CN102702321A - Method for purifying eptifibatide acetate - Google Patents
Method for purifying eptifibatide acetate Download PDFInfo
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- CN102702321A CN102702321A CN201210194644XA CN201210194644A CN102702321A CN 102702321 A CN102702321 A CN 102702321A CN 201210194644X A CN201210194644X A CN 201210194644XA CN 201210194644 A CN201210194644 A CN 201210194644A CN 102702321 A CN102702321 A CN 102702321A
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Abstract
The invention discloses a method for purifying eptifibatide acetate by a reversed phase high-performance liquid chromatography (RP-HPLC) method, and aims to mainly solve the technical problems that the conventional purification process has toxic raw materials and a high cost. The method for purifying the eptifibatide acetate comprises the following steps: 1) filtering crude eptifibatide acetate solution with a filtration membrane of 0.45 micron for further use, gradiently eluting and purifying the obtained solution by a stationary phase and a moving phase, wherein the stationary phase is a reverse phase silica gel column of octadecyl silane, and the moving phase is an A phase composed of pure water and trifluoroacetic acid with a volume percentage of 0.1%, and a B phase composed of chromatographically pure methanol and trifluoroacetic acid with a volume percentage of 0.1%, and collecting the peptide solution reaching a target peak; 2)exchanging and converting the peptide solution into acetate by an HPLC salt conversion method; and 3) depressurizing, rotatably steaming, concentrating, cooling and drying the finally obtained high-purity peptide solution to obtain powder peptide. The method is mainly used for the industrial purification of eptifibatide acetate.
Description
Technical field
The invention belongs to the reversed-phase HPLC technical field, especially relate to a kind of method of mass industrialization purifying Integrilin.
Background technology
Integrilin has another name called dust for non-crust skin, English name: Eptifibatide, molecular formula: C
35H49N
LlO
9S
2, its structure is:
Integrilin is an anti-platelet aggregation agent, is developed jointly by U.S. COR Therapeuties 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.Integrilin is annular skin; 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, what unstable angina pectoris, no Q ripple myocardial infarction, coronary artery PCI caused is broken dead.The clinical study result shows; Patient injects dust and treats for non-crust skin; 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) dust reduced 37% for non-crust skin than other agent groups.Death or incidence rate of myocardial infarction, dust replace non-crust skin group than other agent groups low 40%.
Integrilin 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 Adenosine diphosphate former times activatory thrombocyte.To the acute coronary syndrome patient, Adenosine diphosphate former times 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 hour of administration phase.This article 180 quiet notes of μ g/kg dosage and all can reach with PM 2 μ g/kg rate venoclysises and to suppress thrombocyte and focus on more than 80%.
Utilize the evaluation of structure rafter hydrochlorate antithrombotics, these article suppress the thrombocyte focussing force than the strong 2-4 of other non-huge legendary turtle mould assembly 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.Integrilin is applicable to the treatment acute coronary syndrome, comprises unstable angina pectoris or does not have Q ripple 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 Integrilin: 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.In document of having delivered and patent, about the technology of purifying seldom, nearly all be to be raw material with the acetonitrile, and acetonitrile toxicity is stronger, cost is higher, is used for large-scale industrial production and does not have advantage.
Summary of the invention
The object of the present invention is to provide a purification technique that is suitable for the large-scale industrial production Integrilin, mainly solve the existing toxic and cost technical problems of high of purifying process raw material.
For realizing above-mentioned purpose, technical scheme of the present invention is following: a kind of method of purifying Integrilin comprises the steps:
1) with the Integrilin crude product solution with membrane filtration, be the reverse phase silica gel post of octadecyl silane with the stationary phase, moving phase has two phases; Wherein trifluoroacetic acid aqueous solution is the A phase; The trifluoroacetic acid methanol solution is the B phase, carries out the gradient elution purifying, collects the peptide solution of purpose peak value;
2) adopt HPLC to change the exchange of salt method the high purity peptide solution behind the purifying and change into acetate;
3) final high purity peptide solution vacuum rotary steam concentrated frozen is dry, get Powdered finished product peptide.
More preferred scheme is: said filter membrane aperture is 0.45 μ m.
More preferred scheme is: the trifluoroacetic acid methanol solution of said B phase is 0.1% trifluoroacetic acid for chromatographically pure methyl alcohol adds volume percent.
More preferred scheme is: during the gradient elution purifying, the volume percent of B phase is 35-85%, and the detection wavelength is 220nm.
More preferred scheme is: the volume percent of described mobile phase A phase trifluoroacetic acid is 0.1%.
More preferred scheme is: described HPLC changes the salt method, the two phase liquid of the acetum of volume percent 0.5% and chromatographically pure methyl alcohol, and chromatographic column is the reverse phase silica gel post of tetraalkyl silane group silica gel or octadecyl silane.
The invention has the beneficial effects as follows: the purification technique that is suitable for the industrialization Integrilin that the present invention proposes; In HPLC method purge process with methyl alcohol as organic solvent moving phase; And change the exchange of salt method with HPLC and change salt, HPLC purity can not only be obtained greater than 99.0% smart peptide, and the toxicity of organic solvent in the production process can be effectively reduced; Reduce cost, thereby reach the purpose of large-scale industrial production.
Embodiment
Embodiment 1
1. sample preparation: it is ultrasonic to the liquid clarification in Integrilin oxidation liquid, to add a little methyl alcohol, collects filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 5cm * 25cm. moving phase: A mutually: the trifluoroacetic acid aqueous solution of volume percent 0.1%; B phase: the trifluoroacetic acid methanol solution of volume percent 0.1%.Flow velocity: 60-80ml/min.Detect wavelength: 220nm.Gradient: B%:35-85%, 30-50min.Sample size is 3.5-5g;
Purge process: chromatographic column is gone up appearance with 35% the B back of balancing each other, and applied sample amount is the 1.5-2.0L sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. commentaries on classics salt: use the deionized water rinsing stationary phase more than 60% to be the reverse phase silica gel post of octadecyl silane; Wash to effluent be again with the acetate aqueous solution and the chromatographically pure methyl alcohol two phase liquid balance chromatographic column of volume percent 0.5% after the neutrality; Treat that beginning to go up appearance after the chromatographic column balance thoroughly changes salt; The purpose peptide solution of applied sample amount for having collected; Linear gradient elution: the methanol solution of 60-90min volume percent 30-90% again behind the acetate aqueous solution of 10-60min volume percent 90-95%, collect whole target peaks;
4. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, and lyophilize gets purity greater than 99.0% Integrilin, purification yield 78.2% then.
Embodiment 2
1. sample preparation: it is ultrasonic to the liquid clarification in Integrilin oxidation liquid, to add a little methyl alcohol, collects filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 10cm * 25cm. moving phase: A mutually: the trifluoroacetic acid aqueous solution of volume percent 0.1%; B phase: the trifluoroacetic acid methanol solution of volume percent 0.1%.Flow velocity: 180-220ml/min.Detect wavelength: 220nm.Gradient: B%:35-85%, 30-50min.Sample size is 10-15g;
Purge process: chromatographic column is gone up appearance with 35% the B back of balancing each other, and applied sample amount is the 4-5L sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. commentaries on classics salt: use the deionized water rinsing stationary phase more than 60% to be the reverse phase silica gel post of octadecyl silane; Wash to effluent be again with the acetate aqueous solution and the chromatographically pure methyl alcohol two phase liquid balance chromatographic column of volume percent 0.5% after the neutrality; Treat that beginning to go up appearance after the chromatographic column balance thoroughly changes salt; The purpose peptide solution of applied sample amount for having collected; Linear gradient elution: the methanol solution of 60-90min volume percent 30-90% again behind the acetate aqueous solution of 10-60min volume percent 90-95%, collect whole target peaks;
4. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, and lyophilize gets purity greater than 99.0% Integrilin, purification yield 77.8% then.
Embodiment 3
1. sample preparation: it is ultrasonic to the liquid clarification in Integrilin oxidation liquid, to add a little methyl alcohol, collects filtrate for later use after using the aperture as the 0.45um membrane filtration;
2. purifying
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 15cm * 25cm. moving phase: A mutually: the trifluoroacetic acid aqueous solution of volume percent 0.1%; B phase: the trifluoroacetic acid methanol solution of volume percent 0.1%.Flow velocity: 650-850ml/min.Detect wavelength: 220nm.Gradient: B%:35-85%, 30-50min.Sample size is 15-20g;
Purge process: chromatographic column is gone up appearance with the B of volume percent 35% back of balancing each other, and applied sample amount is the 8-10L sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution;
3. commentaries on classics salt: use the deionized water rinsing stationary phase more than 60% to be the reverse phase silica gel post of octadecyl silane; Wash to effluent be again with the acetate aqueous solution and the chromatographically pure methyl alcohol two phase liquid balance chromatographic column of volume percent 0.5% after the neutrality; Treat that beginning to go up appearance after the chromatographic column balance thoroughly changes salt; The purpose peptide solution of applied sample amount for having collected; Linear gradient elution: the methanol solution of 60-90min volume percent 30-90% again behind the acetate aqueous solution of 10-60min volume percent 90-95%, collect whole target peaks;
4. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, and lyophilize gets purity greater than 99.0% Integrilin, purification yield 77.5% then.
Claims (5)
1. the method for a purifying Integrilin is characterized in that comprising the steps:
1) with the Integrilin crude product solution with membrane filtration, be the reverse phase silica gel post of octadecyl silane with the stationary phase, moving phase has two phases; Wherein trifluoroacetic acid aqueous solution is the A phase; The trifluoroacetic acid methanol solution is the B phase, carries out the gradient elution purifying, collects the peptide solution of purpose peak value;
2) adopt HPLC to change the exchange of salt method the high purity peptide solution behind the purifying and change into acetate;
3) final high purity peptide solution vacuum rotary steam concentrated frozen is dry, get Powdered finished product peptide.
2. the method for a kind of purifying Integrilin according to claim 1 is characterized in that: said step 1) filter membrane aperture is 0.45 μ m.
3. the method for a kind of purifying Integrilin according to claim 1 is characterized in that: Mobile phase B adds the trifluoroacetic acid solution of volume percent 0.1% in the said step 1) for chromatographically pure methyl alcohol.
4. the method for a kind of purifying Integrilin according to claim 1 is characterized in that: the volume percent of said step 1) A phase trifluoroacetic acid is 0.1%, and during the gradient elution purifying, the volume percent of B phase is 35-85%, and the detection wavelength is 220nm.
5. the method for a kind of purifying Integrilin according to claim 1; It is characterized in that: described step 2) HPLC changes the salt method; Moving phase is the acetum of volume percent 0.5% and the two phase liquid of chromatographically pure methyl alcohol, and chromatographic column is the reverse phase silica gel post of octadecyl silane.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104829695A (en) * | 2015-05-04 | 2015-08-12 | 吉尔生化（上海）有限公司 | Purifying method for alarelin |
| 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 |
Citations (4)
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| CN101538314A (en) * | 2009-01-13 | 2009-09-23 | 深圳市翰宇药业有限公司 | Method for purifying Eptifibatide |
| CN101787071A (en) * | 2010-02-26 | 2010-07-28 | 深圳翰宇药业股份有限公司 | Purification method of vapreotide |
| CN101981048A (en) * | 2008-02-06 | 2011-02-23 | 拜康有限公司 | A method of purifying a peptide |
| CN102471368A (en) * | 2009-08-11 | 2012-05-23 | 拜康有限公司 | Chromatographic processes and purified compounds thereof |
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2012
- 2012-06-14 CN CN201210194644XA patent/CN102702321A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101981048A (en) * | 2008-02-06 | 2011-02-23 | 拜康有限公司 | A method of purifying a peptide |
| CN101538314A (en) * | 2009-01-13 | 2009-09-23 | 深圳市翰宇药业有限公司 | Method for purifying Eptifibatide |
| CN102471368A (en) * | 2009-08-11 | 2012-05-23 | 拜康有限公司 | Chromatographic processes and purified compounds thereof |
| CN101787071A (en) * | 2010-02-26 | 2010-07-28 | 深圳翰宇药业股份有限公司 | Purification method of vapreotide |
Non-Patent Citations (1)
| Title |
|---|
| 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）》, vol. 46, no. 1, 31 January 2007 (2007-01-31), pages 100 - 103 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104829695A (en) * | 2015-05-04 | 2015-08-12 | 吉尔生化（上海）有限公司 | Purifying method for alarelin |
| CN104829695B (en) * | 2015-05-04 | 2018-09-07 | 吉尔生化（上海）有限公司 | A method of purifying alarelin |
| 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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Application publication date: 20121003 |