WO2011071799A2 - Purification de la bivalirudine - Google Patents

Purification de la bivalirudine Download PDF

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Publication number
WO2011071799A2
WO2011071799A2 PCT/US2010/059045 US2010059045W WO2011071799A2 WO 2011071799 A2 WO2011071799 A2 WO 2011071799A2 US 2010059045 W US2010059045 W US 2010059045W WO 2011071799 A2 WO2011071799 A2 WO 2011071799A2
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Prior art keywords
bivalirudin
column
acetonitrile
fractions
purity
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PCT/US2010/059045
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English (en)
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WO2011071799A3 (fr
Inventor
Pratap Reddy Padi
Santhana Krishnan Srinivasan
Venugopal Rao Dama
Basanthi Devi
Chakravarthula Kalyan Narasimham Nalla
Ramesh Bochha
Parameswara Reddy Koche
Yagna Kiran Kumar Komaravolu
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Dr. Reddy's Laboratories Ltd.
Dr. Reddy's Laboratories, Inc.
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Publication of WO2011071799A2 publication Critical patent/WO2011071799A2/fr
Publication of WO2011071799A3 publication Critical patent/WO2011071799A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin

Definitions

  • aspects of the present application relate to processes for the purifying bivalirudin. Further aspects of the application relate to substantially pure
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • 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 Ci 8 RP-HPLC column, to obtain fractions containing bivalirudin at a purity >97.5%, containing not more than 0.5% Asp 9 -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 Ci 8 or C 8 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.
  • TFA trifluoroacetic acid
  • 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.
  • 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:
  • orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;
  • the present application provides process for the purification of bivalirudin, embodiments comprising:
  • step e) 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.
  • the present application provides process for the purification of bivalirudin, embodiments comprising:
  • acetonitrile and orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;
  • the present application provides substantially pure bivalirudin having purity greater than about 98.5% and each of the impurities [Asp 9 ]-bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGly]-bivalirudin, [D-Asn 9 ]- bivalirudin, and [D-phe 12 ]-bivalirudin being present at less than about 1%.
  • the present application provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [D-Asn 9 ]-bivalirudin.
  • the present application provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [-Gly]-bivalirudin.
  • 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:
  • orthophosphoric acid buffer having a pH value from about 2.9 to about 3.1 ;
  • 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.
  • RP reverse phase
  • 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.
  • 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.
  • 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.
  • all the fractions of similar purity from each of the cycle are pooled and taken forward to the next step of the purification process.
  • 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.
  • 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.
  • 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.
  • the present invention provides processes for the purification of bivalirudin, embodiments comprising:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the purification by RP-HPLC using a composition of acetonitrile and ammonium acetate buffer is performed on a crude sample having [D-phe 12 ]-bivalirudin more than about 1 %, by HPLC, to provide bivalirudin having [D-phe 12 ]-bivalirudin at less than about 0.5% by HPLC.
  • 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.
  • Bivalirudin having [D-phe 12 ]-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.
  • the present application provides process for the purification of bivalirudin comprising:
  • bivalirudin obtained by the above processes has a purity greater than about 98.5%.
  • bivalirudin obtained by the above processes has not more than about 0.5% [D-Asn 9 ]-bivalirudin impurity.
  • the present application provides a process for the purification of bivalirudin comprising:
  • 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;
  • the present application provides process for the purification of bivalirudin, embodiments comprising:
  • Step a) involves loading crude bivalirudin onto an Ion chromatography column.
  • a column with Q-sepharose Fast Flow resin (a strong anion exchange resin with a matrix active group of -0-CH 2 CHOHCH20CH 2 CHOHCH 2 N + (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.
  • 0.05% formic acid in water (pH 4.1 ) is used as formic acid buffer.
  • 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.
  • 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%.
  • 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.
  • the ion chromatographic purification facilitates in reducing the impurities like [+Gly]-bivalirudin, [-Gly]-bivalirudin, [D-phe 12 ]- bivalirudin, from bivalirudin.
  • the present invention provides processes for preparing bivalirudin substantially free of the process related impurities using the ion chromatography purification as described above.
  • 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.
  • the ion chromatography step acts as a pre-purification step for the next Reverse-Phase purification step.
  • 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 Ci 8 RP-HPLC column (5 ⁇ , 100 A).
  • Step b) involves eluting bivalirudin fractions from a RP-HPLC column with a composition of acetonitrile and ammonium acetate buffer.
  • ammonium acetate buffer used in the process of the present invention is prepared by dissolving ammonium acetate in water.
  • 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.
  • 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.
  • 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 fraction of desired bivalirudin purity.
  • 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.
  • step g) 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%.
  • the Bivalirudin obtained by the above purification process has the content of [D-phe 12 ]-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.
  • 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.
  • 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 .
  • the base used is triethylamine.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present application provides processes for the purification of bivalirudin, embodiments comprising:
  • the present application provides a process for purifying bivalirudin comprising:
  • 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;
  • 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.
  • the present application provides bivalirudin having purity greater than about 98.5%, and each of the impurities [Asp 9 ]- bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGlyJ-bivalirudin, [D-Asn 9 ]- bivalirudin, [D-phe 12 ]-bivalirudin being present in amounts less than about 0.5% as determined using HPLC.
  • the present application provides bivalirudin having purity greater than about 98.5% and less than about 0.5% [D-Asn 9 ]-bivalirudin, less than about [D-phe 12 ]-bivalirudin, obtained by the purification processes as described in the present application.
  • the present application provides substantially pure bivalirudin having purity greater than about 98.5%, and each of the impurities [Asp 9 ]-bivalirudin, [+Gly]-bivalirudin, [-Gly]-bivalirudin, [DiGlyJ-bivalirudin, [D-Asn 9 ]- bivalirudin, [D-phe 12 ]-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%.
  • the present application provides substantially pure bivalirudin having purity greater than about 98.5% and not more than about 0.5% [D-Asn 9 ]-bivalirudin.
  • 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.
  • the present application provides an HPLC method for the analysis of bivalirudin samples, wherein the analysis is performed using a Waters system, equipped with ZorbaxTM SB C-18, 200x4-6 mm, 1 .8 ⁇ or equivalent column with a guard column of HypersilTM Gold 10 mmx4.6 mm, 3 ⁇ . 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 QTM 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 ⁇ membrane filter.
  • Mobile phase B a mixture of methanol and acetonitrile in the volume ratio of 750:250 and filtered through a 0.22 ⁇ membrane filter.
  • the [D-Asn 9 ]-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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ filter paper.
  • 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.
  • 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.
  • 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-phe 12 ]- bivalirudin: 0.54%; [+Gly]-bivalirudin: 2.3%; [-Gly]-bivalirudin: 2.4%; [Di-Gly]- bivalirudin: 0.2%; [Asp 9 ]-bivalirudin: 0.8%) in buffer A, stir for 10-15 minutes and filter the solution.
  • 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-phe 12 ]-bivalirudin: 0.54%; [+Gly]-bivalirudin: 2.3%; [- Gly]-bivalirudin: 2.4%; [Di-Gly]-bivalirudin: 0.2%; [Asp 9 ]-bivalirudin-0.8%) in 100 ml_ of buffer A, sonicate for 10-15 minutes and filter.
  • 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.
  • 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.

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Abstract

La présente invention concerne des procédés de purification de la bivalirudine. L'invention concerne également la bivalirudine essentiellement pure.
PCT/US2010/059045 2009-12-11 2010-12-06 Purification de la bivalirudine WO2011071799A2 (fr)

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IN3077CH2009 2009-12-11
IN3077/CHE/2009 2009-12-11
US31286510P 2010-03-11 2010-03-11
US61/312,865 2010-03-11

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WO2011071799A3 WO2011071799A3 (fr) 2011-10-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104877024A (zh) * 2015-06-29 2015-09-02 海南中和药业有限公司 一种比伐芦定原料药的纯化工艺
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
CN116087389A (zh) * 2022-12-28 2023-05-09 江苏诺泰澳赛诺生物制药股份有限公司 一种注射用比伐芦定有关物质的hplc测定方法
CN117088966A (zh) * 2022-12-29 2023-11-21 江苏诺泰澳赛诺生物制药股份有限公司 一种比伐芦定杂质的合成方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070093423A1 (en) * 2005-09-14 2007-04-26 Avi Tovi Process for production of Bivalirudin
US20080051558A1 (en) * 2006-03-10 2008-02-28 Yiming Zhou Method of preparing bivalirudin
US20080287650A1 (en) * 2007-03-01 2008-11-20 Avi Tovi High purity peptides
US20090062511A1 (en) * 2007-09-05 2009-03-05 Raghavendracharyulu Venkata Palle Process for the preparation of bivalirudin and its pharmaceutical compositions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070093423A1 (en) * 2005-09-14 2007-04-26 Avi Tovi Process for production of Bivalirudin
US20080051558A1 (en) * 2006-03-10 2008-02-28 Yiming Zhou Method of preparing bivalirudin
US20080287650A1 (en) * 2007-03-01 2008-11-20 Avi Tovi High purity peptides
US20090062511A1 (en) * 2007-09-05 2009-03-05 Raghavendracharyulu Venkata Palle Process for the preparation of bivalirudin and its pharmaceutical compositions

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
CN104877024A (zh) * 2015-06-29 2015-09-02 海南中和药业有限公司 一种比伐芦定原料药的纯化工艺
CN104877024B (zh) * 2015-06-29 2018-09-18 海南中和药业股份有限公司 一种比伐芦定原料药的纯化工艺
CN116087389A (zh) * 2022-12-28 2023-05-09 江苏诺泰澳赛诺生物制药股份有限公司 一种注射用比伐芦定有关物质的hplc测定方法
CN116087389B (zh) * 2022-12-28 2023-11-10 江苏诺泰澳赛诺生物制药股份有限公司 一种注射用比伐芦定有关物质的hplc测定方法
CN117088966A (zh) * 2022-12-29 2023-11-21 江苏诺泰澳赛诺生物制药股份有限公司 一种比伐芦定杂质的合成方法

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