by reverse phase (RP)-HPLC or both according to the present application methods, as described below for decreasing the content of process related impurities and to obtain a semi-purified bivalirudin.

In an embodiment, a sample solution of the bivalirudin provided by dissolving crude or semi-purified bivalirudin in orthophosphoric acid buffer, optionally in combination with acetonitrile, is loaded onto the column.

Step b) involves eluting bivalirudin from the column with acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1.

Orthophosphoric acid buffer used in the process of the present invention may be prepared by dissolving orthophosphoric acid in water and adjusting the pH of the buffer solution with a base to a pH value of about 2.9 to about 3.1. In one of the embodiments, the base used is triethylamine. The concentration of orthophosphoric acid used may be about 0.1 % to about 1 % in water. In a specific embodiment, about 0.3% orthophosphoric acid buffer with a pH about 3±0.05 is used.

The solution of bivalirudin loaded onto the column is eluted with a gradient composition of acetonitrile and orthophosphoric acid buffer with an increasing composition of acetonitrile, from 95% orthophosphoric acid buffer and 5% acetonitrile at about 0 minutes to about 70% orthophosphoric acid buffer and 30% acetonitrile at 180 minutes. The flow rate used for elution may depend on the diameter of the column used. In an embodiment a flow rate of 360 mL/min is utilized for elution.

Step c) involves collecting the fractions of desired bivalirudin purity and pooling.

During elution, desired fractions are collected at regular intervals and analyzed for purity. The suitable collected fractions containing the product of similar purities may be pooled together and optionally subjected to removal of acetonitrile solvent. Optionally, after completing the desired number of cycles of purification by repeating the steps a) and b), all the fractions of similar purity from each of the cycle are pooled and taken forward to the next step of the purification process. In an embodiment, pooled fractions having a purity of more than about 98% may be taken forward to the next step of the purification process

Step d) involves loading the pooled fractions onto the column.

The pooled fractions obtained in step c) may be diluted with water and loaded onto the column. The column may be stabilized by washing with a composition of about 0.1 % trifluoroacetic acid and acetonitrile, followed by washing with about 0.1 % trifluoroacetic acid, before the sample loading.

Step e) involves washing the column with about 0.1 % trifluoroacetic acid.

The column may be washed with about 0.1 % trifluoroacetic acid in water until the effluent becomes acidic at the end of the wash, so that residual phosphate is removed.

Step f) involves eluting the product from the column with a composition of water and acetonitrile. The product is eluted from the column using a linear gradient of water and acetonitrile as mobile phase, with an increasing composition of acetonitrile from 100% water and 0% acetonitrile at 0 minutes to 20% water and 80% acetonitrile at 40 minutes, and optionally continued the elution using the same composition up to 100 minutes run time. During elution, fractions are collected at regular intervals, and the collected fractions are analyzed by HPLC to determine the purity, and fraction with desired purities may be pooled together.

In one embodiment, desired fractions having purity greater than about 98% by HPLC are pooled.

Step g) involves isolating purified bivalirudin.

The pooled fractions obtained in step f) are analyzed for their trifluoroacetic acid content. If required, the trifluoroacetic acid content may be adjusted in accordance with the pharmacopeial requirement. Further, the solution may be evaporated under vacuum at temperatures from about 15 to about 20°C to remove acetonitrile and maintain its content as per ICH requirement. The concentrate solution thus obtained may be lyophilized to provide a lyophilized powder of bivalirudin.

In another aspect, the present invention provides processes for the purification of bivalirudin, embodiments comprising:

i) loading crude bivalirudin onto a C-18 RP-HPLC column (5 μιτι, 100

A);

ii) eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer; and

iii) collecting the fractions of desired bivalirudin purity and pooling.

iv) optionally isolating purified bivalirudin.

Step i) involves loading bivalirudin onto a C-18 RP-HPLC column (5 pm, 100 A).

Crude bivalirudin prepared by any process may be used as the input.

The crude bivalirudin is dissolved in appropriate amount of ammonium acetate buffer to provide a sample solution which may be loaded onto the column.

The ammonium acetate buffer used in the process of the present invention is prepared by dissolving ammonium acetate in water.

The concentration of ammonium acetate buffer that may be utilized ranges from about 0.1 M to about 0.3M, in water. In one of the embodiments, about 0.2M ammonium acetate buffer is used for the dissolution of the crude bivalirudin. Optionally, the column may be washed with 100% acetonitrile and may be stabilized with 95% water-5% acetonitrile, by volume, before loading the sample onto the column.

Step ii) involves eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer.

The ammonium acetate buffer used in the step i) for dissolution and acetonitrile are utilized for eluting bivalirudin from the column.

The product is eluted from the column with a mobile phase composition of from 80% to about 85% ammonium acetate buffer and from about 20% to about 15% acetonitrile, by volume, for about 190 minutes.

In an embodiment, the product is eluted isocratically from the column with a mobile phase composition of about 84% ammonium acetate buffer and about 16% acetonitrile, by volume, for about 190 minutes.

The flow rate used for elution may depend on the diameter of the column used. In an embodiment a flow rate of 360 mL/min is utilized for elution.

Step iii) involves collecting fractions of desired bivalirudin purity and pooling the desired fractions;

During elution, desired fractions are collected at regular intervals and analyzed for purity. The collected fractions containing the product and of similar purity may be pooled together.

Step iv) involves optionally isolating purified bivalirudin.

The pooled fractions may be optionally subjected to evaporation for the removal of acetonitrile solvent and product may be isolated or the pooled fractions obtained in step iii) may also be taken as the sample solution for the next level of purification.

In an embodiment, the purification by RP-HPLC using a composition of acetonitrile and ammonium acetate buffer is performed on a crude sample to provide semi-purified Bivalirudin.

In a particular embodiment, the purification by RP-HPLC using a composition of acetonitrile and ammonium acetate buffer is performed on a crude sample having [D-phe¹²]-bivalirudin more than about 1 %, by HPLC, to provide bivalirudin having [D-phe¹²]-bivalirudin at less than about 0.5% by HPLC. In an embodiment, the present application provides process for the purification of bivalirudin comprising at least one step of purification by eluting bivalirudin from a RP-HPLC column with a composition of acetonitrile and ammonium acetate buffer.

In a particular embodiment, Bivalirudin having [D-phe¹²]-bivalirudin at less than about 0.5% by HPLC may be obtained by a process comprising at least one step of purification by eluting bivalirudin from a RP-HPLC column with a composition of acetonitrile and ammonium acetate buffer.

In another embodiment, the present application provides process for the purification of bivalirudin comprising:

a) eluting bivalirudin from a RP-HPLC column with acetonitrile and ammonium acetate buffer; and

b) eluting bivalirudin fractions from a RP-HPLC column with acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1.

In one of the embodiments, bivalirudin obtained by the above processes has a purity greater than about 98.5%.

In a particular embodiment, bivalirudin obtained by the above processes has not more than about 0.5% [D-Asn⁹]-bivalirudin impurity.

In a particular embodiment, the present application provides a process for the purification of bivalirudin comprising:

a) loading crude bivalirudin onto a C-18 RP-HPLC column;

b) eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer;

c) collecting the fractions of desired bivalirudin purity and pooling; d) optionally concentrating the pooled fractions; and

e) loading the pooled fractions onto a C-18 RP-HPLC column;

f) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ; g) collecting the fractions of desired bivalirudin purity and pooling; h) loading the pooled fractions onto the column;

i) washing the column with about 0.1 % trifluoroacetic acid;

j) eluting the product from the column with a composition of water and acetonitrile; and k) isolating purified bivalirudin.

I. Purification by Ion Chromatography

a) loading crude bivalirudin onto an ion chromatography column;

c) collecting fractions of desired bivalirudin purity;

II. Purification by Reverse Phase Chromatography

a) loading the bivalirudin fractions onto a RP-HPLC column;

b) eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and ammonium acetate buffer;

c) collecting the fraction of desired bivalirudin purity;

and/or

d) loading the bivalirudin fractions onto a RP-HPLC column;

e) eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;

f) collecting the fraction of desired bivalirudin purity; and

g) isolating purified bivalirudin.

All of the steps of the above process are individually described herein below.

I. Purification by Ion Chromatography

Step a) involves loading crude bivalirudin onto an Ion chromatography column. In embodiments, a column with Q-sepharose Fast Flow resin (a strong anion exchange resin with a matrix active group of -0-CH₂CHOHCH20CH₂CHOHCH₂N⁺(CH3)3) is used for the purification of bivalirudin.

Bivalirudin prepared by any process known in the art, including a process disclosed in U.S. Patent Application Publication No. 2009/0062511 A1 may be used as the input in the purification procedure. The first step of the purification process comprises dissolution of bivalirudin in a formic acid buffer, adjusting the pH to 4.1 using ammonia and loading the solution onto the ion chromatography column. The formic acid buffer used for dissolution may be prepared by dissolving formic acid in water.

In an embodiment, about 0.05% formic acid in water (pH 4.1 ) is used as formic acid buffer.

The clear solution of crude bivalirudin prepared by using appropriate quantities formic acid buffer is loaded onto the column.

Step b) involves eluting bivalirudin from the column with a gradient of about 0.05% formic acid and a mixture of about 0.05% formic acid with about 0.5 M ammonium formate.

The product is eluted from the ion chromatographic column with about 0.05% formic acid and a mixture of about 0.05% formic acid with about 0.5 M ammonium formate, with a gradient composition of 100% about 0.05% formic acid at about 0 minutes to 70% about 0.05% formic acid and 30% about 0.05% formic acid with about 0.5 M ammonium formate mixture at 190 minutes. The flow rate used for elution may depend on the diameter of the column used. In an embodiment a flow rate of 25 mL/min is utilized for elution.

Step c) involves collecting fractions of desired bivalirudin purity.

Fractions are collected at regular intervals during elution and analyzed for purity. The collected fractions containing the product of similar purity may be pooled together and optionally subjected to evaporation for the removal of acetonitrile solvent. After completing the desired number of cycles of purification, repeating the steps a) and b), all the fraction of similar purity from each of the cycle are pooled and taken forward for the next step of the purification process.

The ion chromatographic purification provides semi-purified bivalirudin having purity up to about 85%, if performed starting with a crude bivalirudin having purity not more than about 70%.

According to present invention, the ion chromatographic purification process facilitates in reducing some of the process-related impurities formed in the various synthetic processes including the prior processes or due to amino acid quality variations. In particular embodiment, the ion chromatographic purification facilitates in reducing the impurities like [+Gly]-bivalirudin, [-Gly]-bivalirudin, [D-phe¹²]- bivalirudin, from bivalirudin.

In yet another embodiment, the present invention provides processes for preparing bivalirudin substantially free of the process related impurities using the ion chromatography purification as described above.

Further, the inventors of the present application have also found that purifying crude bivalirudin by an ion chromatography purification process is advantageous in reducing the maximum amount of process impurities or undesired components, thereby decreasing the load on a subsequent RP-HPLC column, and also helps to promote increased lifetime of the expensive preparative columns as compared to the processes that involve direct purification using RP- HPLC.

In other words, the ion chromatography step acts as a pre-purification step for the next Reverse-Phase purification step.

II. Purification by Reverse Phase Chromatography

Step a) involves loading the bivalirudin fractions onto a RP-HPLC column. The IC pooled fractions obtained in step c) of the above purification Stage I may be optionally mixed with ammonium acetate buffer and loaded onto a Ci₈ RP-HPLC column (5 μιτι, 100 A).

Step b) involves eluting bivalirudin fractions from a RP-HPLC column with a composition of acetonitrile and ammonium acetate buffer.

The concentrations of ammonium acetate used may be about 0.1 M to about 0.3M in water. In one of the specific embodiment of the present invention, about 0.2M ammonium acetate buffer is used.

Optionally, the column may be washed with 100% acetonitrile and may be stabilized with 95% water-5% acetonitrile, by volume, before loading the sample onto the column.

The product is eluted from the column with a mobile phase composition of from 80% to about 85% ammonium acetate buffer and from about 20 to about 15% acetonitrile, by volume, for about 190 minutes. ln an embodiment, the product is eluted isocratically from the column with a mobile phase composition of about 84% ammonium acetate buffer and about 16% acetonitrile, by volume, for about 190 minutes.

Step c) involves collecting the fraction of desired bivalirudin purity.

During elution, fractions are collected at regular intervals and analyzed for purity. The collected fractions containing the product of similar purity may be pooled together and optionally subjected to evaporation for the removal of acetonitrile solvent.

After completing the desired number of cycles of purification, repeating the steps a) and b) of Stage II, all the fraction of similar purity from each of the cycle are pooled and may be optionally be taken forward for the next step of the purification process or may be subjected to the steps resulting in product isolation as detailed in step g).

The purification process described above provides bivalirudin having purity up to about 98.5%.

In an embodiment, the Bivalirudin obtained by the above purification process has the content of [D-phe¹²]-bivalirudin in amounts less than about 0.5% or in amounts less than about 0.2% by HPLC.

Step d) involves loading the bivalirudin fraction onto a RP-HPLC column.

The pooled fractions obtained from the step c) of Stage I purification using ion chromatography may be taken as the input for this purification step or the pooled fractions obtained from the step c) of Stage II purification by RP-HPLC using ammonium acetate buffer may be taken as the input sample solution for loading onto the column in step d).

Step e) involves eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 .

The pooled fractions of the crude bivalirudin is loaded onto the column and is eluted with a gradient of acetonitrile and orthophosphoric acid buffer, with an increasing composition of acetonitrile from 95% orthophosphoric acid buffer and 5% acetonitrile at about 0 minutes to about 70% orthophosphoric acid buffer and 30% acetonitrile at 180 minutes.

Orthophosphoric acid buffer used in the process may be prepared by dissolving appropriate quantities of orthophosphoric acid in water and adjusting the pH with a base to a pH value from about 2.9 to about 3.1 . In one of the embodiments the base used is triethylamine.

In embodiments, the concentration of orthophosphoric acid used may be about 0.3% to about 1 % in water. In embodiments, about 0.3% orthophosphoric acid buffer with a pH about 3±0.05 is used.

Step f) involves collecting the fraction of desired bivalirudin purity.

Fractions are collected at regular intervals and analyzed for purity. The collected fractions containing the product and of similar kind may be pooled together and optionally subjected to evaporation for the removal of acetonitrile solvent.

Step g) involves isolating purified bivalirudin.

The pooled fractions obtained in step c) of stage II may be directly subjected to salt exchange if the purification steps d) and e) are not performed or if performed the fractions collected in step f) may be subjected to salt exchange.

After completing the desired number of cycles of purification by repeating the process steps, all the fractions of similar purity from each of the cycles are pooled.

The pooled fractions obtained in step c) of stage II or step f) of stage II may be subjected to salt exchange. The pooled fractions obtained may be diluted with water and loaded onto the column and the column may be washed with about 0.1 % trifluoroacetic acid in water until the effluent becomes acidic at the end of the wash, so that residual phosphate is removed.

Further, the product is eluted from the column using a linear gradient of water and acetonitrile as mobile phase, with an increasing composition of acetonitrile from 100% water and 0% acetonitrile at 0 minutes to 20% water and 80% acetonitrile at 40 minutes, and optionally continued the elution using the same composition up to 100 minutes run time. During elution, fractions are collected at regular intervals, and the collected fractions are analyzed by HPLC to determine the purity, and fraction with desired purities may be pooled together.

The pooled fractions obtained are analyzed for their trifluoroacetic acid content. If required, the trifluoroacetic acid content may be adjusted in accordance with the pharmacopeial requirement. Further, the solution may be evaporated under vacuum at temperatures from about 15°C to about 20°C to remove acetonitrile and maintain its content as per ICH requirement. The concentrated solution thus obtained may be lyophilized to provide a lyophilized powder of bivalirudin.

In an aspect, the present application provides processes for the purification of bivalirudin, embodiments comprising:

a) loading bivalirudin onto an ion chromatography column;

c) collecting fractions of desired bivalirudin purity;

d) loading the bivalirudin fractions onto a RP-HPLC column and eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and ammonium acetate buffer;

e) collecting fractions of desired bivalirudin purity;

f) loading the bivalirudin fractions onto a RP-HPLC column and eluting bivalirudin fractions from the RP-HPLC column with acetonitrile and orthophosphoric acid buffer having pH about 2.9 to about 3.1 ;

g) collecting fractions of desired bivalirudin purity; and

h) isolating purified bivalirudin.

In a particular embodiment, the present application provides a process for purifying bivalirudin comprising:

a) loading crude bivalirudin onto an ion chromatography column;

c) collecting fractions of desired bivalirudin purity;

d) loading IC purified bivalirudin onto an RP-HPLC column; e) eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer;

f) collecting the fractions of desired bivalirudin purity and pooling;

g) optionally concentrating the pooled fractions; and/or

h) loading the bivalirudin fractions onto an RP-HPLC column;

i) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ; j) collecting the fractions of desired bivalirudin purity and pooling;

k) optionally loading the pooled fractions onto the column;

I) washing the column with about 0.1 % trifluoroacetic acid;

m) eluting the product from the column with a composition of water and acetonitrile; and

n) isolating purified bivalirudin.

In embodiments, the present application provides substantially pure bivalirudin having purity greater than about 98.5% or greater than about 99.0%, obtained by the purification processes as described in the present application performed in any order.

In yet other embodiments, the present application provides bivalirudin having purity greater than about 98.5%, and each of the impurities [Asp⁹]- bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGlyJ-bivalirudin, [D-Asn⁹]- bivalirudin, [D-phe¹²]-bivalirudin being present in amounts less than about 0.5% as determined using HPLC.

In another embodiment, the present application provides bivalirudin having purity greater than about 98.5% and less than about 0.5% [D-Asn⁹]-bivalirudin, less than about [D-phe¹²]-bivalirudin, obtained by the purification processes as described in the present application.

In yet another aspect, the present application provides substantially pure bivalirudin having purity greater than about 98.5%, and each of the impurities [Asp⁹]-bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGlyJ-bivalirudin, [D-Asn⁹]- bivalirudin, [D-phe¹²]-bivalirudin being present at less than about 1%, or less than about 0.5%, and all of the foregoing impurities together being present at less than about 1.5%. In a further aspect, the present application provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [D-Asn⁹]-bivalirudin.

In another aspect, the invention provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [-Gly]- bivalirudin.

Bivalirudin obtained by processes of the present invention may be analyzed for purity using HPLC. In an embodiment, the present application provides an HPLC method for the analysis of bivalirudin samples, wherein the analysis is performed using a Waters system, equipped with Zorbax™ SB C-18, 200x4-6 mm, 1 .8 μητι or equivalent column with a guard column of Hypersil™ Gold 10 mmx4.6 mm, 3 μιη. The column is maintained at 45-50°C and a UV detector at 210 nm. Analyses are performed using the following mobile phase, with flow rate of about 0.4 mUminute and a run time of 150 minutes.

Mobile phase A: dissolve 0.5 g of sodium 1 -butanesulphonate in 1000 mL of Milli Q™ water, add 3 mL orthophosphoric acid, and adjust the pH to 2.8+0.05 with trimethylamine. Add 5 mL methanol and filter through a 0.22 μητι membrane filter. Mobile phase B: a mixture of methanol and acetonitrile in the volume ratio of 750:250 and filtered through a 0.22 μιη membrane filter.

Elution gradient program:

The [D-Asn⁹]-bivalirudin impurity frequently elutes adjacent to the bivalirudin peak and the HPLC analytical methods disclosed in the art are not capable of separating and detecting all the above-mentioned process related peptide impurities. Therefore, the HPLC method of the present invention is robust enough and provides enhanced capability to resolve and detect all these process related peptide impurities.

Moisture content (determined, for example, by the Karl Fischer method) of bivalirudin obtained by a process of the present invention may range from about 4% to 8%, or about 5% to 6%.

SEQUENCES LISTINGS

[D-phe¹²]-bivalirudin: D-phenylalanyl-L-prolyl-L-arginyl-L-prolyl-glycyl-glycyl- glycyl-glycyl-L-asparagyl-glycyl-L-aspartyl-D-phenylalanyl-L-glutamyl-L-glutamyl- L-isoleucyl-L-prolyl-L-glutamyl-L-glutamyl-L-tyrosyl-L-leucine trifluoroacetate (salt) [DiGly]-bivalirudin: D-Phenylalanyl-L-Prolyl-L-arginyl-L-Prolyl-glycyl-glycyl-L- aspargyl-glycyl-L-aspartyl-L-phenylalanyl-L-glutamyl- L-glutamyl-L-isoleucyl-L- prolyl- L-glutamyl-L-glutamyl-L-tyrosyl-L-leucine trifluoroacetate (salt)

[Asp⁹]-bivalirudin: D-Phenylalanyl-L-Prolyl-L-arginyl-L-Prolyl-glycyl-glycyl-glycyl- glycyl-L- aspartyl-glycyl-L-aspartyl-L-phenylalanyl-L-glutamyl- L-glutamyl-L- isoleucyl-L-prolyl- L-glutamyl- L-glutamyl-L-tyrosyl-L-leucine trifluoroacetate (salt) Pentagly impurity or [+Gly]-bivalirudin: D-Phenylalanyl-L-Prolyl-L-arginyl-L- Prolyl-glycyl-glycyl-glycyl-glycyl-glycyl-L-aspargyl-glycyl-L-aspartyl-L-phenylalanyl- L-glutamyl- L-glutamyl-L-isoleucyl-L-prolyl- L-glutamyl- L-glutamyl-L-tyrosyl-L- leucine trifluoroacetate (salt)

Trigly impurity or [-Glyj-bivalirudin: D-Phenylalanyl-L-Prolyl-L-arginyl-L-Prolyl- glycyl-glycyl-glycyl-L-aspargyl-glycyl-L-aspartyl-L-phenylalanyl-L-glutamyl- L- glutamyl-L-isoleucyl-L-prolyl- L-glutamyl-L-glutamyl-L-tyrosyl-L-leucine

trifluoroacetate (salt)

[D-Asn⁹]-bivaiirudin: D-phenylalanyl-L-prolyl-L-arginyl-L-prolyl-glycyl-glycyl- glycyl-glycyl-D-asparagyl-glycyl-L-aspartyl-L-phenylalanyl-L-glutamyl-L-glutamyl- L-isoleucyl-L-prolyl-L-glutamyl-L-glutamyl-L-tyrosyl-L-leucine trifluoroacetate (salt) Certain specific aspects and embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the application in any manner. EXAMPLES

EXAMPLE 1 : Purifying bivalirudin by RP-HPLC using orthophosphoric acid buffer. Bivalirudin is purified using a high-pressure column packed with C18 reverse phase media (5 pm, 100A, high purity) under a dynamic axial compression mode.

PART A: Purification by RP-HPLC using orthophosphoric acid buffer.

Buffer A: 0.3% orthophosphoric acid buffer (orthophosphoric acid solution in water, with pH adjusted to about 3 using triethylamine). Buffer B: acetonitrile. Wavelength: 210 nm. Sample preparation: dissolve 40 g of bivalirudin (purity by HPLC: 58.77%) in 3800 mL of Buffer A and 200 mL of Buffer B, sonicate for 20 minutes and filter the solution.

Procedure: Stabilize the column by washing with a composition of 95% Buffer A and 5% Buffer B for 30 minutes, with a flow rate of 360 mUminute. Load the sample solution onto the column with a flow rate of 280 mL/minute. Elute with a gradient program of Buffer A and Buffer B with a composition of 95% Buffer A and 5% Buffer B at 0 minutes to 74% Buffer A and 26% Buffer B in 180 minutes, at a flow rate of 360 mL/minute. The desired fractions are collected and analyzed for bivalirudin purity. Fractions of similar purity are pooled and separated to obtain pure pool 1 (purity at least 98%) and pure pool 2 (purity at least 97.5% and up to 98%). After completing four cycles of purification, with each cycle of 40 g input, all the fractions of similar purity from each of the cycle are pooled and separated to obtain four lots of pure pool 1 and pure pool 2. Purity of each pool is analyzed and a composite is provided by mixing both of the pools. Yield by HPLC: 21 .84 g. Purity by HPLC: 98.04%.

PART B: Salt exchange. Buffer: 0.1 % trifluoroacetic acid solution in water. Solution A: water. Solution B: acetonitrile, Wavelength: 210 nm. Sample preparation: add 4.2 L of water to the 4.2 L of the composite pool obtained in PART A.

Procedure: Stabilize the column by washing with a composition of 50% buffer and 50% solution B for 30 minutes, followed by washing with 0.1 % TFA buffer for 30 minutes at a flow rate of 40 mL/minute. Load the sample solution onto the column at a flow rate of 40 mL/minute. Wash the column with Solution A for 60 minutes until the pH of the effluent is neutral. Wash the column with 0.1 % TFA buffer for 60 minutes until the pH of the effluent is acidic (pH about 2). Elute with a gradient program of Solution A and Solution B with a composition of 100% solution A at 0 minutes to 20% solution A and 80% solution B at 100 minutes, at a flow rate of 40 mlJminute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled and separated to give a pure pool of bivalirudin trifluoroacetate. Yield by HPLC: 32 g. Purity of pure pool by HPLC: 98.0%; [D-phe¹²]-bivalirudin: 0.03%; [+Gly]-bivalirudin: 0.15%; [D-Asn⁹]- bivalirudin: 0.74%; [-Gly]-bivalirudin: 0.32%; [Di-Gly]-bivalirudin: not detected; [Asp⁹]-bivalirudin: not detected.

EXAMPLE 2: Preparation of bivalirudin trifluoroacetate.

120 g of bivalirudin trifluoroacetate pure pool (purity: 98.0%) (obtained by a process similar to that disclosed in Example 1 ) is placed into a round bottom flask, trifluoroacetic acid (5 ml_) is added and the mixture is diluted with UF water to a volume of 3500 ml_. Evaporate the solution at a temperature of 15-19°C under high vacuum using a Biichi® rotary evaporator to remove acetonitrile. Filter the residual solution using a sterile filter and lyophilize to give bivalirudin trifluoroacetate. Yield: 105 g. Purity by HPLC: 98.1 %; [D-phe¹²]-bivalirudin: 0.03%; [+Gly]-bivalirudin: 0.16%; [D-Asn⁹]-bivalirudin: 0.76%; [-Gly]-bivalirudin: 0.29%; [Di-Gly]-bivalirudin: 0.14%; [Asp⁹]-bivalirudin: not detected.

EXAMPLE 3: Purifying bivalirudin by ion chromatography.

Resin: Q Sepharose FF, Resin volume: 588 mL, Mobile phase A: 0.05% formic acid, Mobile phase B: 0.05% formic acid + 0.5 M ammonium formate, Wavelength: 220 nm. Sample preparation: 20 g of crude bivalirudin (purity: 69.84%; [+Gly]-bivalirudin: 2.3%; [-Gly]-bivalirudin: 2.4%) is dissolved in 1000 mL of mobile phase A and pH is adjusted to 4.1 with ammonia, sonicate for 20 minutes and filter the solution.

Procedure: Stabilize the column by washing with mobile phase A for 40-45 minutes with a flow rate of 25 mL/minute. Load the bivalirudin sample onto the column using mobile phase A at a flow rate of 20 mL/minute in 50 minutes. Wash the column with mobile phase A for 50 minutes. Elute with a gradient program of mobile phase A and mobile phase B with a composition of 100% mobile phase A at 0 minutes to 30% mobile phase B at 190 minutes, at a flow rate of 25 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled. Yield by HPLC: 8.29 g, Purity by HPLC: 84.18%; [+Gly]-bivalirudin: 0.22%; [-Gly]-bivalirudin: 0.24%.

EXAMPLE 4: Purifying bivalirudin by ion chromatography followed RP-HPLC.

PART A: Purifying bivalirudin by ion chromatography. Resin: Q Sepharose FF, Resin volume: 588 mL, Mobile phase A: 0.05% formic acid, Mobile phase B: 0.05% formic acid + 0.5 M ammonium formate, Wavelength: 220 nm. Sample preparation: 20 g of crude bivalirudin (purity: 73.0%) is dissolved in 800 mL of mobile phase A and pH is adjusted to 4.1 with ammonia solution, sonicate for 20 minutes and filter the solution through a 0.45 μιη filter paper.

Procedure: Stabilize the column by washing with mobile phase A for 40-45 minutes with a flow rate of 30 mUminute. Load the bivalirudin sample onto the 588 mL column using mobile phase A at a flow rate of 20 mL/minute in 50 minutes. Wash the column with mobile phase A for 50 minutes with a flow rate of 25 mL/minute. Elute with a gradient program of mobile phase A and mobile phase B with a composition of 100% mobile phase A at 0 minutes to 30% mobile phase B at 190 minutes, at a flow rate of 25 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled. Yield by HPLC: 9.78 g. Purity by HPLC: 82.24%.

PART B: RP-HPLC purification using ammonium acetate buffer. Buffer A: 0.2 M ammonium acetate, Buffer B: Acetonitrile, Wavelength: 210 nm. Sample preparation: pooled fractions (240 mL) obtained in Part A are mixed with Buffer A (240 mL) and stirred.

Procedure: Stabilize the column by washing with buffer B, followed by washing with water. Load the sample onto the column at a flow rate of 40 mL/minute. Elute with an isocratic composition of 84% buffer A and 16% buffer B for 100 minutes, at a flow rate of 40 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled and stored at -20°C. Purity of pure fraction: 96.68%; [D-phe¹²]-bivalirudin: not detected; [+Gly]-bivalirudin: 0.29%; [-Gly]-bivalirudin: 1 .67%; [Di-Gly]-bivalirudin: not detected; [Asp⁹]-bivalirudin: not detected.

PART C: RP-HPLC purification using orthophosphoric acid buffer. Mobile phase A: 0.3% orthophosphoric acid buffer (orthophosphoric acid solution in water, with pH adjusted to about 3 using triethylamine). Mobile phase B: acetonitrile, Wavelength: 210 nm. Sample preparation: Pooled fractions (240 ml_) obtained in part B are diluted with 240 mL of mobile phase A and mixed.

Procedure: Stabilize the column by washing with a composition of 95% mobile phase A and 5% mobile phase B for 30 minutes. Load the sample solution onto the column. Elute with a gradient program of mobile phase A and mobile phase B with a composition of 95% mobile phase A and 5% mobile phase B at 0 minutes to 0% mobile phase A and 100% mobile phase B at 190 minutes, at a flow rate of 40 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled to give bivalirudin bis- triethylammonium phosphate salt (bivalirudin TEAP salt). Purity: 99.47%; [D- phe¹²]-bivalirudin: not detected; [+Gly]-bivalirudin: 0.24%; [D-Asn⁹]-bivalirudin: not detected; [-Gly]-bivalirudin: 0.29%; [Di-Gly]-bivalirudin: not detected; [Asp⁹]- bivalirudin: not detected.

The purified bivalirudin pooled fractions obtained in part C is subjected to evaporation for the removal of acetonitrile solvent. The concentrated pure pool is treated with trifluoroacetic acid solution to obtain a solution of bivalirudin trifluoroacetic acid salt. The pure solution is subjected to lyophilization to obtain a powder of bivalirudin trifluoroacetate.

EXAMPLE 5: Purifying bivalirudin by RP-HPLC, using ammonium acetate buffer.

Buffer A: 0.2 M ammonium acetate, Buffer B: acetonitrile, Wavelength: 210 nm. Sample preparation: Dissolve 1 g of bivalirudin (purity: 69.84%; [D-phe¹²]- bivalirudin: 0.54%; [+Gly]-bivalirudin: 2.3%; [-Gly]-bivalirudin: 2.4%; [Di-Gly]- bivalirudin: 0.2%; [Asp⁹]-bivalirudin: 0.8%) in buffer A, stir for 10-15 minutes and filter the solution.

Procedure: Stabilize the column by washing with a composition of 84% buffer A and 16% buffer B. Load the sample onto the column at a flow rate of 40 mL/minute. Elute with an isocratic composition of 84% buffer A and 16% buffer B for 100 minutes at a flow rate of 40 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled and separated to give bivalirudin ammonium acetate salt. Purity by HPLC: 97.91%; [D-phe¹²]-bivalirudin: not detected, [+Gly]-bivalirudin: 0.28%, [D-Asn⁹]-bivalirudin: not detected; [-Gly]-bivalirudin: not detected; [Di-Gly]-bivalirudin: not detected; [Asp⁹]-bivalirudin: not detected. EXAMPLE 6: Purifying bivalirudin by RP-HPLC using orthophosphoric acid buffer.

Mobile phase A: 0.3% orthophosphoric acid buffer (orthophosphoric acid solution in water, with pH adjusted to about 3 using triethylamine). Mobile phase B: acetonitrile, Wavelength: 210 nm. Sample preparation: Dissolve 2 g of crude bivalirudin (purity: 69.84%; [D-phe¹²]-bivalirudin: 0.54%; [+Gly]-bivalirudin: 2.3%; [- Gly]-bivalirudin: 2.4%; [Di-Gly]-bivalirudin: 0.2%; [Asp⁹]-bivalirudin-0.8%) in 100 ml_ of buffer A, sonicate for 10-15 minutes and filter.

Procedure: Stabilize the column by washing with a composition of 95% Buffer A and 5% solution B for 30 minutes. Load the sample solution onto the column with a low flow rate. Elute with a gradient program of buffer A and buffer B with a composition of 95% buffer A and 5% Buffer B at 0 minutes to 0% buffer A and 100% buffer B at 190 minutes, at a flow rate of 40 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled to give bivalirudin TEAP salt. Purity by HPLC: 98.32%; [D-phe¹²]- bivalirudin: not detected; [+Gly]-bivalirudin: 0.24%; [-Gly]-bivalirudin: 0.92%; [Di- Gly]-bivalirudin: 0.17%; [Asp⁹]-bivalirudin: 0.1 1 %.

EXAMPLE 7: Purifying bivalirudin by RP-HPLC, using ammonium acetate buffer followed by RP-HPLC using orthophosphoric acid buffer.

PART A: Purifying bivalirudin by RP-HPLC using ammonium acetate buffer. Buffer A: 0.2 M ammonium acetate, Buffer B: acetonitrile, Wavelength: 210 nm. Sample preparation: Dissolve 1 g of bivalirudin (purity: 70.84%) in buffer A, stir for 10-15 minutes and filter the solution.

Procedure: Stabilize the column by washing with a composition of 84% buffer A and 16% buffer B. Load the sample onto the column at a flow rate of 360 mL/minute. Elute with an isocratic composition of 84% buffer A and 16% buffer B for 100 minutes at a flow rate of 40 mL/minute. The desired fractions are collected and analyzed for purity. Fractions of similar purity are pooled to give bivalirudin ammonium acetate salt. Purity by HPLC: 85.59%; [D-phe¹²]- bivalirudin: not detected; [+Gly]-bivalirudin: 3.03%; [-Gly]-bivalirudin: not detected; [Di-Gly]-bivalirudin: not detected; [Asp⁹]-bivalirudin: not detected.

PART B: Purifying bivalirudin by RP-HPLC using orthophosphoric acid buffer. Buffer A: 0.3% orthophosphoric acid buffer (orthophosphoric acid solution in water, with pH adjusted to about 3.0 using triethylamine). Buffer B: acetonitrile, Wavelength: 210 nm. Sample preparation: The product fractions obtained in part A are diluted with an equal volume of buffer A and stirred.

Procedure: Stabilize the column by washing with a composition of 95% buffer A and 5% buffer B for 30 minutes. Load the sample solution onto the column. Elute with a gradient program of buffer A and buffer B with a composition of 95% buffer A and 5% buffer B at 0 minutes to 0% buffer A and 100% buffer B at 190 minutes, at a flow rate of 40 mL/minute. The desired fraction are collected and analyzed for their purity. Fractions of similar purity are pooled to give bivalirudin TEAP salt. Purity by HPLC: 99.38%; [D-phe¹²]-bivalirudin: not detected; [+Gly]-bivalirudin: 0.61 %; [D-Asn⁹]-bivalirudin: not detected; [-Gly]- bivalirudin: not detected; [Di-Gly]-bivalirudin: not detected; [Asp⁹]-bivalirudin: not detected.

While particular aspects of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Claims

CLAIMS:

1. A process for purifying bivalirudin comprising:

a) loading crude or semi-purified bivalirudin onto a column;

b) eluting bivalirudin from the column with acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;

c) collecting fractions of desired bivalirudin purity and pooling;

d) loading the pooled fractions onto the column;

e) washing the column with 0.1 % trifluoroacetic acid; and

f) eluting the product from the column with a composition of water and acetonitrile.

2. The process of claim , wherein the column is a preparative HPLC column packed with high pure C-18 reverse phase media under a dynamic axial compression mode with operating pressures up to about 200 bar.

3. The process of claim 1 , wherein the elution in step b) is carried out using a gradient composition of acetonitrile and orthophosphoric acid buffer.

4. The process of claim 1 , wherein the elution in step b) is carried out using a gradient composition of acetonitrile and orthophosphoric acid buffer with an increasing composition of acetonitrile from about 95% orthophosphoric acid buffer and about 5% acetonitrile at 0 minutes to a composition of about 70%

orthophosphoric acid buffer and about 30% acetonitrile at about 180 minutes.

5. The process of claim 1 , wherein fractions having a bivalirudin purity greater than about 98% are collected and pooled in step c).

6. The process of claim , wherein the elution in step f) is carried out using a linear gradient of water and acetonitrile.

7. The process of claim 1 , wherein the elution in step f) is carried out using a linear gradient of water and acetonitrile with an increasing composition of acetonitrile from about 100% water and about 0% acetonitrile at 0 minutes, to a composition of about 20% water and about 80% acetonitrile at about 40 minutes.

8. The process of claim 1 , wherein step f) further comprises pooling collected fractions of desired bivalirudin purity.

9. The process of claim 8, further comprising: g) analyzing the fractions of step f) for their content of trifluoroacetic acid, and if required adjusting the content;

h) evaporating the solution under vacuum at temperatures from about 15°C to about 20°C, to obtain a concentrated solution; and

i) lyophilizing the concentrated solution to obtain a lyophilized powder of bivalirudin.

10. The process of claim 1 , wherein the crude bivalirudin has purity less than about 70% by HPLC.

1 1 . The process of claim 1 , wherein the semi-purified bivalirudin has purity less than about 98% by HPLC and an individual impurity greater than about 1 %.

12. The process of any one of claims 1 to 1 1 , wherein the semi-purified bivalirudin in step a) is obtained by a process comprising:

i) loading crude bivalirudin onto a C- 8 RP-HPLC column;

ii) eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer;

iii) collecting the fractions of desired bivalirudin purity and pooling; and iv) optionally, concentrating the pooled fractions.

13. The process of claim 12, wherein step i) includes loading a solution of bivalirudin obtained by dissolving bivalirudin in an ammonium acetate buffer.

14. The process of claim 12, wherein the elution in step ii) is carried out using a mobile phase composition of about 84% ammonium acetate buffer and about 16% acetonitrile, by volume.

15. The process of claim 12, wherein the pooled fractions obtained in step iii) have a content of [D-phe¹²]-bivalirudin less than about 0.5% as determined using HPLC.

16. The process of any one of claims 1 to 1 1 , wherein the semi-purified bivalirudin in step a) is obtained by a process comprising:

i) loading crude bivalirudin onto an ion chromatography column;

ii) eluting bivalirudin from the column with a gradient of about 0.05% formic acid and a mixture of about 0.05% formic acid with about 0.5 M ammonium formate;

iii) collecting fractions of desired bivalirudin purity; and

iv) optionally concentrating the pooled fractions.

17. The process of claim 16, wherein step i) includes loading a solution of bivalirudin obtained by dissolving bivalirudin in a formic acid buffer, and adjusting the pH to about 4.1 using ammonia.

18. The process of claim 16, wherein the elution in step ii) is carried out using a gradient of about 0.05% formic acid and a mixture of about 0.05% formic acid with about 0.5 M ammonium formate.

19. The process of claim 16, wherein the column used in step i) is an ion chromatography column packed with a strong anion exchange resin with an matrix active group of -0-CH₂CHOHCH₂OCH2 CHOHCH₂N⁺(CH3)3.

20. A process for purifying bivalirudin, comprising:

a) loading crude bivalirudin onto a C-18 RP-HPLC column;

c) collecting the fractions of desired bivalirudin purity and pooling; d) optionally, concentrating the pooled fractions;

e) loading the pooled fractions of step d) onto a C-18 RP-HPLC column;

f) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ; g) collecting the fractions of desired bivalirudin purity and pooling; h) loading the pooled fractions of step g) onto a C-18 RP-HPLC column;

i) washing the column with about 0.1 % trifluoroacetic acid; and j) eluting the product from the column with a composition of water and acetonitrile.

21. A process for purifying bivalirudin comprising:

a) loading crude bivalirudin onto an ion chromatography column;

c) collecting fractions of desired bivalirudin purity;

d) loading the fractions from step c) onto a RP-HPLC column; e) eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer;

f) collecting the fractions of desired bivalirudin purity and pooling; g) optionally, concentrating the pooled fractions;

h) loading the pooled fractions from f) or g) onto a RP-HPLC column; i) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ; j) collecting the fractions of desired bivalirudin purity and pooling; k) optionally loading the pooled fractions onto a RP-HPLC column; I) washing the column with about 0.1 % trifluoroacetic acid; and m) eluting bivalirudin from the column with a composition of water and acetonitrile; and

n) isolating purified bivalirudin.

22. A process for purifying bivalirudin comprising:

a) loading bivalirudin onto an ion chromatography column;

b) eluting bivalirudin from the column with a gradient of 0.05% formic acid and a mixture of 0.05% formic acid with about 0.5 M ammonium formate;

c) collecting fractions of desired bivalirudin purity;

d) loading IC purified bivalirudin onto a C-18 RP-HPLC column;

e) eluting bivalirudin from the column with acetonitrile and ammonium acetate buffer;

f) collecting the fractions of desired bivalirudin purity and pooling; g) optionally concentrating the pooled fractions;

h) loading the pooled fractions from f) or g) onto a C-18 RP-HPLC column;

i) eluting bivalirudin from the column with acetonitrile and

orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ; j) collecting the fractions of desired bivalirudin purity and pooling; k) loading the pooled fractions onto the column;

I) washing the column with about 0.1 % trifluoroacetic acid;

m) eluting the product from the column with a composition of water and acetonitrile; and n) isolating purified bivalirudin.

23. Bivalirudin trifluoroacetate having purity greater than about 98.5%, and each of the impurities [Asp⁹]-bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGly]- bivalirudin, [D-Asn⁹]-bivalirudin, [D-phe¹²]-bivalirudin being present in amounts less than about 0.5% as determined using HPLC.

24. Bivalirudin having purity greater than about 98.5% and not more than about 0.5% of [D-Asn⁹]-bivalirudin.

25. Bivalirudin having purity greater than about 98.5% and not more than about 0.5% of [-Gly]-bivalirudin.

26. Bivalirudin trifluoroacetate, obtained by the process of any one of claims 1 , 20, 21 , or 22, having purity greater than about 98.5%, and each of the impurities

[Asp⁹]-bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGly]-bivalirudin, [D-Asn⁹]- bivalirudin, [D-phe¹²]-bivalirudin being present in amounts less than about 0.5% as determined using HPLC.