CN102977192A - Purification method of carbetocin - Google Patents
Purification method of carbetocin Download PDFInfo
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- CN102977192A CN102977192A CN2012105189078A CN201210518907A CN102977192A CN 102977192 A CN102977192 A CN 102977192A CN 2012105189078 A CN2012105189078 A CN 2012105189078A CN 201210518907 A CN201210518907 A CN 201210518907A CN 102977192 A CN102977192 A CN 102977192A
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Abstract
The invention discloses a purification method of carbetocin. The method comprises the following steps in sequence: purifying and synthesizing rough titanium through reversed-phase ion-pair chromatography, and removing an ion-pair agent through ion exchange resin, so as to obtain fine carbetocin peptide with purity of 99.5%. According to the purification method of carbetocin, the reversed-phase ion-pair chromatography is adopted, so that the purification yield and the purity of the carbetocin can be greatly improved.
Description
Technical field
The present invention relates to the purification technique field of polypeptide, relate in particular to the purification technique of carbetocin.
Background technology
Carbetocin (Carbetocin) is a kind of synthetic long-acting pitocin nonapeptide analogue with agonist character, to the not effect of nogestational uterus, but the uterus of gestation and the uterus of harsh product are had effective uterine contraction effect, and its pitocin clinical and pharmacological property and natural generation is similar.As pitocin, carbetocin is combined with the ocytocin receptor of uterine smooth muscle, causes that the rhythmicity in uterus is shunk, and on original contraction basis, increases its frequency and increases uterus tension force.Under non pregnant state, the ocytocin receptor content in uterus is very low, increases at pregnancy duration, reaches the peak during childbirth.Single dose intravenously administrable immediately after the cesarean section under epidural or the lumbar anesthesia is with prevention uterus tension force deficiency and postpartum hemorrhage.
No matter after being intravenous injection or intramuscular injection carbetocin, shrink rapidly in the uterus, can in two minutes, reach a clear and definite intensity.Therefore single dose intravenous injection carbetocin sustainable about hour to the active function in uterus is enough to prevent the postpartum hemorrhage in harsh postpartum.After giving carbetocin postpartum, all long than pitocin aspect amplitude in the frequency of shrinking, and to bleeding tendency being arranged, need extra use oxytocin person, the carbetocin better tolerance is the same with oxytocin effective even more effective.The medicine ergotocine is received at present clinical promotion uterus commonly used contracting, can cause feeling sick, vomiting, hypertension and coronary spasm etc.Carbetocin is a kind of long-acting class pitocin medicine.Compare with ergotocine, use carbetocin effective equally aspect prevention vaginal delivery women's postpartum hemorrhage, and feel sick, vomiting, hypertensive proportion significantly reduce.Therefore, carbetocin has wide market outlook in clinical application.
Carbetocin purification process commonly used generally includes reversed-phased high performace liquid chromatographic, gel method, ion exchange method etc., adopts any single method, all is difficult to separate obtaining high purity product (＜99%), perhaps yield lower (less than 50%).At present, seldom report relevant for the research of its separation and purification aspect, Cheng Shengqiang, the inferior equality of horse report in its research as elutriant, adopts the reversed-phased high performace liquid chromatographic purifying Carbetocin with phosphoric acid buffer, and adopt ion exchange method to turn salt, obtain Carbetocin, yield is 70%-75%, and purpose peptide purity is 98%.
About the purification process of synthetic carbetocin, mainly have 2 problems: 1. adopt single purification process, product purity is low both at home and abroad for analysis-by-synthesis; 2. adopt multiple means of purification, product yield is low.
Summary of the invention
The present invention is directed to the deficiency of existing technique, propose a kind of separation purification method of carbetocin, product purity is high, and purification yield is high.
In order to realize the foregoing invention purpose, the invention provides following technical scheme: adopt reversed phase ion to liquid phase chromatography, the advantage of reverse-phase chromatography and chromatography of ions is combined, reduce purification step, improve product purity, its method comprises following steps:
Step 1, adopt reversed phase ion to liquid phase chromatography, take reverse-phase chromatographic column as stationary phase, the finite concentration ion pair reagent aqueous solution is the mobile phase A phase, methyl alcohol or acetonitrile or ethanol are the Mobile phase B phase, carry out gradient elution, gradient B% is: 5-40%, preferred 25%-35%, minutes three sections collect the purpose peaks, be respectively the peak before, salable product, behind the peak, salable product are that purity reaches 99.5% part, peak fore portion retention time is less than the retention time of salable product, purity is 90.0%-99.5%, and rear section, peak retention time is greater than the retention time of salable product, and purity is 90.0%-99.5%.
Step 2, the above 1. step obtained the peak before, rear section, peak purifying respectively, adopt reversed phase ion to liquid phase chromatography, take reverse-phase chromatographic column as stationary phase, the finite concentration ion pair reagent aqueous solution is the mobile phase A phase, and methyl alcohol or acetonitrile or ethanol are the Mobile phase B phase, carry out gradient elution, gradient B% is: 5-40%, preferred 25%-35% merges the salable product that obtain, and obtaining purity is 99.5% the above object peptide.
Step 3, the salable product that obtain more than the general, adopt reversed phase ion to liquid phase chromatography, take reverse-phase chromatographic column as stationary phase, the certain density counterion aqueous solution is the A phase, and methyl alcohol or acetonitrile or ethanol are the Mobile phase B phase, carry out gradient elution, gradient B% is: 10-80%, preferred 30%-70% merges the salable product that obtain, and obtaining purity is 99.5% the above object peptide.
Elutriant is added counterion, comprise acetic acid, phosphoric acid, hydrochloric acid, trifluoroacetic acid etc., obtain the carbetocin of different salt forms.
Compare with existing technique, the present invention is simple to operate, and single step purification reaches 99.5% purity, and yield reaches 70%-75%, has obvious cost advantage.
Embodiment
Described herein, term " reversed-phase high-performance liquid chromatography " refers to take reverse phase filler as stationary phase, ion pair reagent is the chromatographic behavior of moving phase, or take ion-exchange packing as stationary phase, organic solvent is the chromatographic behavior of moving phase, purpose is the advantage of coupled ion chromatogram and reverse-phase chromatography, and the high efficiency separation purifying synthesizes carbetocin.
Described herein, term " ion pair reagent " comprises R-SO
3Na or R-SO
4Na, wherein R is selected from methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl.
Describe the present invention below in conjunction with specific embodiment, the description of this part only is exemplary and explanatory, should any restriction not arranged to protection scope of the present invention.
Embodiment 1:
1, pre-treatment: carbetocin to be purified is mixed with the water that concentration is 200-300mg/ml
Solution, via hole diameter are the filtering with microporous membrane of 0.45um, collect filtrate.
Reverse-phase chromatographic column pre-treatment: use 60% acetonitrile solution flushing chromatographic column 6min, wash 6min with 5% acetonitrile solution again.
The ion-exchange chromatography pre-treatment: concentration is the NaOH aqueous solution regeneration 6min of 2mol/L, is washed till neutrality with pure water again.
2, separation and purification: get filtrate, inject the chromatographic column of handling well and carry out separation and purification, to the purpose wash-out
It is totally three sections collections behind front, salable product, the peak that liquid is divided the peak, peak fore portion purity 90%-99.5%, and salable product part purity is greater than 99.5%, rear section, peak purity 90%-99.5%; Respectively with before the peak, the elutriant separation and purification of rear section, peak, chromatographic condition is identical with the separation and purification of primary sample.Merge salable product, for subsequent use.
Separation and purification chromatographic condition: chromatographic column: the chromatographic column take anti-phase C18 as stationary phase, the pillar specification is: 50mm * 250mm; The sodium heptanesulfonate of mobile phase A phase: 10mmol/L, phosphoric acid is transferred pH to 2.0, and the Mobile phase B phase: acetonitrile, flow velocity are 80ml/min; Gradient: in acetonitrile concentration, 25%-35%, the time is 40min; The detection wavelength is 220nm; Applied sample amount 1-1.2g
3, desalination, drying: will inject the good chromatographic column of pre-treatment by the carbetocin salable product after separation and purification, gradient elution is collected the purpose peptide; Carbetocin purpose peptide after the desalination is concentrated into 120-150mg/ml, and frozen drying obtains lyophilized powder.Product purity is 99.7%, and yield is 72.8%.
Desalination chromatographic condition: chromatographic column: Zeo-karb: pul resin D201, pillar diameter and length: 50mm * 400mm; Mobile phase A phase: 0.1% aqueous acetic acid, Mobile phase B phase: ethanol; Gradient: in alcohol concn, 30-70%, the time is 70min; The detection wavelength is 220nm; Applied sample amount 4.5-5.0g.
Embodiment 2:
1, pre-treatment: carbetocin to be purified is mixed with the water that concentration is 200-300mg/ml
Solution, via hole diameter are the filtering with microporous membrane of 0.45um, collect filtrate.
Reverse-phase chromatographic column pre-treatment: use 60% acetonitrile solution flushing chromatographic column 6min, wash 6min with 5% acetonitrile solution again.
The ion-exchange chromatography pre-treatment: concentration is the NaOH aqueous solution regeneration 6min of 2mol/L, is washed till neutrality with pure water again.
2, separation and purification: get filtrate, inject the chromatographic column of handling well and carry out separation and purification, to the purpose wash-out
It is totally three sections collections behind front, salable product, the peak that liquid is divided the peak, peak fore portion purity 90%-99.5%, and salable product part purity is greater than 99.5%, rear section, peak purity 90%-99.5%; Respectively with before the peak, the elutriant separation and purification of rear section, peak, chromatographic condition is identical with the separation and purification of primary sample.Merge salable product, for subsequent use.
Separation and purification chromatographic condition: chromatographic column: the chromatographic column take anti-phase C18 as stationary phase, the pillar specification is: 50mm * 250mm; The sodium laurylsulfonate of mobile phase A phase: 10mmol/L, phosphoric acid is transferred pH to 2.0, and the Mobile phase B phase: acetonitrile, flow velocity are 80ml/min; Gradient: in acetonitrile concentration, 25%-35%, the time is 40min; The detection wavelength is 220nm; Applied sample amount 1-1.2g
3, desalination, drying: will inject the good chromatographic column of pre-treatment by the carbetocin salable product after separation and purification, gradient elution is collected the purpose peptide; Carbetocin purpose peptide after the desalination is concentrated into 120-150mg/ml, and frozen drying obtains lyophilized powder.Product purity is 99.7%, and yield is 75.1%.
Desalination chromatographic condition: chromatographic column: Zeo-karb: pul resin D201, pillar diameter and length: 50mm * 400mm; Mobile phase A phase: 0.1% aqueous acetic acid, Mobile phase B phase: ethanol; Gradient: in alcohol concn, 30-70%, the time is 70min; The detection wavelength is 220nm; Applied sample amount 4.5-5.0g.
Embodiment 3:
1, pre-treatment: carbetocin to be purified is mixed with the water that concentration is 200-300mg/ml
Solution, via hole diameter are the filtering with microporous membrane of 0.45um, collect filtrate.
Reverse-phase chromatographic column pre-treatment: use 60% acetonitrile solution flushing chromatographic column 6min, wash 6min with 5% acetonitrile solution again.
The ion-exchange chromatography pre-treatment: concentration is the NaOH aqueous solution regeneration 6min of 2mol/L, is washed till neutrality with pure water again.
2, separation and purification: get filtrate, inject the chromatographic column of handling well and carry out separation and purification, to the purpose wash-out
It is totally three sections collections behind front, salable product, the peak that liquid is divided the peak, peak fore portion purity 90%-99.5%, and salable product part purity is greater than 99.5%, rear section, peak purity 90%-99.5%; Respectively with before the peak, the elutriant separation and purification of rear section, peak, chromatographic condition is identical with the separation and purification of primary sample.Merge salable product, for subsequent use.
Separation and purification chromatographic condition: chromatographic column: the chromatographic column take anti-phase C18 as stationary phase, the pillar specification is: 50mm * 250mm; The sodium heptanesulfonate of mobile phase A phase: 10mmol/L, phosphoric acid is transferred pH to 7.0, and the Mobile phase B phase: acetonitrile, flow velocity are 80ml/min; Gradient: in acetonitrile concentration, 26%-36%, the time is 40min; The detection wavelength is 220nm; Applied sample amount 1-1.2g
3, desalination, drying: will inject the good chromatographic column of pre-treatment by the carbetocin salable product after separation and purification, gradient elution is collected the purpose peptide; Carbetocin purpose peptide after the desalination is concentrated into 120-150mg/ml, and frozen drying obtains lyophilized powder.Product purity is 99.7%, and yield is 71.6%.
Desalination chromatographic condition: chromatographic column: Zeo-karb: pul resin D301, pillar diameter and length: 50mm * 400mm; Mobile phase A phase: 0.1% aqueous acetic acid, Mobile phase B phase: ethanol; Gradient: in alcohol concn, 30-70%, the time is 70min; The detection wavelength is 220nm; Applied sample amount 4.5-5.0g.
To sum up, reversed phase ion is to the synthetic carbetocin of liquid phase chromatography separation and purification, and product purity is high, and yield is high, is fit to the separation and purification of synthetic polypeptide.
It below only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. the purification process of a carbetocin may further comprise the steps:
1., adopt reversed phase ion to the thick peptide of liquid phase chromatography separation and purification carbetocin: take the reverse-phase chromatography filler as stationary phase, the certain density ion pair reagent aqueous solution is the mobile phase A phase, methyl alcohol or acetonitrile or ethanol are the Mobile phase B phase, obtain purity behind the purifying and be the smart peptide more than 99.5%; 2., adopt ion exchange chromatography to remove smart peptide intermediate ion to reagent and other salt: smart peptide solution is transferred to suitable pH, so that carbetocin is combined on the ion exchange resin, remove inorganic salt and most of ion pair reagent with pure water rinsing, re-use a certain proportion of aqueous ethanolic solution gradient elution, ion pair reagent and carbetocin are separated, obtain salt-free carbetocin aqueous ethanolic solution.
2. method as claimed in claim 1, it is characterized in that: described reverse-phase chromatography filler comprises C18, C8, C4, but is not limited only to C18, C8, C4.
3. method as claimed in claim 1, it is characterized in that: described ion pair reagent comprises R-SO
3Na or R-SO
4Na, wherein R is selected from methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl.
4. such as method as described in claim 1 or 3, it is characterized in that: the ion pair reagent concentration of aqueous solution is 0.002-0.100mol/L, and mobile phase A phase pH value is 1.5-8.0.
5. method as claimed in claim 1, it is characterized in that: ion exchange resin comprises Zeo-karb and anionite-exchange resin.
6. such as method as described in claim 1 or 5, it is characterized in that: when adopting Zeo-karb, smart peptide solution pH value transfers to 1.0-3.5; When adopting anionite-exchange resin, smart peptide solution pH value transfers to 5.0-10.0.
7. method as claimed in claim 1, it is characterized in that: behind the ion exchange column, adopt the method for gradient elution on the smart peptide solution, wash-out phase A is aqueous acetic acid mutually, aqueous hydrochloric acid, trifluoroacetic acid aqueous solution, phosphate aqueous solution, wash-out phase B are methyl alcohol mutually, ethanol.
8. method as claimed in claim 1 is characterized in that: collect carbetocin purpose peak, obtain carbetocin.
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| CN2012105189078A CN102977192A (en) | 2012-12-06 | 2012-12-06 | Purification method of carbetocin |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435687A (en) * | 2013-09-05 | 2013-12-11 | 杭州诺泰制药技术有限公司 | Method for purifying carbetocin |
| 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 |
| WO2019032946A1 (en) | 2017-08-11 | 2019-02-14 | Ferring B.V. | Method of manufacturing of oxytocin |
| WO2019030412A1 (en) | 2017-08-11 | 2019-02-14 | Ferring B.V. | Method of manufacturing a pharmaceutical composition |
| CN110016072A (en) * | 2019-05-07 | 2019-07-16 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin |
| CN110308222A (en) * | 2019-07-17 | 2019-10-08 | 武汉赛沃医药科技有限公司 | A kind of related substance detecting method of carbetocin bulk pharmaceutical chemicals |
| CN111060635A (en) * | 2019-11-27 | 2020-04-24 | 吉尔生化（上海）有限公司 | Analysis and detection method of carbetocin |
| CN112175045A (en) * | 2020-10-28 | 2021-01-05 | 合肥亿帆生物制药有限公司 | Method for purifying oxytocin |
| US10967040B2 (en) | 2018-09-20 | 2021-04-06 | Levo Therapeutics, Inc. | Methods of treating prader-willi syndrome with carbetocin |
| CN113252807A (en) * | 2021-04-15 | 2021-08-13 | 武汉大安制药有限公司 | High performance liquid chromatography separation method for oxytocin raw material medicine related substances |
| US11207373B2 (en) | 2018-09-20 | 2021-12-28 | Levo Therapeutics, Inc. | Agitation process for preparing a carbetocin drug product |
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| CN101531705A (en) * | 2009-04-21 | 2009-09-16 | 深圳市翰宇药业有限公司 | Method for purifying Carbetocin |
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| CN101531705A (en) * | 2009-04-21 | 2009-09-16 | 深圳市翰宇药业有限公司 | Method for purifying Carbetocin |
| CN102146122A (en) * | 2010-11-12 | 2011-08-10 | 深圳市健元医药科技有限公司 | Process for producing medicament with uterine contraction effect |
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435687B (en) * | 2013-09-05 | 2015-01-28 | 杭州阿德莱诺泰制药技术有限公司 | Method for purifying carbetocin |
| CN103435687A (en) * | 2013-09-05 | 2013-12-11 | 杭州诺泰制药技术有限公司 | Method for purifying carbetocin |
| 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 |
| WO2019032946A1 (en) | 2017-08-11 | 2019-02-14 | Ferring B.V. | Method of manufacturing of oxytocin |
| WO2019030412A1 (en) | 2017-08-11 | 2019-02-14 | Ferring B.V. | Method of manufacturing a pharmaceutical composition |
| US10967040B2 (en) | 2018-09-20 | 2021-04-06 | Levo Therapeutics, Inc. | Methods of treating prader-willi syndrome with carbetocin |
| US11844764B2 (en) | 2018-09-20 | 2023-12-19 | Acadia Pharmaceuticals, Inc. | Agitation process for preparing a carbetocin drug product |
| US11298399B2 (en) | 2018-09-20 | 2022-04-12 | Levo Therapeutics, Inc. | Carbetocin drug product and process for preparing same |
| US11207373B2 (en) | 2018-09-20 | 2021-12-28 | Levo Therapeutics, Inc. | Agitation process for preparing a carbetocin drug product |
| CN110016072B (en) * | 2019-05-07 | 2022-03-15 | 上海上药第一生化药业有限公司 | Method for refining oxytocin |
| CN110016072A (en) * | 2019-05-07 | 2019-07-16 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin |
| CN110308222A (en) * | 2019-07-17 | 2019-10-08 | 武汉赛沃医药科技有限公司 | A kind of related substance detecting method of carbetocin bulk pharmaceutical chemicals |
| CN111060635A (en) * | 2019-11-27 | 2020-04-24 | 吉尔生化（上海）有限公司 | Analysis and detection method of carbetocin |
| CN112175045A (en) * | 2020-10-28 | 2021-01-05 | 合肥亿帆生物制药有限公司 | Method for purifying oxytocin |
| CN113252807A (en) * | 2021-04-15 | 2021-08-13 | 武汉大安制药有限公司 | High performance liquid chromatography separation method for oxytocin raw material medicine related substances |
| CN113252807B (en) * | 2021-04-15 | 2022-05-06 | 远大生命科学(武汉)有限公司 | High performance liquid chromatography separation method for oxytocin raw material medicine related substances |
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Application publication date: 20130320 |