CN101538314A - Method for purifying Eptifibatide - Google Patents
Method for purifying Eptifibatide Download PDFInfo
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- CN101538314A CN101538314A CN200910104986A CN200910104986A CN101538314A CN 101538314 A CN101538314 A CN 101538314A CN 200910104986 A CN200910104986 A CN 200910104986A CN 200910104986 A CN200910104986 A CN 200910104986A CN 101538314 A CN101538314 A CN 101538314A
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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 is
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 causes by various activator capable of blocking, it is the strongest known specificity platelet aggregation inhibitor, be mainly used in and prevent the myocardial oxygen delivery arterial occlusion, heart attack, the sudden death that unstable angina pectoris, non-q wave myocardial infarction, coronary artery interventional therapy 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, the healthy volunteer be and can suppress among the patient of PCI art that fiber is former to be combined with adenosine diphosphate (ADP) activatory thrombocyte.To the acute coronary syndrome patient, adenosine diphosphate (ADP) inductive platelet aggregation is injected as far back as this product and promptly was suppressed in back 5 minutes, and it recovered normal in lasting 4～8 hours of administration phase.This product 180 quiet notes of μ g/kg dosage and all can reach with per minute 2 μ g/kg speed venoclysises and to suppress thrombocyte and focus on more than 80%.
Utilize the evaluation of citrate antithrombotics, this product suppresses the thrombocyte focussing force than the strong 2-4 of other non-chelating type antithrombotics doubly.This product can make the acute coronary syndrome patient and carry out PCI art patient's bleeding time 2-4 times of prolongation (comparing with its baseline value), recovers normal after stopping infusion this product in 1 hour.This product is not seen the sign of toxic reaction through the safety evaluation test of rat, rabbit and monkey, gives 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 GP IIb/IIIa receptor antagonist has been represented one of maximum progress of present intervention Cardiology.On a large scale, at random, contrast clinical trial has been confirmed its status in the heart trouble interventional therapy 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.Under special messenger's supervision, this product can be assisted with acetylsalicylic acid or heparin and be 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. the selectivity height acts on strong.3. no antigen almost itself can not cause allergic reaction.But in document of having delivered and patent, find no scale operation and have the report or the application of the purifying process of higher yields.
Summary of the invention
The object of the present invention is to provide a processing method that is suitable for the industrialization purifying leuprorelin, use reversed-phased high performace liquid chromatographic to change the salt purifying Eptifibatide in conjunction with anion exchange method, purity height and yield are good, reach industrialized requirement, solve the defective that prior art exists.
For achieving the above object, 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): with stationary phase is the alkyl silane bonded silica gel, moving phase comprise A mutually and 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): trifluoroacetate is changed into acetate and obtains 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 invention provides a processing method that is suitable for industrialization purifying Eptifibatide (Eptifibatide), use reversed-phased high performace liquid chromatographic to change the salt purifying Eptifibatide in conjunction with anion exchange method, 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 described in further details:
Embodiment 1
1, the first step purifying: get the B of a suitable size, pad one deck filter paper, the diatomite 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: will go up sample after the chromatographic column usefulness mobile phase A balance, 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 standby 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 sample 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 diatomite 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: will go up sample after the chromatographic column usefulness mobile phase A balance, 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 standby 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 sample 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 diatomite 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: will go up sample after the chromatographic column usefulness mobile phase A balance, 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 standby 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 sample 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 be in conjunction with concrete preferred implementation to further describing that the present invention did, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (4)
1, 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): with stationary phase is the alkyl silane bonded silica gel, moving phase comprise A mutually and 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): trifluoroacetate is changed into acetate and obtains satisfactory acetic acid Eptifibatide bulk drug with anion exchange method.
2, the method for purifying Eptifibatide as claimed in claim 1 is characterized in that: it is 2-3cm that diatomaceous thickness is housed in the described step 1).
3, as the method for right 1 described purifying Eptifibatide, it is characterized in that: 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.
4, the method for purifying Eptifibatide as claimed in claim 1 is characterized in that: the aqueous acetic acid concentration of anionresin in the described step 3) is 1-2%; Detect wavelength: 280nm.
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| CN 200910104986 CN101538314B (en) | 2009-01-13 | 2009-01-13 | Method for purifying Eptifibatide |
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| CN 200910104986 CN101538314B (en) | 2009-01-13 | 2009-01-13 | Method for purifying Eptifibatide |
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| CN101538314B CN101538314B (en) | 2012-10-03 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702321A (en) * | 2012-06-14 | 2012-10-03 | 吉尔生化（上海）有限公司 | Method for purifying eptifibatide acetate |
| CN102702320A (en) * | 2012-06-01 | 2012-10-03 | 深圳翰宇药业股份有限公司 | Method for preparing eptifibatide |
| 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 |
| CN106749526A (en) * | 2016-12-22 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of method of nine victory peptides 1 of low cost purifying |
| CN106831943A (en) * | 2016-12-22 | 2017-06-13 | 陕西慧康生物科技有限责任公司 | A kind of method of low cost purifying transdermal peptide |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222537C (en) * | 2002-11-14 | 2005-10-12 | 吉尔生化（上海）有限公司 | Preparing process for Eptifibatide |
| AU2005233603B2 (en) * | 2004-04-08 | 2011-07-21 | Millennium Pharmaceuticals, Inc. | Processes for preparing eptifibatide and pertinent intermediate compounds |
| CN1858060B (en) * | 2005-05-08 | 2010-09-29 | 周达明 | Process for preparing solid phase polypeptide synthetic eptifibatide |
-
2009
- 2009-01-13 CN CN 200910104986 patent/CN101538314B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102702320A (en) * | 2012-06-01 | 2012-10-03 | 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 |
| CN106749526A (en) * | 2016-12-22 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of method of nine victory peptides 1 of low cost purifying |
| CN106831943A (en) * | 2016-12-22 | 2017-06-13 | 陕西慧康生物科技有限责任公司 | A kind of method of low cost purifying transdermal peptide |
| 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 |
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| CN101538314B (en) | 2012-10-03 |
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