CN101538314B - Method for purifying Eptifibatide - Google Patents
Method for purifying Eptifibatide Download PDFInfo
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- CN101538314B CN101538314B CN 200910104986 CN200910104986A CN101538314B CN 101538314 B CN101538314 B CN 101538314B CN 200910104986 CN200910104986 CN 200910104986 CN 200910104986 A CN200910104986 A CN 200910104986A CN 101538314 B CN101538314 B CN 101538314B
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Abstract
The invention discloses a method for purifying Eptifibatide, which comprises the following steps of: 1) conducting suction filtration with a Buchner funnel containing diatomite to remove slightly soluble impurities; 2) conducting gradient elution and purification with the fixed phase being alkylsilane bonded silica, the phase A of a mobile phase being trifluoroacetic acid aqueous solution and the phase B thereof being chromatographic grade acetonitrile, wherein the mobile phase includes phase A and phase B; and collecting the peptide solution at a target peak value, and conducting reduced pressure distillation and concentration; and 3) using anion exchange method for converting the trifluoroacetate into acetate, thus obtaining the acetic acid Eptifibatide bulk drug which meets the requirement. In the invention, the method which is applicable to the industrialized purification of Eptifibatide is provided; reversed phase high-performance liquid chromatography is used and combined with the anion exchange method for converting the trifluoroacetate into acetate so as to purify the Eptifibatide; and the purity is high, and the purification yield of the yield reaches more than 70 percent and meets the industrialization requirement.
Description
Technical field
The invention belongs to the HPLC technical field, especially relate to the method for a kind of mass-producing purifying Eptifibatide (Eptifibatide).
Background technology
Eptifibatide has another name called Integrilin, Eptifibatide, English name: Eptifibatide, molecular formula: C
35H
49N
11O
9S
2, CAS accession number 148031-34-9, its structure does
Eptifibatide is an anti-platelet aggregation agent, is developed jointly by U.S. COR Therapeutics company and U.S. Schering-Plough company.In July, 1998 by Schering-Plough company with trade(brand)name Intrifiban in U.S.'s Initial Public Offering, went on the market in Europe with trade(brand)name Intrifiban in 1999.Different name IntrifibanTM, Sch-60936, C68-22, SB-1.
Eptifibatide is a cyclic peptide; It is platelet glycoprotein GPIIb/IIIa receptor antagonist; The platelet aggregation reaction that is caused by various activator capable of blocking is a strongest known specificity platelet aggregation inhibitor, is mainly used in and prevents the myocardial oxygen delivery arterial occlusion; Heart attack, the sudden death that unstable angina pectoris, non-q wave myocardial infarction, coronary artery PCI cause.The clinical study result shows; Patient injects Eptifibatide and treats; 48 hours after surgery, the associating terminal point incidence of death or serious problems (main terminal point index comprises: heart attack, case fatality rate and needs are emergency treatment intervention or thromboembolism treatment rate once more) Eptifibatide reduced 37% than other agent groups.Death or incidence rate of myocardial infarction, the Eptifibatide group is than other agent groups low 40%.
Eptifibatide is dose-dependently ground and suppresses external hematoblastic gathering in the acute coronary syndrome patient, can suppress in healthy volunteer and the patient who does the PCI art that fiber is former to be combined with ADP activatory thrombocyte.To the acute coronary syndrome patient, ADP inductive platelet aggregation promptly was suppressed as far back as the injection of these article in back 5 minutes, and it recovered normal in lasting 4～8 hours of administration phase.This article 180 quiet notes of μ g/kg dosage and all can reach with PM 2 μ g/kg speed venoclysises and to suppress thrombocyte and focus on more than 80%.
Utilize the evaluation of citrate antithrombotics, these article suppress the thrombocyte focussing force than the strong 2-4 of other non-chelating type antithrombotics doubly.The bleeding time that these article can make the acute coronary syndrome patient and carry out PCI art patient prolongs 2-4 doubly (comparing with its baseline value), stop infusion originally article recover normal in back 1 hour.These article are not seen the sign of toxic reaction through the safety evaluation test of rat, rabbit and monkey, give the continuous venoclysis of monkey 28 days, and every day, dosage was not seen the toxicity performance up to 7.2mg/kg yet.
Platelet glycoprotein GPIIb/IIIa receptor antagonist has been represented one of maximum progress of present intervention property Cardiology.On a large scale, at random, contrast clinical trial has been confirmed its status in the heart trouble PCI undisputedly; It can approximately reduce patients'perioperative ischemic complication 50%～60%; And, can not obvious increase bleeding episode incidence when the heparin using dosage is suitable.
Eptifibatide is applicable to the treatment acute coronary syndrome, comprises unstable angina pectoris or non-q wave myocardial infarction (both pectoralgia continued outbreak, ECG change and/or the rising of cardiovascular systems enzyme in 24 hours); Also can be used for comprising angioplasty or atherosclerotic plaque surgical blanking through skin coronary artery interventional therapy.Generally be not used in the angioplasty of no acute coronary syndrome.Supervise down the special messenger, these article can be assisted with Frosst) or heparin and used.The advantage of Eptifibatide: 1. the retardance to platelet glycoprotein IIb/IIIa acceptor is a reversible, in case untoward reaction drug withdrawal immediately occurs, untoward reaction is light.2. selectivity is high, and effect is strong.3. no antigen almost itself can not cause allergic reaction.But in document of having delivered and patent, do not find the report or the application that scale operation are arranged and have the purifying process of higher yields.
Summary of the invention
The object of the present invention is to provide a process method that is suitable for the industrialization purifying leuprorelin; Use reversed-phased high performace liquid chromatographic to combine anion exchange method to change the salt purifying Eptifibatide; Purity height and yield are good, reach industrialized requirement, solve the defective that prior art exists.
For realizing above-mentioned purpose, the present invention takes following technical scheme:
A kind of method of purifying Eptifibatide may further comprise the steps:
1): carry out suction filtration to remove insoluble impurity with diatomaceous B is housed;
2): use stationary phase to be the alkyl silane bonded silica gel, moving phase comprise A mutually with B mutually, described A is that trifluoroacetic acid aqueous solution, B are trifluoroacetic acid aqueous solution mutually mutually, carries out the gradient elution purifying; The peptide solution vacuum rotary steam of collecting the purpose peak value concentrates;
3): change into trifluoroacetate acetate and obtain satisfactory acetic acid Eptifibatide bulk drug with anion exchange method.
Preferred scheme is: it is 2-3cm that diatomaceous thickness is housed in the described step 1).
More preferred scheme is: trifluoroacetic acid aqueous solution concentration is 0.1% described step 2); The B of described gradient elution is 15%～30% mutually; The detection wavelength is 280nm.
More preferred scheme is: the aqueous acetic acid concentration of anionresin in the described step 3) is 1-2%; Detect wavelength: 280nm.
The present invention compared with prior art has following advantage and beneficial effect:
The present invention provides a process method that is suitable for industrialization purifying Eptifibatide (Eptifibatide); Use reversed-phased high performace liquid chromatographic to combine anion exchange method to change the salt purifying Eptifibatide; Purity height and yield purification yield reach industrialized requirement up to more than 70%.
Embodiment
Below in conjunction with specific embodiment the present invention is explained further details:
Embodiment 1
1, the first step purifying: get the B of a suitable size, pad one deck filter paper, the zeyssatite of the thick 2cm-3cm of layer overlay fills up one deck filter paper above more then.Pour Eptifibatide oxidation liquid into funnel and carry out decompress filter, collect filtrate for later use.
2, the second step purifying: purification condition: chromatographic column: with the alkyl silane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 70-80ml/min.Detect wavelength: 280nm.Gradient: B%:15%～30% (30min), sample size are 1.0-1.2g.
Purge process: chromatographic column is gone up appearance after with the mobile phase A balance, and applied sample amount is the 2.0-2.4L sample solution.Linear gradient elution 30min collects the purpose peak, and the purpose peptide solution of collecting is subsequent use after water temperature is no more than 37 ℃ of following vacuum rotary steams to be concentrated into about 50-70mg/mL.
3, the 3rd step was changeed salt: purification condition: chromatographic column filler is an anionite-exchange resin: Amberlite IRA-93, pillar diameter and length are: 5cm * 25cm.Moving phase: 1-2% aqueous acetic acid.Flow velocity: 50-60ml/min.Detect wavelength: 280nm.Sample size is 1.5-2.0g.
Change the salt process: chromatographic column is gone up appearance after with deionized water balance, and applied sample amount is the 30-40ml sample solution.1-2% aqueous acetic acid wash-out 50min collects the purpose peak, the purpose peptide solution of collecting is merged to revolve go to suitable big small vessels after steaming is concentrated into about 100-150mg/mL.After carry out lyophilize, can obtain purity greater than 98.5% satisfactory Eptifibatide, purification yield reaches 73.2%.
Embodiment 2
1, the first step purifying: get the B of a suitable size, pad one deck filter paper, the zeyssatite of the thick 2cm-3cm of layer overlay fills up one deck filter paper above more then.Pour Eptifibatide oxidation liquid into funnel and carry out decompress filter, collect filtrate for later use.
2, the second step purifying: purification condition: chromatographic column: with the alkyl silane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 15cm * 25cm.Moving phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 400-500ml/min.Detect wavelength: 280nm.Gradient: B%:15%～30% (30min).Sample size is 15-18g.
Purge process: chromatographic column is gone up appearance after with the mobile phase A balance, and applied sample amount is the 15-20L sample solution.Linear gradient elution 30min collects the purpose peak, and the purpose peptide solution of collecting is subsequent use after water temperature is no more than 37 ℃ of following vacuum rotary steams to be concentrated into about 50-70mg/mL.
3, the 3rd step was changeed salt: purification condition: chromatographic column filler is an anionite-exchange resin: Amberlite IRA-93, pillar diameter and length are: 15cm * 45cm.Moving phase: 1-2% aqueous acetic acid.Flow velocity: 150-170ml/min.Detect wavelength: 280nm.Sample size is 6.0-8.0g.
Change the salt process: chromatographic column is gone up appearance after with deionized water balance, and applied sample amount is the 160-180ml sample solution.1-2% aqueous acetic acid wash-out 75min collects the purpose peak, the purpose peptide solution of collecting is merged to revolve go to suitable big small vessels after steaming is concentrated into about 100-150mg/mL.After carry out lyophilize, can obtain purity greater than 98.5% satisfactory Eptifibatide, purification yield reaches 72.7%.
Embodiment 3
1, the first step purifying: get the B of a suitable size, pad one deck filter paper, the zeyssatite of the thick 2cm-3cm of layer overlay fills up one deck filter paper above more then.Pour Eptifibatide oxidation liquid into funnel and carry out decompress filter, collect filtrate for later use.
2, the second step purifying: purification condition: chromatographic column: with the alkyl silane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 30cm * 25cm.Moving phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 1500-2000ml/min.Detect wavelength: 280nm.Gradient: B%:15%～30% (30min).Sample size is 60-80g.
Purge process: chromatographic column is gone up appearance after with the mobile phase A balance, and applied sample amount is the 60-80L sample solution.Linear gradient elution 30min collects the purpose peak, and the purpose peptide solution of collecting is subsequent use after water temperature is no more than 37 ℃ of following vacuum rotary steams to be concentrated into about 50-70mg/mL.
3, the 3rd step was changeed salt: the 3rd step was changeed salt: purification condition: chromatographic column filler is an anionite-exchange resin: Amberlite IRA-93, pillar diameter and length are: 30cm * 70cm.Moving phase: 1-2% aqueous acetic acid.Flow velocity: 750-850ml/min.Detect wavelength: 280nm.Sample size is 40.0-50.0g.
Change the salt process: chromatographic column is gone up appearance after with deionized water balance, and applied sample amount is the 1000-1100ml sample solution.1-2% aqueous acetic acid wash-out 160min collects the purpose peak, the purpose peptide solution of collecting is merged to revolve go to suitable big small vessels after steaming is concentrated into about 100-150mg/mL.After carry out lyophilize, can obtain purity greater than 98.5% satisfactory Eptifibatide, purification yield reaches 71.3%.
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, can also make some simple deduction or replace, all should be regarded as belonging to protection scope of the present invention.
Claims (3)
1. the method for a purifying Eptifibatide may further comprise the steps:
Step 1, purifying are got the B of a suitable size, pad one deck filter paper, and the zeyssatite of thick 2 cm of layer overlay-3cm fills up one deck filter paper above more then, pours Eptifibatide oxidation liquid into funnel and carries out decompress filter, collects filtrate for later use;
Step 2, being further purified, is the chromatographic column of stationary phase with the alkyl silane bonded silica gel, and pillar diameter and length are: 5 cm * 25 cm; As the mobile phase A phase, is 70-80 ml/min with the flow velocity with 0.1% trifluoroacetic acid aqueous solution, and detecting wavelength is 280 nm; Gradient is in 30 min 15%～30%, sample size be the trifluoroacetic acid aqueous solution of 1.0-1.2 g as the Mobile phase B phase, chromatographic column is gone up after with the mobile phase A balance kind; Applied sample amount is the 2.0-2.4L sample solution; Linear gradient elution 30min collects the purpose peak, and the purpose peptide solution of collecting is subsequent use after water temperature is no more than 37 ℃ of following vacuum rotary steams to be concentrated into about 50-70 mg/mL;
Step 3, change salt, chromatographic column is gone up appearance after with deionized water balance, applied sample amount is the 30-40ml sample solution; With 1-2% aqueous acetic acid wash-out 50min, collect the purpose peak, the purpose peptide solution of collecting is merged to revolve to steam go to suitable big small vessels after being concentrated into about 100-150 mg/mL; Carry out lyophilize then; Can obtain purity greater than 98.5% satisfactory Eptifibatide, purification yield reaches 73.2%, and the purification condition of wherein said step 3 is:
Chromatographic column filler is an anionite-exchange resin: Amberlite IRA-93;
Pillar diameter and length are: 5 cm * 25 cm;
Moving phase: 1-2% aqueous acetic acid;
Flow velocity: 50-60 ml/min;
Detect wavelength: 280 nm;
Sample size is 1.5-2.0g.
2. the method for a purifying Eptifibatide may further comprise the steps:
Step 1, purifying are got the B of a suitable size, pad one deck filter paper, and the zeyssatite of thick 2 cm of layer overlay-3cm fills up one deck filter paper above more then, pours Eptifibatide oxidation liquid into funnel and carries out decompress filter, collects filtrate for later use;
Step 2, being further purified, is the chromatographic column of stationary phase with the alkyl silane bonded silica gel, and pillar diameter and length are: 15 cm * 25 cm; As the mobile phase A phase, is 400-500 ml/min with the flow velocity with 0.1% trifluoroacetic acid aqueous solution, and detecting wavelength is 280 nm; Gradient is in 30 min 15%～30%, sample size be the trifluoroacetic acid aqueous solution of 15-18 g as the Mobile phase B phase, chromatographic column is gone up after with the mobile phase A balance kind; Applied sample amount is the 15-20L sample solution; Linear gradient elution 30min collects the purpose peak, and the purpose peptide solution of collecting is subsequent use after water temperature is no more than 37 ℃ of following vacuum rotary steams to be concentrated into about 50-70 mg/mL;
Step 3, change salt, chromatographic column is gone up appearance after with deionized water balance, applied sample amount is the 160-180ml sample solution; With 1-2% aqueous acetic acid wash-out 75 min, collect the purpose peak, the purpose peptide solution of collecting is merged to revolve to steam go to suitable big small vessels after being concentrated into about 100-150 mg/mL; Carry out lyophilize then; Can obtain purity greater than 98.5% satisfactory Eptifibatide, purification yield reaches 72.7%, and the purification condition of wherein said step 3 is:
Chromatographic column filler is an anionite-exchange resin: Amberlite IRA-93;
Pillar diameter and length are: 15 cm * 45 cm;
Moving phase: 1-2% aqueous acetic acid;
Flow velocity: 150-170ml/min;
Detect wavelength: 280 nm;
Sample size is 6.0-8.0g.
3. the method for a purifying Eptifibatide may further comprise the steps:
Step 1, purifying are got the B of a suitable size, pad one deck filter paper, and the zeyssatite of thick 2 cm of layer overlay-3cm fills up one deck filter paper above more then, pours Eptifibatide oxidation liquid into funnel and carries out decompress filter, collects filtrate for later use;
Step 2, being further purified, is the chromatographic column of stationary phase with the alkyl silane bonded silica gel, and pillar diameter and length are: 30cm * 25 cm; As the mobile phase A phase, is 1500-2000 ml/min with the flow velocity with 0.1% trifluoroacetic acid aqueous solution, and detecting wavelength is 280 nm; Gradient is in 30 min 15%～30%, sample size be the trifluoroacetic acid aqueous solution of 60-80g as the Mobile phase B phase, chromatographic column is gone up after with the mobile phase A balance kind; Applied sample amount is the 60-80L sample solution; Linear gradient elution 30min collects the purpose peak, and the purpose peptide solution of collecting is subsequent use after water temperature is no more than 37 ℃ of following vacuum rotary steams to be concentrated into about 50-70 mg/mL;
Step 3, change salt, chromatographic column is gone up appearance after with deionized water balance, applied sample amount is the 1000-1100ml sample solution; With 1-2% aqueous acetic acid wash-out 160min, collect the purpose peak, the purpose peptide solution of collecting is merged to revolve to steam go to suitable big small vessels after being concentrated into about 100-150 mg/mL; Carry out lyophilize then; Can obtain purity greater than 98.5% satisfactory Eptifibatide, purification yield reaches 71.3%, and the purification condition of wherein said step 3 is:
Chromatographic column filler is an anionite-exchange resin: Amberlite IRA-93;
Pillar diameter and length are: 30 cm * 70 cm;
Moving phase: 1-2% aqueous acetic acid;
Flow velocity: 750-850 ml/min;
Detect wavelength: 280 nm;
Sample size is 40.0-50.0g.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102702320B (en) * | 2012-06-01 | 2013-08-21 | 深圳翰宇药业股份有限公司 | Method for preparing eptifibatide |
| CN102702321A (en) * | 2012-06-14 | 2012-10-03 | 吉尔生化（上海）有限公司 | Method for purifying eptifibatide acetate |
| CN102875639A (en) * | 2012-09-26 | 2013-01-16 | 深圳翰宇药业股份有限公司 | Solid-phase synthetic method of peptide and peptide synthesized by same |
| CN106831943B (en) * | 2016-12-22 | 2020-05-19 | 陕西慧康生物科技有限责任公司 | Method for purifying transdermal peptide at low cost |
| CN106749526B (en) * | 2016-12-22 | 2020-06-19 | 陕西慧康生物科技有限责任公司 | Method for purifying nonapeptide-1 at low cost |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500805A (en) * | 2002-11-14 | 2004-06-02 | 吉尔生化（上海）有限公司 | Preparing process for Eptifibatide |
| CN1858060A (en) * | 2005-05-08 | 2006-11-08 | 周达明 | Process for preparing solid phase polypeptide synthetic eptifibatide |
| CN1972960A (en) * | 2004-04-08 | 2007-05-30 | 米伦纽姆医药公司 | Processes for preparing eptifibatide |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500805A (en) * | 2002-11-14 | 2004-06-02 | 吉尔生化（上海）有限公司 | Preparing process for Eptifibatide |
| CN1972960A (en) * | 2004-04-08 | 2007-05-30 | 米伦纽姆医药公司 | Processes for preparing eptifibatide |
| CN1858060A (en) * | 2005-05-08 | 2006-11-08 | 周达明 | Process for preparing solid phase polypeptide synthetic eptifibatide |
Non-Patent Citations (2)
| Title |
|---|
| Stéphane roux, et al..Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches.《Journal of Peptide Science》.2008,第14卷354-359. * |
| 熊瑛等.Eptifibatide的固相合成及分离纯化.《厦门大学学报(自然科学版)》.2007,第46卷(第1期),100-103. * |
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