CN104250289A - Method for separating and purifying Pneumocandins B0 - Google Patents
Method for separating and purifying Pneumocandins B0 Download PDFInfo
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- CN104250289A CN104250289A CN201310265836.XA CN201310265836A CN104250289A CN 104250289 A CN104250289 A CN 104250289A CN 201310265836 A CN201310265836 A CN 201310265836A CN 104250289 A CN104250289 A CN 104250289A
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Abstract
The invention provides a method for separating and purifying Pneumocandins B0, which comprises the steps of acidifying and extracting a broth, separating a leaching solution by resin adsorption, concentrating, and then drying to obtain the Pneumocandins B0. The obtained product with high purity is a good raw material to prepare antifungal drug Pneumocandins B0. The prepared end product has the advantages of high yield, low production cost, and simple operation, and is especially suitable for industrial production.
Description
Technical field
The present invention relates to the separation purification method of Pneumocandin B0.
Background technology
Pneumocandin B0 (pneumocandinB0) is by mycetogenetic secondary metabolite, is compound before the precursor of synthetic antifunguses thing Caspofungin (Caspofungin), has following structural formula:
Pneumocandin B0 is the precursor compound of the synthesis of caspofungin, if Pneumocandin B0 purity is too low, the yield of chemical reaction product is often walked after direct impact, and sizable difficulty is brought to the polishing purification of final product Caspofungin, therefore prepare the key that high purity Pneumocandin B0 is Caspofungin synthesis, purifying.
Because Pneumocandin B0 is obtained by fermentable, can produce in fermenting process and the akin by product of Pneumocandin B0 structure and a large amount of pigments, and microbial fermentation product composition is more, add Pneumocandin B0 complex structure, to heat, alkali is all unstable, so add difficulty to the highly purified Pneumocandin B0 of preparation.
At present, the method to Pneumocandin B0 purifying reported, the effect that mostly there is purifying is undesirable, or
Operation is extremely complicated, causes product yield to reduce, is difficult to the defect of accomplishing scale production.
Summary of the invention
The object of this invention is to provide a kind of method of Pneumocandin B0 separation and purification, overcome the Pneumocandin B0 extraction purification existed in prior art and be difficult to industrialization, the defect of purifying technique complexity, pigment in effective removing Pneumocandin B0, improve its purity and content, and make its suitability for industrialized production become easier.
The invention provides a kind of extracting and purifying method of Pneumocandin B0, comprise the following steps:
Step 1): the fermented liquid of acidifying Pneumocandin B0 also crosses leaching mycelium;
Described acidifying refers to use weak acid, regulates fermented liquid pH to be 3-4.
Described weak acid is oxalic acid, carbonic acid or acetic acid, is preferably oxalic acid; Regulate fermented liquid pH to be 3-4, preferably regulate fermented liquid pH to be 4.
In fermented liquid, add appropriate amount of acid agent regulates fermented liquid pH to be 3-4, stirs 1-3 hour; Filter, wash mycelium with water, after drying, collect mycelium.
Step 2): mycelium is used organic solvent lixiviate, filter and concentrated filtrate;
Described organic solvent is selected from alcohol or ketone solution.
Described alcohol is methyl alcohol, ethanol, Virahol, propyl carbinol, ethylene glycol or 1,3-PD, is preferably methyl alcohol or ethanol; Described ketone is acetone, butanone or pentanone.
The 2-4 that the organic solvent quality used in described leaching process is mycelium quality times, preferably 3 times; Soak mycelium while stirring after 3-5 hour, preferably 4 hours, refilter and collect filtrate.
Further, leaching process can repeat 1-3 time, preferably 2 times, to reach abundant extraction, to reduce dissolvent residual and cost-saving object.
Described concentrated be carry out under temperature is less than 50 DEG C of conditions, be concentrated into organic solvent content in solution and be less than 30%.
Step 3): filtrate be separated by resin, concentrates to obtain Pneumocandin B0;
After polymeric adsorbent absorption vat liquor, with 25-40%(V/V) acetonitrile-aqueous solution rinses resin, is then the 40-60%(V/V of 4-6 with pH value) acetonitrile-acid solution rinses resin, the elutriant of Pneumocandin B0 is rich in collection; Concentrated, dry Pneumocandin B0 in conventional manner.
In described acetonitrile-aqueous solution, acetonitrile concentration is preferably 30-35%(V/V).
Described pH value is the 40-60%(V/V of 4-6) in acetonitrile-acid solution, pH value is preferably 5; Acetonitrile concentration is preferably 45-55%(V/V).
Described acid is acetic acid, trifluoracetic acid, perchloric acid or sulfuric acid; Preferred acetic acid.
Described resin refers to that being selected from granularity is more than or equal to 60 orders; Aperture is 70-120, preferred 90-100.The resin of preferred AB-8, HPD722, HPD-100, D-101, DM-301 or DM130 model, preferred AB-8 model resin.
Separation condition gentleness of the present invention is easy to operate, method of the present invention solves the problem that a small amount of impurity can not be removed, make the target product purity after separation reach more than 95%, it is high that the quality not only increasing product also avoid separation costs in prior art, the problem of working method condition harshness.Secondly, in the present invention, the peracid process of fermentation liquor, makes Pneumocandin B0 be leached out fully from fermented liquid, improves the yield of product.The present invention is simple to operate, and product yield is high, purity is high, is applicable to scale operation.
embodiment:
In order to make technical problem solved by the invention, technical scheme and beneficial effect clearly understand, below in conjunction with specific embodiment, the present invention is further illustrated.The fermented liquid preparation method of Pneumocandin B0 prepares according to method disclosed in ZL200910133118.0.
embodiment 1:
Getting Pneumocandin B0 fermented liquid 1000ml, is 4 with careless acid for adjusting pH, stirs 2 hours, filters, with 500 ml water washing mycelium, collects mycelium after drying.Get mycelium, add 1000ml ethanolic soln, immersion limit, limit stirs 4 hours, filters, obtains vat liquor and be about 980ml, and at 50 DEG C, concentrated vat liquor to volume is 100ml; Join in 750ml ethanolic soln again by filtering the mycelium obtained after lixiviate, immersion limit, limit stirs 4 hours, filter, obtain vat liquor and be about 730ml, at 50 DEG C, concentrated vat liquor to volume is 75ml, be cooled to room temperature, merge the vat liquor of twice, at 25 DEG C, concentrated vat liquor to the ethanol content in solution is 25%.
Get 200mlAB-8 resin (Cangzhou Bao En Chemical Co., Ltd.) and load resin column (Φ 20 × 500mm), by above-mentioned ethanol content be 25% Pneumocandin B0 vat liquor adsorb, HPLC detects, use 400ml water rinse resin post after having adsorbed again, then use 1000ml40%(V/V) acetonitrile-aqueous solution flushing resin column.Then being 4 by acetic acid adjust ph, then configuring 50%(V/V) acetonitrile-acid solution 500ml carries out wash-out to resin, and elutriant receives 20ml/ bottle, collects the elutriant 300ml being altogether rich in Pneumocandin B0.Through Liquid Detection, merge the elutriant that purity is greater than 95%, be concentrated into dry, obtain white powder Pneumocandin B0 954mg, it is 97.2% that HPLC detects purity.
embodiment 2:
Getting Pneumocandin B0 fermented liquid 1000ml, is 3 with vinegar acid for adjusting pH, stirs 1 hour, filters, with 500 ml water washing mycelium, collects mycelium after drying.Get mycelium, add 1000ml methanol solution, immersion limit, limit stirs 5 hours, filters, obtains vat liquor and be about 950ml, and at 50 DEG C, concentrated vat liquor to volume is 100ml; Join in 750ml methanol solution again by filtering the mycelium obtained after lixiviate, immersion limit, limit stirs 3 hours, filter, obtain vat liquor and be about 725ml, at 50 DEG C, concentrated vat liquor to volume is 72ml, be cooled to room temperature, merge the vat liquor of twice, at 25 DEG C, concentrated vat liquor to the methanol content in solution is 20%.
Get 200ml HPD-100 resin (Cangzhou Bao En Chemical Co., Ltd.) and load resin column (Φ 30 × 400mm), by above-mentioned methanol content be 20% Pneumocandin B0 vat liquor adsorb, HPLC detects, use 500ml water rinse resin post after having adsorbed again, then use 1000ml25%(V/V) acetonitrile-aqueous solution flushing resin column.Then being 5 by trifluoracetic acid adjust ph, then configuring 60%(V/V) acetonitrile-acid solution 500ml carries out wash-out to resin, and elutriant receives 20ml/ bottle, collects the elutriant 260ml being altogether rich in Pneumocandin B0.Through Liquid Detection, merge the elutriant that purity is greater than 95%, be concentrated into dry, obtain white powder Pneumocandin B0 947mg, it is 96.8% that HPLC detects purity.
embodiment 3:
Getting Pneumocandin B0 fermented liquid 1000ml, is 4 with carbon acid for adjusting pH, stirs 2 hours, filters, with 500 ml water washing mycelium, collects mycelium after drying.Get mycelium, add 1000ml acetone soln, immersion limit, limit stirs 3 hours, filters, obtains vat liquor and be about 985ml, and at 50 DEG C, concentrated vat liquor to volume is 100ml; Join in 700ml acetone soln again by filtering the mycelium obtained after lixiviate, immersion limit, limit stirs 3 hours, filter, obtain vat liquor and be about 735ml, at 50 DEG C, concentrated vat liquor to volume is 75ml, be cooled to room temperature, merge the vat liquor of twice, at 25 DEG C, concentrated vat liquor to the acetone content in solution is 28%.
Get 200mlHPD722 resin (Cangzhou Bao En Chemical Co., Ltd.) and load resin column (Φ 20 × 500mm), by above-mentioned acetone content be 28% Pneumocandin B0 vat liquor adsorb, HPLC detects, use 380ml water rinse resin post after having adsorbed again, then use 1000ml30%(V/V) acetonitrile-aqueous solution flushing resin column.Then being 5 by perchloric acid adjust ph, then configuring 40%(V/V) acetonitrile-acid solution 400ml carries out wash-out to resin, and elutriant receives 20ml/ bottle, collects the elutriant 275ml being altogether rich in Pneumocandin B0.Through Liquid Detection, merge the elutriant that purity is greater than 95%, be concentrated into dry, obtain white powder Pneumocandin B0 952mg, it is 97.0% that HPLC detects purity.
embodiment 4:
Getting Pneumocandin B0 fermented liquid 1000ml, is 4 with careless acid for adjusting pH, stirs 2 hours, filters, with 500 ml water washing mycelium, collects mycelium after drying.Get mycelium, add 800ml ethanolic soln, immersion limit, limit stirs 4 hours, filters, obtains vat liquor and be about 980ml, and at 50 DEG C, concentrated vat liquor to volume is 100ml; Join in 750ml ethanolic soln again by filtering the mycelium obtained after lixiviate, immersion limit, limit stirs 3 hours, filter, obtain vat liquor and be about 735ml, at 50 DEG C, concentrated vat liquor to volume is 75ml, be cooled to room temperature, merge the vat liquor of twice, at 25 DEG C, concentrated vat liquor to the ethanol content in solution is 26%.
Get 200ml D-101 resin (Anhui Samsung resin Science and Technology Ltd.) and load resin column (Φ 30 × 400mm), by above-mentioned ethanol content be 26% Pneumocandin B0 vat liquor adsorb, HPLC detects, use 400ml water rinse resin post after having adsorbed again, then use 1000ml35%(V/V) acetonitrile-aqueous solution flushing resin column.Then being 6 by perchloric acid adjust ph, then configuring 60%(V/V) acetonitrile-acid solution 600ml carries out wash-out to resin, and elutriant receives 20ml/ bottle, collects the elutriant 310ml being altogether rich in Pneumocandin B0.Through Liquid Detection, merge the elutriant that purity is greater than 95%, be concentrated into dry, obtain white powder Pneumocandin B0 961mg, it is 96.9% that HPLC detects purity.
embodiment 5:
Getting Pneumocandin B0 fermented liquid 1000ml, is 4 with carbon acid for adjusting pH, stirs 2 hours, filters, with 500 ml water washing mycelium, collects mycelium after drying.Get mycelium, add 800ml ethylene glycol solution, immersion limit, limit stirs 4 hours, filters, obtains vat liquor and be about 980ml, and at 50 DEG C, concentrated vat liquor to volume is 100ml; Join in 750ml ethylene glycol solution again by filtering the mycelium obtained after lixiviate, immersion limit, limit stirs 3 hours, filter, obtain vat liquor and be about 735ml, at 50 DEG C, concentrated vat liquor to volume is 75ml, be cooled to room temperature, merge the vat liquor of twice, at 25 DEG C, concentrated vat liquor to the ethylene glycol content in solution is 25%.
Get 200ml DM-301 resin (Shanghai Hua Ling resin company limited) and load resin column (Φ 30 × 400mm); by above-mentioned ethylene glycol content be 25% Pneumocandin B0 vat liquor adsorb; HPLC detects; use 400ml water rinse resin post after having adsorbed again, then use 1000ml40%(V/V) acetonitrile-aqueous solution flushing resin column.Then being 6 by perchloric acid adjust ph, then configuring 60%(V/V) acetonitrile-acid solution 500ml carries out wash-out to resin, and elutriant receives 20ml/ bottle, collects the elutriant 285ml being altogether rich in Pneumocandin B0.Through Liquid Detection, merge the elutriant that purity is greater than 95%, be concentrated into dry, obtain white powder Pneumocandin B0 957mg, it is 96.8% that HPLC detects purity.
embodiment 6:
Getting Pneumocandin B0 fermented liquid 1000ml, is 3 with careless acid for adjusting pH, stirs 2 hours, filters, with 500 ml water washing mycelium, collects mycelium after drying.Get mycelium, add 1000ml pentanone solution, immersion limit, limit stirs 4 hours, filters, obtains vat liquor and be about 980ml, and at 50 DEG C, concentrated vat liquor to volume is 100ml; Join in 750ml pentanone solution again by filtering the mycelium obtained after lixiviate, immersion limit, limit stirs 3 hours, filter, obtain vat liquor and be about 730ml, at 45 DEG C, concentrated vat liquor to volume is 75ml, be cooled to room temperature, merge the vat liquor of twice, at 25 DEG C, concentrated vat liquor is 25% to the pentanone content in solution.
Get 200ml DM130 resin (Shanghai Hua Ling resin company limited) and load resin column (Φ 30 × 400mm); the Pneumocandin B0 vat liquor being 25% by above-mentioned pentanone content adsorbs; HPLC detects; use 400ml water rinse resin post after having adsorbed again, then use 1000ml25%(V/V) acetonitrile-aqueous solution flushing resin column.Then being 6 by acetic acid adjust ph, then configuring 40%(V/V) acetonitrile-acid solution 400ml carries out wash-out to resin, and elutriant receives 20ml/ bottle, collects the elutriant 270ml being altogether rich in Pneumocandin B0.Through Liquid Detection, merge the elutriant that purity is greater than 95%, be concentrated into dry, obtain white powder Pneumocandin B0 963mg, it is 97.1% that HPLC detects purity.
It should be noted that and the foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a separation purification method for Pneumocandin B0, is characterized in that comprising the following steps:
1) fermented liquid of acidifying Pneumocandin B0 also crosses leaching mycelium;
2) by the organic solvent lixiviate of the mycelium in step 1), filter and concentrated filtrate;
3) by step 2) in filtrate be separated by resin, obtain Pneumocandin B0.
2. the method as described in claim 1, is characterized in that the acidification described in step 1) refers to use weak acid, regulates fermented liquid pH to be 3-4.
3. the method as described in claim 2, is characterized in that described weak acid is oxalic acid, carbonic acid or acetic acid; Fermented liquid pH is regulated to be 4.
4. the method as described in claim 1, is characterized in that step 2) described in organic solvent be selected from alcohol or ketone solvent.
5. the method as described in claim 4, is characterized in that described alcohol is methyl alcohol, ethanol, Virahol, propyl carbinol, ethylene glycol or 1,3-PD; Described ketone is acetone, butanone or pentanone.
6. the method as described in claim 4, it is characterized in that described alcohol is methyl alcohol or ethanol, described ketone is acetone.
7. the method as described in claim 1, is characterized in that the resin described in step 3) is that granularity is more than or equal to 60 orders; Aperture is 70-120.
8. method as claimed in claim 7, is characterized in that resin is the resin of AB-8, HPD722, HPD-100, D-101, DM-301 or DM130 model.
9. the method as described in claim 1, it is characterized in that the resin described in step 3) carries out in sepn process, acetonitrile-aqueous solution ethane nitrile content is 25-40%(V/V), acetonitrile-acid solution ethane nitrile content is 40-60%(V/V), pH value is 4-6.
10. the method as described in claim 9, is characterized in that in described acetonitrile-acid solution, acid is acetic acid, trifluoracetic acid, perchloric acid or sulphuric acid soln.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820213A (en) * | 2016-04-15 | 2016-08-03 | 中国医药集团总公司四川抗菌素工业研究所 | Method for efficiently separating and purifying pnemocandin |
| CN107674116A (en) * | 2016-08-02 | 2018-02-09 | 北大方正集团有限公司 | A kind of purification process of Pneumocandin B0 |
| CN109400680A (en) * | 2018-12-07 | 2019-03-01 | 成都雅途生物技术有限公司 | A kind of caspofungin precursor pneumocandinB0Crystallization purifications |
| CN112321682A (en) * | 2020-11-24 | 2021-02-05 | 苏州纳微科技股份有限公司 | Purification method of pneumocandin B0 |
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| CN101659693A (en) * | 2008-08-27 | 2010-03-03 | 上海医药工业研究院 | Method for preparing pneumocandin B0 |
| WO2011120842A1 (en) * | 2010-03-29 | 2011-10-06 | Dsm Ip Assets B.V. | Purification of caspofungin intermediates |
| CN102295686A (en) * | 2011-09-09 | 2011-12-28 | 杭州华东医药集团生物工程研究所有限公司 | Method for extracting and purifying pneumocandin B0 |
| CN102335596A (en) * | 2010-07-19 | 2012-02-01 | 上海天伟生物制药有限公司 | Stationary phase and method for purifying lipopeptide by using same |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101659693A (en) * | 2008-08-27 | 2010-03-03 | 上海医药工业研究院 | Method for preparing pneumocandin B0 |
| WO2011120842A1 (en) * | 2010-03-29 | 2011-10-06 | Dsm Ip Assets B.V. | Purification of caspofungin intermediates |
| CN102335596A (en) * | 2010-07-19 | 2012-02-01 | 上海天伟生物制药有限公司 | Stationary phase and method for purifying lipopeptide by using same |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820213A (en) * | 2016-04-15 | 2016-08-03 | 中国医药集团总公司四川抗菌素工业研究所 | Method for efficiently separating and purifying pnemocandin |
| CN105820213B (en) * | 2016-04-15 | 2019-01-22 | 中国医药集团总公司四川抗菌素工业研究所 | The method for efficiently separating purifying knob not Kangding |
| CN107674116A (en) * | 2016-08-02 | 2018-02-09 | 北大方正集团有限公司 | A kind of purification process of Pneumocandin B0 |
| CN109400680A (en) * | 2018-12-07 | 2019-03-01 | 成都雅途生物技术有限公司 | A kind of caspofungin precursor pneumocandinB0Crystallization purifications |
| CN112321682A (en) * | 2020-11-24 | 2021-02-05 | 苏州纳微科技股份有限公司 | Purification method of pneumocandin B0 |
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Address after: Suzhou City, Jiangsu Province, Suzhou Industrial Park 215123 Xinghu Street No. 218 Nano Technology Park building C25 Applicant after: Borui Pharmaceutical (Suzhou) Limited by Share Ltd Applicant after: Xintai Pharmaceutical (Suzhou) Co., Ltd. Address before: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 No. 218 BioBAY building C27 Applicant before: Borui Bio-medical Technology (Jiangsu) Co., Ltd. Applicant before: Xintai Pharmaceutical (Suzhou) Co., Ltd. |
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Application publication date: 20141231 |