CN103539841A - Separation and purification method of cyclohexanol peptide compound and salt thereof - Google Patents
Separation and purification method of cyclohexanol peptide compound and salt thereof Download PDFInfo
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- CN103539841A CN103539841A CN201210240054.6A CN201210240054A CN103539841A CN 103539841 A CN103539841 A CN 103539841A CN 201210240054 A CN201210240054 A CN 201210240054A CN 103539841 A CN103539841 A CN 103539841A
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- caspofungin
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- nvp
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Abstract
The invention relates to a purification method of a heterocyclic nitrogen peptide compound or salt thereof. The purification method comprises the following steps of loading a coarse compound to resin which is prepared by the polymerization of divinylbenzene and N-vinyl pyrrolidone; and eluting the resin with an aqueous solution and an organic solvent or a mixed solution of the organic solvent and water to obtain the pure heterocyclic nitrogen peptide compound or the pure salt thereof.
Description
Technical field
The present invention relates to the separation purification method of a kind of cyclohexyl peptide compounds or its pharmacy acceptable salt, relate to more specifically the separation purification method of Caspofungin.
Background technology
Caspofungin (Caspofungin) is that lung is read rhzomorph B
0(knob is Kangding B not
0) semi-synthetic derivative, there is spectrum anti-mycotic activity.Its acetate February calendar year 2001 in U.S.'s Initial Public Offering.The structural formula of Caspofungin is suc as formula shown in I:
Caspofungin is semi-synthetic lipopeptid (echinocandin) compound synthetic by Glarea Lozoyensis tunning and that come, and its preparation method is open in the many pieces of patents such as WO9421677, EP620232, WO9624613.By fermentation with semi-synthetic after Caspofungin must just can reach medicinal standard through separation and purification.
US5552521 discloses the method with preparation HPLC purifying Caspofungin altogether, take C18 post as stationary phase, take acetonitrile/water/acetic acid as eluent.It is 78% product that the method only can obtain purity.
US2009291996, also discloses the method for utilizing reversed-phase HPLC purifying Caspofungin in US2009324635.
The disclosed method of utilizing reversed-phase HPLC purifying Caspofungin in prior art, owing to using preparing chromatography system, expensive, and mostly be high-pressure system, be difficult for amplifying and produce.Caspofungin after purifying need to be again by the mode purifying of chromatogram or crystallization toward contact, separation and purification operation so repeatedly, too much more than refrigerating temperature, carry out, increased the possibility that Caspofungin impurity produces, be unfavorable for very much quality and the security control of the finished product.
Therefore it is a kind of more convenient to need, and purification efficiency is high, is suitable for the purification process of industrialized Caspofungin and salt thereof.
Summary of the invention
The separation purification method that the object of this invention is to provide a kind of cyclohexyl peptide compounds or its pharmacy acceptable salt, is characterized in that adopting the resin that divinylbenzene and NVP are polymerized to carry out separation and purification.
The preferred Caspofungin of described cyclohexyl peptide compounds.
The salt that described pharmacy acceptable salt comprises and following acid forms: hydrochloric acid, Hydrogen bromide, phosphoric acid, sulfuric acid, toxilic acid, citric acid, acetic acid, tartrate, succsinic acid, oxysuccinic acid etc., the salt preferably forming with acetic acid.
In the resin that described divinylbenzene and NVP are polymerized, the mol ratio of Vinylstyrene and NVP is 80-20:20-80; The mol ratio of Vinylstyrene and NVP is preferably 70-85:15-30.Described resin particle diameter is 10 μ m-100 μ m, preferably 20-50 μ m.The aperture of resin is 50-500nm, preferably 100-300mn.Resin specific surface area is 100-1000m
2/ gram, preferred 300-600m
2/ gram.
Further, separation and purification of the present invention comprises step:
(1) resin crude product loading of Caspofungin or its pharmacy acceptable salt to Ethenylbenzene and NVP being polymerized;
(2) with the mixed solvent of the aqueous solution, organic solvent or organic solvent and water, carry out wash-out, separating impurity, Caspofungin or its pharmacy acceptable salt of acquisition purifying.
In above-mentioned steps (1), described Caspofungin or the crude product of its pharmacy acceptable salt can be according to the disclosed method preparations of WO9421677.Described crude product refers under the testing conditions of high performance liquid chromatography (HPLC), the mixture of the content <90% of Caspofungin or its pharmacy acceptable salt, it can be the solution mixture that the damping fluid of Caspofungin dissolving crude product in water or PH≤7 obtains, also can be the reaction solution of the Caspofungin that obtains of any process, or this reaction solution be dissolved in the solution mixture that the damping fluid of water or PH≤7 obtains.For example, can be the direct reaction solution that contains Caspofungin preparing by the method for WO9421677.
Described loading, refers to crude product is contacted with resin, can adopt wet method loading or dry method loading.In the resin that described divinylbenzene and NVP are polymerized, the mol ratio of Vinylstyrene and NVP is 80-20:20-80; The mol ratio of Vinylstyrene and NVP is preferably 70-85:15-30.Described resin particle diameter is 10 μ m-100 μ m, preferably 20-50 μ m.The aperture of resin is 50-500nm, preferably 100-300mn.Resin specific surface area is 100-1000m
2/ gram, preferred 300-600m
2/ gram.The weight of crude product loading and the amount ratio of resin (w/v, grams per milliliter) are 1:1-1:300, preferably 1:50-1:150.
If adopt wet method loading, be that crude product is placed in to acidic aqueous solution, then contact with resin.Described acidic aqueous solution can be aqueous hydrochloric acid, aqueous acetic acid, trifluoroacetic acid aqueous solution etc.Preferred aqueous acetic acid.Aqueous acetic acid is selected from the aqueous acetic acid of 0.01%-5%, preferably the aqueous acetic acid of 0.1-2%.
In above-mentioned steps (2), the described aqueous solution is that acidic aqueous solution can be aqueous hydrochloric acid, aqueous acetic acid, trifluoroacetic acid aqueous solution etc.Preferred aqueous acetic acid.Described organic solvent is selected from C
1-C
4alcohol, C
1-C
4ketone, acetonitrile or tetrahydrofuran (THF); There is several C
1-C
4alcohol be selected from following one or more: methyl alcohol, ethanol, propyl alcohol, butanols, Virahol; C
1-C
4ketone be selected from acetone or butanone.Described organic solvent is preferably from ethanol, acetonitrile or acetone.Described elution process, the mixed solvent of the preferred acidic aqueous solution and organic solvent, wherein the content of organic solvent is 5%-95%(v/v), preferred 10%-60%(v/v), more preferably 10%-20%(v/v).
Described elution process, can carry out gradient elution by the different elutriant of concentration, or uses the elutriant wash-out of fixed concentration.
Further, after the elutriant that above-mentioned steps is obtained concentrates, can obtain Caspofungin or its pharmacy acceptable salt of purifying.
Of the present invention simple to operate, operating process is short, mild condition, and yield and the purity of purifying are high, have alleviated the difficulty of technological operation, have reduced production cost.Shorten the step of the purification process time of Caspofungin or its pharmacy acceptable salt, be conducive to stablize the effect that the finished product reach quality control.And the equipment that purge process is used is glass column, equipment requirements is low, simple to operate, is more suitable in suitability for industrialized production.Particularly applicant finds unexpectedly, when the resin that uses Ethenylbenzene of the present invention and NVP to be polymerized carries out the separation and purification of Caspofungin, the efficiency of purifying is very high, only, by once crossing column purification, just can reach more than 96% purity.
Embodiment
Below will further set forth the present invention by specific embodiment, but be not limited to scope of the present invention.In following embodiment, except as otherwise noted, the actual conditions of described experimental technique is conventionally according to the condition of normal condition or manufacturer's suggestion; Described raw material all obtains by commercially available purchase; Described per-cent, ratio, ratio or umber etc. calculate according to weight.
Embodiment 1
Adopt the disclosed synthetic route of US5552521, with knob Kangding B not
0for raw material, prepare Caspofungin crude product, HPLC detects purity approximately 50%.
Embodiment 2
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 0.1% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (Waters Oasis HLB, the 85:15) 100ml that process in advance, flow velocity 1L/h.With 0.1% acetum 400ml, rinse, then with 0.1% the aqueous acetic acid wash-out that contains 10% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.48g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 3
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 0.1% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (Waters Oasis HLB, the 80:20) 100ml that process in advance, flow velocity 1L/h.With 0.1% acetum 400ml, rinse, then with 0.1% the aqueous acetic acid wash-out that contains 10% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.48g caspofungin acetate, high performance liquid phase shows that purity purity is greater than 99%.
Embodiment 4
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 0.1% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (Waters Oasis HLB, the 70:30) 100ml that process in advance, flow velocity 1L/h.With 0.1% acetum 400ml, rinse, then with 0.1% the aqueous acetic acid wash-out that contains 10% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.49g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 5
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 0.5% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (Waters Oasis HLB, the 70:30) 100ml that process in advance, flow velocity 1L/h.With 0.5% acetum 400ml, rinse, then with 0.5% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.47g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 6
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 1% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (Waters Oasis HLB, the 70:30) 100ml that process in advance, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.48g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 7
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 2% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (Waters Oasis HLB, the 70:30) 100ml that process in advance, flow velocity 1L/h.With 2% acetum 400ml, rinse, then with 2% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.45g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 8
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 1% acetum (20ml), loading is to the divinylbenzene and the NVP fluoropolymer resin (70:30 that process in advance, according to the disclosed method preparation of CN102382227, particle diameter 30 μ m, specific surface area 600m
2/ gram, aperture 300nm) 100ml is upper, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.47g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 9
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 1% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (ProElut PLS, 70:30, the Dikma) 100ml that process in advance, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.48g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 10
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 1% acetum (20ml), loading is to the divinylbenzene and the NVP fluoropolymer resin (70:30 that process in advance, purchased from Suzhou Nano-Micro Bio-technology Co., Ltd., Uni 30BPC) 100ml is upper, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 10% acetone.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.46g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 11
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 1% acetum (20ml), and loading is upper to the divinylbenzene and NVP fluoropolymer resin (70:30, the Uni 30BPC) 100ml that process in advance, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.49g caspofungin acetate, high performance liquid phase shows that purity is greater than 99%.
Embodiment 12
The Caspofungin crude product 1g of preparation preparation in embodiment 1 is dissolved in 1% acetum (20ml), loading is to the divinylbenzene of processing in advance and NVP fluoropolymer resin (50:50, self-control, particle diameter 30 μ m, specific surface area 600m
2/ gram, aperture 300nm) 100ml is upper, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.49g caspofungin acetate, high performance liquid phase shows that purity is greater than 97%.
Embodiment 13
The Caspofungin crude product 1g of preparation in embodiment 1 is dissolved in 1% acetum (20ml), and loading is to the divinylbenzene of processing in advance and NVP fluoropolymer resin (20:80, self-control, particle diameter 30 μ m, specific surface area 600m
2/ gram, aperture 300nm) 100ml is upper, flow velocity 1L/h.With 1% acetum 400ml, rinse, then with 1% the aqueous acetic acid wash-out that contains 17.5% acetonitrile.Through Liquid Detection, merge elutriant, be concentrated into dryly, obtain 0.45g caspofungin acetate, high performance liquid phase shows that purity is greater than 95%.
Claims (11)
1. a separation purification method for Caspofungin or its pharmacy acceptable salt, is characterized in that adopting the resin that divinylbenzene and NVP are polymerized to carry out separation and purification.
2. method according to claim 1, is characterized in that, in the resin that described divinylbenzene and NVP are polymerized, the mol ratio of Vinylstyrene and NVP is 80-20:20-80.
3. method according to claim 2, is characterized in that, described Vinylstyrene and the mol ratio of NVP are 70-85:15-30.
4. method according to claim 3, is characterized in that, described resin particle diameter is 10 μ m-100 μ m; Aperture is 50-500nm; Specific surface area is 100-1000m
2/ gram.
5. method according to claim 4, is characterized in that, described resin particle diameter is 20-50 μ m; Aperture is 100-300nm; Specific surface area is 300-600m
2/ gram.
6. method according to claim 1, is characterized in that, described method comprises:
The resin that the crude product loading of Caspofungin or its pharmacy acceptable salt to Ethenylbenzene and NVP is polymerized;
With the mixed solvent of the aqueous solution, organic solvent or organic solvent and water, carry out wash-out, separating impurity, Caspofungin or its pharmacy acceptable salt of acquisition purifying.
7. method according to claim 6, is characterized in that, in step (1), the weight of crude product loading and the amount ratio of resin (w/v) are 1:1-1:300.
8. according to the method for claim 7, it is characterized in that, the weight of crude product loading and the amount ratio of resin (w/v) are 1:50-1:150.
9. method according to claim 6, is characterized in that, step (2) is carried out wash-out with the mixed solvent of organic solvent and water, and the content of organic solvent is 5%-95%(v/v).
10. according to the method described in claim 6 or 9, it is characterized in that, the aqueous solution described in step (2) is acidic aqueous solution; Described organic solvent is selected from C
1-C
4alcohol, C
1-C
4ketone, acetonitrile or tetrahydrofuran (THF).
11. methods according to claim 10, is characterized in that, the aqueous solution described in step (2) is aqueous acetic acid; Described organic solvent is selected from ethanol, acetonitrile or acetone.
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|---|---|---|---|
| CN201210240054.6A CN103539841A (en) | 2012-07-12 | 2012-07-12 | Separation and purification method of cyclohexanol peptide compound and salt thereof |
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|---|---|---|---|
| CN201210240054.6A CN103539841A (en) | 2012-07-12 | 2012-07-12 | Separation and purification method of cyclohexanol peptide compound and salt thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104910258A (en) * | 2015-06-23 | 2015-09-16 | 苏州纳微科技有限公司 | Method for finely purifying caspofungin |
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| CN101648994A (en) * | 2009-08-06 | 2010-02-17 | 上海天伟生物制药有限公司 | Azepine argireline or pharmaceutically acceptable salt thereof and preparation method and application thereof |
| CN102213656A (en) * | 2011-04-08 | 2011-10-12 | 江苏省农业科学院 | Method for extracting and purifying and detecting type B trichothecene toxin from cereals |
| CN102488886A (en) * | 2011-09-26 | 2012-06-13 | 上海天伟生物制药有限公司 | Caspofungin preparation with low impuritycontent and preparation method and application thereof |
-
2012
- 2012-07-12 CN CN201210240054.6A patent/CN103539841A/en active Pending
Patent Citations (3)
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| CN101648994A (en) * | 2009-08-06 | 2010-02-17 | 上海天伟生物制药有限公司 | Azepine argireline or pharmaceutically acceptable salt thereof and preparation method and application thereof |
| CN102213656A (en) * | 2011-04-08 | 2011-10-12 | 江苏省农业科学院 | Method for extracting and purifying and detecting type B trichothecene toxin from cereals |
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Application publication date: 20140129 |