CN107759667A - A kind of isolation and purification method of Cetrorelix - Google Patents
A kind of isolation and purification method of Cetrorelix Download PDFInfo
- Publication number
- CN107759667A CN107759667A CN201711231965.1A CN201711231965A CN107759667A CN 107759667 A CN107759667 A CN 107759667A CN 201711231965 A CN201711231965 A CN 201711231965A CN 107759667 A CN107759667 A CN 107759667A
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- China
- Prior art keywords
- cetrorelix
- isolation
- purification method
- solution
- purifying
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
Abstract
The invention provides a kind of method for efficiently separating purifying Cetrorelix, comprise the following steps, Cetrorelix crude extract is dissolved, filtered；Solution after above-mentioned filtering is loaded in the chromatographic column equipped with equal grain reverse phase silica gel and chromatographed；Target product is eluted as mobile phase using acetic acid aqueous solution and organic solvent；The solution of Fractional Collections purpose peak value, satisfactory component liquid is collected.The depth that the present invention is used for Cetrorelix is consummate, it is only necessary to a step chromatographic purifying can meet Cetrorelix list it is miscellaneous be less than 0.1% requirement, purify high income and stablize, while separation method of the present invention is simple and convenient, available for large-scale production, substantially reduces production cost.
Description
Technical field
The present invention relates to medicine purification art, particularly a kind of method for efficiently separating purifying Cetrorelix.
Background technology
Cetrorelix (Cetrorelix) is a kind of luteinizing hormone-releasing hormone (LHRH) antagonist.LHRH can be thin with hypophysis
The membrane receptor of born of the same parents combines.Cetrorelix and these acceptors of the emulative combinations of endogenous LH RH, so as to control promoting sexual gland hormone
Secretion.Cetrorelix can be by the secretion of HPG approach dependence hormone always, so as to reach indirect suppression
Growth of cancers or prevention, alleviation, the purpose for treating other illnesss, prostate cancer and breast available for treatment treatment to hormone-sensitive
Gland cancer and some benign gynecological disorders (endometriosis, fibroid and endometrium are thinning).Cetrorelix and GNRH
Activator is compared, and has the advantages that short treating period, adverse reaction are few, can effectively reduce OHSS incidences.
Cetrorelix is decapeptide：
Ac-D-2Nal1-D-4Cl-Phe2-D-3Pal3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-D-Ala10-
NH2, structural formula is as follows：
Patent, document etc. are mostly synthetic method patent to the report of Cetrorelix both at home and abroad at present, and isolation and purification method is fresh
Have been reported that.CN201510332163.4 provides a kind of synthetic method of Cetrorelix, which describes anti-phase using C18 or C8
Column chromatography separating purification, mobile phase are 0.05%-0.5% hydrochloric acid or phosphoric acid or the trifluoroacetic aqueous solution and acetonitrile solution, sample
Product peak turns salt after merging, but this method process is more, causes the reduction of complex operation yield.It is therefore desirable to Cetrorelix
Further research is done in separation, purifying and preparation.
The content of the invention
It is an object of the invention to provide a kind of method for efficiently separating purifying Cetrorelix, it is only necessary to which a step chromatographic purifying is
Can meet Cetrorelix list it is miscellaneous be less than 0.1% requirement, purify high income and stablize, while separation method of the present invention is simply square
Just, available for large-scale production, production cost is substantially reduced.
To reach above-mentioned purpose, technical scheme is as follows：A kind of isolation and purification method of Cetrorelix, its feature
It is, Cetrorelix crude extract is dissolved, filtered；Cetrorelix solution after above-mentioned filtering is loaded to and is equipped with
Chromatographed in the chromatographic column of UniSil microballoons；Using acidic aqueous solution and organic solvent as mobile phase to Cetrorelix solution
Carry out gradient elution；The Cetrorelix solution of Fractional Collections purpose peak value, satisfactory component liquid is collected.
Preferably, the acidic aqueous solution is aqueous acetic acid.
Preferably, the model of the UniSil microballoons is C18 or C8.
Preferably, the organic solvent is methanol, ethanol, acetonitrile, acetone, isopropanol.
Preferably, before loading, chromatographic column is balanced, first high concentration organic phase is cleaned, after be less than with concentration
20% organic phase is balanced.
Preferably, the gradient elution process is that the mobile phase for being first 10-20% with second eyeball content rinses 1-5 cylinder
Product, then the mobile phase main elution peak for being 20-70% with second eyeball content.
UniSil C18 or UniSil C8 are that particle diameter is respectively 10 μm, aperturePolymer microsphere, the microballoon tool
There are the particle size strictly controlled and aperture structure (such as Fig. 1), for microballoon monodispersed, that there is pore passage structure, make its conduct
There is good specific aim during chromatograph packing material.
The present invention using organic solvent and the aqueous solution containing 1% acetic acid as mobile phase, only can with 6-10 column volume
The elution of Cetrorelix main peak is completed, therefore mobile phase used in this process is safe and pollution-free and cost is relatively low, and the stream used
Dynamic phasor is less, and purification cycle is shorter.
The present invention is using monodispersed UniSil C18 or UniSil C8 as stationary phase, it is only necessary to which step chromatography can make
Cetrorelix purity is higher than 99.5%, and list is miscellaneous to be less than 0.1%, and yield is up to 70%.
Therefore, the inventive method isolates and purifies Cetrorelix, and not only purity is high, high income and stably, and simple to operate
Convenient, stationary phase used is repeatable to be utilized, and flowing phasor used is saved less, substantially reduces cost.
Brief description of the drawings
Fig. 1 is the stereoscan photograph for UniSil C18 or UniSil the C8 microballoons that embodiment 1 uses.
Fig. 2 is the efficient liquid phase chromatographic analysis of the Cetrorelix crude extract before embodiment 1 purifies.
Fig. 3 is the efficient liquid phase chromatographic analysis of the Cetrorelix of embodiment 1 after purification.
Embodiment
Technical scheme is further described with reference to specific embodiment, but the present invention is not limited to this
A little embodiments.
A kind of isolation and purification method of Cetrorelix, Cetrorelix crude extract is dissolved, filtered；By above-mentioned filtering
Cetrorelix solution afterwards, which is loaded in the chromatographic column equipped with UniSil microballoons, to be chromatographed；Using acidic aqueous solution and organic molten
Agent carries out gradient elution as mobile phase to Cetrorelix solution；The Cetrorelix solution of Fractional Collections purpose peak value, to meeting
It is required that component liquid collected.
Preferably, the acidic aqueous solution is aqueous acetic acid.
Preferably, the model of the UniSil microballoons is C18 or C8.
Preferably, the organic solvent is methanol, ethanol, acetonitrile, acetone, isopropanol.
Preferably, before loading, chromatographic column is balanced, first high concentration organic phase is cleaned, after be less than with concentration
20% organic phase is balanced.
Preferably, the gradient elution process is that the mobile phase for being first 10-20% with second eyeball content rinses 1-5 cylinder
Product, then the mobile phase main elution peak for being 20-70% with second eyeball content.
Embodiment 1
The Cetrorelix crude extract for taking purity to be 95.46%, 1% acetic acid is added, dissolving, adds water, adds acetonitrile, solution
Middle Cetrorelix content is 10mg/mL.It is 0.45 μm of membrane filtration with aperture after solution clarification, it is stand-by collects filtrate.Using
4.6 × 250mm chromatographic column, UniSilC18 or C8 microballoons (Suzhou Nano-micro Technology Co., Ltd.'s production) are used as chromatography column packing,
Packed column volume is 4.2ml.To chromatographic column carry out post before pre-process, first cleaned with high concentration organic phase, after be less than with concentration
20% organic phase is balanced.Then ladder is carried out as mobile phase using 1% aqueous acetic acid (A phases) and second eyeball (B phases)
Degree elution, flow control is in 1ml/min.Gradient elution process is first to be rinsed 20 minutes with 20% B phases, after in 14 minutes
Concentration gradient is improved to 70%B by 20%B, the solution of Fractional Collections purpose peak value, satisfactory component liquid is collected,
Through efficient liquid phase chromatographic analysis, the purity of Cetrorelix is more than 99.8% in eluent, and list is miscellaneous to be less than 0.1%, and yield is more than
70%.
Fig. 2 is the efficient liquid phase chromatographic analysis of the Cetrorelix crude extract before purification, it is seen that has certain impurity.Fig. 3
It is the Cetrorelix efficient liquid phase chromatographic analysis after purification, it is seen that impurity is considerably less, and miscellaneous peak is very small.
Embodiment 2
The Cetrorelix crude extract for taking purity to be 95.46%, 1% acetic acid is added, dissolving, adds water, adds acetonitrile, solution
Middle Cetrorelix content is 10mg/mL.It is 0.45 μm of membrane filtration with aperture after solution clarification, it is stand-by collects filtrate.Using
4.6 × 250mm chromatographic column, UniSilC18 or C8 microballoons (Suzhou Nano-micro Technology Co., Ltd.'s production) are used as chromatography column packing,
Packed column volume is 4.2ml.Pre-process before carrying out post to chromatographic column, made using 1% aqueous acetic acid (A phases) and second eyeball (B phases)
Gradient elution is carried out for mobile phase, flow control is in 1ml/min.Gradient elution process is first to be rinsed 8 minutes with 10% B phases,
Eluted, the solution of Fractional Collections purpose peak value, satisfactory component liquid is collected, through efficient liquid phase with 23%B phases afterwards
Chromatography, the purity of Cetrorelix is more than 99.8% in eluent, it is single it is miscellaneous is less than 0.1%, yield is more than 70%.
The depth that the present invention is used for Cetrorelix is consummate, it is only necessary to which a step chromatographic purifying can meet that Cetrorelix list is miscellaneous and be less than
0.1% requirement, purify high income and stablize, while separation method of the present invention is simple and convenient, available for large-scale production, significantly
Reduce production cost.
Above-described is only the preferred embodiment of the present invention, it is noted that for one of ordinary skill in the art
For, without departing from the concept of the premise of the invention, various modifications and improvements can be made, these belong to the present invention
Protection domain.
Claims (10)
1. a kind of isolation and purification method of Cetrorelix, it is characterised in that comprise the following steps：Cetrorelix crude extract is entered
Row dissolving, filtering；Cetrorelix solution after above-mentioned filtering is loaded in the chromatographic column equipped with UniSil microballoons and chromatographed；
Gradient elution is carried out to Cetrorelix solution as mobile phase using acidic aqueous solution and organic solvent；Fractional Collections purpose peak value
Cetrorelix solution, satisfactory component liquid is collected, the Cetrorelix purified.
2. the isolation and purification method of Cetrorelix as claimed in claim 1, it is characterised in that the acidic aqueous solution is acetic acid
The aqueous solution.
3. the isolation and purification method of Cetrorelix as claimed in claim 1, it is characterised in that the model of the UniSil microballoons
It is C18 or C8.
4. the isolation and purification method of Cetrorelix as claimed in claim 1, it is characterised in that the organic solvent be methanol,
Ethanol, acetonitrile, acetone, isopropanol.
5. the isolation and purification method of Cetrorelix as claimed in claim 1, it is characterised in that before loading, carried out to chromatographic column
Balance, first high concentration organic phase cleaned, after be balanced with organic phase of the concentration less than 20%.
6. the isolation and purification method of Cetrorelix as claimed in claim 1, it is characterised in that the gradient elution process is,
The mobile phase for being first 10-20% with second eyeball content rinses 1-5 column volume, then the mobile phase for being 20-70% with second eyeball content is washed
De- main peak.
7. the isolation and purification method of the Cetrorelix as described in claim 1~6 is any, it is characterised in that the Cetrorelix
The purity of crude extract is more than 95%.
8. the isolation and purification method of the Cetrorelix as described in claim 1~6 is any, it is characterised in that the west of the purifying
Qu Ruike purity is more than 99.5%.
9. the isolation and purification method of the Cetrorelix as described in claim 1~6 is any, it is characterised in that the west of the purifying
Qu Ruike purity is more than 99.8%.
10. the isolation and purification method of the Cetrorelix as described in claim 1~6 is any, it is characterised in that the west of the purifying
Qu Ruike yield is more than 70%.
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| CN201711231965.1A CN107759667B (en) | 2017-11-29 | 2017-11-29 | Separation and purification method of cetrorelix |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109651301A (en) * | 2019-01-16 | 2019-04-19 | 苏州纳微科技股份有限公司 | A kind of purification process of orlistat |
| CN114276414A (en) * | 2021-12-30 | 2022-04-05 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying cetrorelix |
| CN114456092A (en) * | 2022-01-14 | 2022-05-10 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for separating and purifying cinacalcet intermediate impurities |
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| CN101284863A (en) * | 2008-06-06 | 2008-10-15 | 吉尔生化（上海）有限公司 | Preparation method of solid phase synthesis cetrorelix |
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| DE19813849A1 (en) * | 1998-03-27 | 1999-09-30 | Degussa | Simultaneous purification of oligopeptide hydrochlorides and conversion to acetate form |
| CN101284863A (en) * | 2008-06-06 | 2008-10-15 | 吉尔生化（上海）有限公司 | Preparation method of solid phase synthesis cetrorelix |
| CN104513286A (en) * | 2013-09-27 | 2015-04-15 | 博瑞生物医药技术(苏州)有限公司 | Method for separating and purifying fidaxomicin |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109651301A (en) * | 2019-01-16 | 2019-04-19 | 苏州纳微科技股份有限公司 | A kind of purification process of orlistat |
| CN109651301B (en) * | 2019-01-16 | 2023-05-12 | 苏州纳微科技股份有限公司 | Purification method of orlistat |
| CN114276414A (en) * | 2021-12-30 | 2022-04-05 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying cetrorelix |
| CN114456092A (en) * | 2022-01-14 | 2022-05-10 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for separating and purifying cinacalcet intermediate impurities |
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| CN107759667B (en) | 2021-07-27 |
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