CN103304640A - Method for extracting echinocandins compound from fermentation liquid - Google Patents
Method for extracting echinocandins compound from fermentation liquid Download PDFInfo
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Abstract
The invention discloses a method for extracting an echinocandins compound from fermentation liquid. The method comprises the following steps of: (a) performing cross flow filtration on the fermentation liquor by using microfiltration membrane equipment, cleaning with water and then adding an extractant for extraction in the filtration process, and filtering to obtain filtrate containing the target object; (b) adsorbing the filtrate containing the target object by using macroporous resin and then prewashing with a second solvent and resolving to obtain resolving liquor containing the target object; (c) concentrating the resolving liquid containing the target object and removing a second solvent; (d) adding a third solvent to separate out the target object to obtain solid-liquid mixture containing the target object; and (e) filtering the solid-liquid mixture from which the target object is separated out and drying to obtain the target object powder. The method has the advantages that the process flow is short, the used solvent system is simple, the extractant can be returned into the microfiltration membrane for continuous extraction through a simple nanofiltration concentration step so as to realize indiscriminate cycle use of the extractant, the using amount of the solvent is greatly reduced and the method is suitable for large-scale industrial production.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of industrialized preparing process that from fermented liquid, extracts the echinocandin compounds.
Background technology
Fungi is conditioned pathogen for healthy human body, and is harmless under normal circumstances; But when Abwehrkraft des Koepers reduces, just might cause part or whole body fungi infestation.In recent years owing to abusing clinically cytotoxic drug, immunosuppressor, Broad spectrum antibiotics and hormone medicine, not only the immunizing power of patient's body is suppressed, and the microecological balance in the body is damaged, so that originally non-pathogenic fungi becomes dominant bacteria, thereby brings out fungi infestation; Add carrying out of the Open Treatment means such as organ transplantation in the modern medical service, interventional therapy, intubation technique, more increased the morbidity risk rate of fungi infestation.
According to the current standard of international medical community, fungi infestation can be divided into shallow fungi infestation, subcutaneous fungi infestation and systemic fungal infection.Wherein front two kinds of fungi infestations are the most common, but not fatal, systemic fungal infection most serious of all, and namely deep fungal infection if can not be controlled timely and effectively after catching an illness, tends to threat to life.Since first antifungal drug amphotericin B comes out, human struggle with the fungi infestation disease has continued over half a century, now, the mankind are making a breakthrough aspect prevention and the fungi infestation for the treatment of shallow, but for subcutaneous fungi infestation and systemic fungal infection, because the existence of the factors such as toxic side effect of the difficulty of its treatment, the resistance of fungi and existing medicine not yet obtains remarkable break-throughs.The medicine that treatment fungi infestation is used on the modern clinic mainly contains amphotericin B and triazole antifungal agent thing (such as fluconazole, itraconazole and Voriconazole etc.), therefore but all there is certain problem in they at aspects such as security, pharmacokinetics or antimicrobial spectrums, demand clinically the appearance of the New-type wide-spectrum antifungal drug of efficient, low toxicity urgently.
Echinocandin (Echinocandins), claim again echinocandin, it is the novel antifungal drug of a class, belong to acetyl six lopps, be glucan synthase inhibitors, β-1 that can noncompetitive ground Antifungi cell walls, synthesizing of 3-D-dextran, thereby destroy the integrity of cell walls and its osmotic pressure is changed and bring into play germicidal action, and the acellular wall of mammalian cell, thus on mammalian cell without impact.The echinocandin class medicine passes through the interior glucan synthase of Antifungi body with killing fungus, to Cytochrome P450 without effect, untoward reaction is few, with amphotericin B and triazole antifungal agent thing without cross resistance, toxicity to human body is low, Main Function also can act on parasite type pathogenic agent such as Pneumocystis carinii (Pneumocystis carinii) etc. in Candida (Candidas) and Eurotium (Aspergillus).
Echinocandin compounds basic structure is similar, all be to be combined with different substituting groups at acetyl six structure of rings, but the compound anti-mycotic activity of different substituents is different, through targetedly screening, the researchist finds that some compound anti-mycotic activity is stronger, comprise that lung reads rhzomorph B0 (Pneumocandin B0), WF11899A and ECB (EchinocandinB), they all obtain by the method for fermentation, carry out semi-syntheticly with it as intermediate, can obtain the antifungal drug Caspofungin respectively, MFG and anidulafungin.Lung is read rhzomorph B0 (Pneumocandin B0) and is carried out the biosynthesizing generation by the fermentation of Zalerion arboricola thalline, Merck ﹠ Co., Inc. carries out semi-synthetic with it as intermediate, obtained the antifungal drug caspofungin acetate, go on the market in the U.S. February calendar year 2001, be used for the treatment of the infection of Candida albicans and aspergillus fumigatus; WF11899A carries out biosynthesizing by the fermentation of Coleophoma empetri F-11899 thalline and produces, rattan pool company carries out semi-synthetic with it as intermediate, obtained the antifungal drug MFG, went on the market in Japan in 2002 months, be mainly used in Candida albicans and aspergillin infection, the anti-candida albicans infection effect is better, and especially the candidiasis activity to anti-fluconazole is stronger; ECB (Echinocandin B) synthesizes generation by Aspergilus nidulans or with Xinchang reality at the bacterium same names Aspergillus of usefulness rugulovalvus thalline fermenting organism, with it as intermediate, through presenting betrothal gifts to the bride's family before marriage company and the joint research and development exploitation of Vicuron Pharmaceuticals company to obtain the antifungal drug anidulafungin, listing in 2006, its anti-mycotic activity is stronger to aspergillus fumigatus, to Candida albicans relatively a little less than.
At present domestic technique for extract the echinocandin compounds from fermented liquid does not almost have report, external mainly is that some large drugmakers have carried out correlative study, but mainly be to concentrate on the small-scale extraction in laboratory, and the extraction process route is longer, the yield of product is very low, the solvent species of using is many, and some solvent is larger on the impact of human body and environment, is not suitable for large-scale industrial production.
WO2011/121599A1 extracts the technique of echinocandin compounds from fermented liquid, main method is: the echinocandin compounds is extracted from fermented liquid with solvent first; Carry out again liquid-liquid extraction remove portion impurity; Behind the activated carbon decolorizing, utilize sorbent material to adsorb, obtain the echinocandin compounds finally by excessively resolving, concentrate, precipitating.The subject matter of this technique is that the usage quantity of solvent is larger in the extraction process; Fermented liquid is carried out liquid-liquid extraction, the extraction liquid that contains target components is carried out all can producing the mixed solvent layer in the process of liquid-liquid extraction, and aqueous phase can dissolve organic phase in various degree, directly discharging can pollute, and reclaims can increase cost again; The sorbent material price that uses is not easy thorough regeneration, and work-ing life is limited.
US5194377 and US5202309 extract the technique of echinocandin compounds from fermented liquid, main method is: the echinocandin compounds is extracted from mycelium with the first solvent first; Carry out again the several times of liquid liquid extraction, make the echinocandin compounds in two-phase, shift remove portion impurity; The extraction liquid that contains the echinocandin compounds carries out chromatography through multiple different chromatographic column again, resolves finally by crossing, concentrated, solvent deposition obtains the echinocandin compounds.The subject matter of this technique is that the usage quantity of solvent is larger in the extraction process; Fermented liquid is carried out liquid-liquid extraction, the extraction liquid that contains target components is carried out all can producing the mixed solvent layer in the process of liquid-liquid extraction, and aqueous phase can dissolve organic phase in various degree, directly discharging can pollute, and reclaims can increase cost again; The sorbent material kind of using simultaneously is many, and price is not easy thorough regeneration, and work-ing life is limited; Operational path is long, and last total recovery is lower.
US661082282 and US2008/0108806A1 extract the technique of echinocandin compounds from fermented liquid, main method is: the echinocandin compounds is extracted from mycelium with isopropylcarbinol first; Contain the isopropylcarbinol of echinocandin compounds and wash after concentrated, add again methyl alcohol-normal heptane and carry out liquid-liquid extraction, form two-phase: organic phase (isopropylcarbinol-normal heptane) and water (methanol-water), the echinocandin compounds changes aqueous phase over to; Water is concentrated to be removed and adds isopropylcarbinol behind the methyl alcohol again and carry out liquid-liquid extraction, and the echinocandin compounds changes in the isopropylcarbinol; Concentrated remove isopropylcarbinol after, add acetonitrile and precipitate and obtain the echinocandin compounds.The subject matter of this technique is that the usage quantity of solvent is larger in the extraction process; The isopropylcarbinol extraction liquid that contains target components is washed, and and add methyl alcohol-normal heptane and carry out can producing the mixed solvent layer in the process of liquid-liquid extraction, and aqueous phase can dissolve organic phase in various degree, directly discharging can pollute, and reclaims can increase cost again; The organic phase that liquid-liquid extraction is used in the leaching process is mixed solvent, and recovery difficult is larger.
Extract the technique of echinocandin compounds among the US2010/0249371A1 from fermented liquid, main method is: the echinocandin compounds is extracted from mycelium with the first solvent first; Then behind the vacuum concentration remove portion solvent, add metallic salt sorbent material (for example calcium phosphate) and obtain echinocandin compounds-calcium phosphate complex; Use again the second solvent from this mixture the echinocandin class compound dissolution out, concentrated remove the second solvent after, add the third solvent and precipitate and obtain the echinocandin compounds.The subject matter of this technique is that the usage quantity of solvent is larger in the extraction process; Added metal salt compound in the leaching process, inevitable meeting have residual in product, need to be further purified and remove metal ion.
Extract the technique of echinocandin compounds among CN101659693 and the CN101659692A from fermented liquid, main method is: the echinocandin compounds is extracted from mycelium with the first solvent first; Concentrated remove the first solvent after, again extract with the second solvent; Concentrated remove the second solvent after, again use the first dissolution with solvents echinocandin compounds, carry out chromatography with acidic alumina column, adsorption resin column, reversed-phase resin post successively and operate; After desorbed solution is concentrated, adds solvent deposition and obtain the echinocandin compounds.The subject matter of this technique is that the usage quantity of solvent is larger; The route that extracts is longer, be through chromatography repeatedly, and total recovery can be lower; The sorbent material kind is many, and regenerative process can be used different kinds of liquid solvents, increases recovery difficult, has improved extraction cost.
Extract the technique of echinocandin compounds among the WO9611210 from fermented liquid, main method is: the echinocandin compounds is extracted from mycelium with acetone first; Concentrated remove acetone after, with using again n-butanol extraction behind the ethyl acetate extraction; Behind the concentrated remove portion propyl carbinol, successively through twice silica gel column chromatography, centrifugal chromatography chromatography, twice silica gel column chromatography again, last desorbed solution is concentrated into dried, transfers pH to 7.0 after adding a small amount of water dissolution again, and freeze-drying obtains the echinocandin compounds.The subject matter of this technique is that the usage quantity of solvent is larger in the extraction process; In ethyl acetate and butanol extraction liquid process, can produce the mixed solvent layer, reclaim, recovery difficult is larger; Chromatographic step is many, and last yield is lower, and a step centrifugal chromatography chromatography is arranged in the middle of adding, and is not suitable for suitability for industrialized production and uses; The chromatography media kind is many, and regenerative process can be used different kinds of liquid solvents, increases recovery difficult, has further improved extraction cost.
From fermented liquid, extract the technique of echinocandin compounds among US4024245, US4024246 and the US4288549, main method is: with methyl alcohol fermented liquid is extracted first, with chloroform methanol extraction liquid is carried out liquid-liquid extraction again, concentrated remove chloroform after, add ether and make the echinocandin compounds generate precipitation; Precipitation is again after the dissolving, and through the reversed-phase resin column chromatography, desorbed solution is again concentrated after with chloroform extraction removes chloroform, adds trimethyl carbinol dissolving again, and then freeze-drying obtains the echinocandin compounds.The subject matter of this technique is, extraction step is many, and last yield is lower; Use chloroform and so on hazardous chemical solvent in the leaching process, be not suitable for use of large-scale production.
Summary of the invention
Present method for echinocandin compounds from fermented liquid, mainly be that several large drugmakers have carried out correlative study both at home and abroad, but mainly be to concentrate on the small-scale extraction in laboratory, and the extraction process route is longer, the yield of product is very low, the solvent species of using is many, and some solvent is larger on the impact of human body and environment, is not suitable for large-scale industrial production.
The present invention discloses a kind of method of new extraction echinocandin compounds, by using membrane technique, the extraction process route is short, the solvent species that uses is also relatively less, and the simple nanofiltration enrichment step of process in the leaching process, extraction agent just can turn back to and continue extraction in the microfiltration membrane, has realized the recycled of solvent in leaching process, greatly reduce the usage quantity of solvent, reduced the cost of solvent recuperation; Simultaneously, use the membrane concentration technology, realized cryoconcentration, not only remove small molecular weight impurity, also reduced temperature drift when the vacuum decompression heating is concentrated and produce the problem of new impurity, improved quality product.Technique of the present invention is extracted the product purity that obtains behind the purifying to target compound high, and yield is high, use quantity of solvent few, and the cyclic utilization rate of solvent is high, is fit to large-scale industrial production.
The invention provides a kind of method of from echinocandin compounds fermented liquid, extracting the echinocandin compounds, the method may further comprise the steps: a) adopt microfiltration equipment that echinocandin compounds fermented liquid is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating, then adding the water that is adjusted to pH=3.0～8.0 cleans mycelium suspended liquid, mycelium suspended liquid after obtaining behind the micro-filtration cleaning, then at adding extraction agent under 5 ℃～50 ℃ temperature the mycelium suspended liquid after cleaning is extracted, obtain containing the filtrate of echinocandin compounds behind the micro-filtration; Wherein, described extraction agent is the mixed solvent of the first solvent and water, and described the first solvent is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone; B) with the echinocandin compounds in the described filtrate of macroporous resin adsorption, then use the prewashing of the second solvent, parsing, obtain containing the desorbed solution of echinocandin compounds; Wherein, described macroporous adsorbent resin is nonpolar macroporous adsorption resin or low-pole macroporous adsorbent resin, and described the second solvent is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone; C) first with the concentrated described desorbed solution of nanofiltration, continue concentrating under reduced pressure to remove the second solvent; D) add after the third solvent separates out the echinocandin compounds, obtain containing the solidliquid mixture of echinocandin compounds; Wherein, described the third solvent is acetone, acetonitrile, ethyl acetate, isobutyl acetate, n-butyl acetate, hexane, ether or water; And e) described solidliquid mixture is filtered, obtain echinocandin compounds dry powder after the wet-milling drying.
Preferably, described echinocandin compounds comprises that lung reads rhzomorph B0 (PneumocandinB0), WF11899A (FR901379) and ECB (Echinocandin B).
Preferably, step a) in, use respectively phosphoric acid, oxalic acid, hydrochloric acid or ammoniacal liquor to regulate water to pH=3.0～8.0, described phosphoric acid, oxalic acid, hydrochloric acid or ammonia concn are 10wt.%～30wt.%.
Preferably, step a) in, described micro-filtration adopts the multiple microfiltration membrane compounding in series in a kind of micro-filtrate membrane filtration in ceramic membrane, stainless steel membrane, the ultra-filtration membrane or ceramic membrane, stainless steel membrane, the ultra-filtration membrane to filter; Described microfiltration membrane is that the aperture is that the ceramic membrane of 0.05～0.5 μ m or stainless steel membrane or molecular weight cut-off are the ultra-filtration membrane of 30K Dalton～3000K Dalton.
Preferably, step a) in, the volume ratio of the first solvent described in the described mixed solvent is 20%～90%, and is preferred 40～60%, more preferably 20%～50%.
Preferably, step a) in, the mycelium suspended liquid after cleaning is extracted adding extraction agent under 20～40 ℃ of temperature.
Preferably, step a) in, before with the echinocandin compounds in the described filtrate of macroporous resin adsorption, the use molecular weight cut-off is that the nanofiltration membrane of 200 Dalton～800 Dalton concentrates, nanofiltration sees through liquid and turns back in the solvent extraction, and the nanofiltration concentrated solution then carries out step b) operation.
Preferably, at step b) in, described low-pole macroporous adsorbent resin is AmberliteXAD-7HP or Diaion SP-207; Described nonpolar macroporous adsorption resin is AmberliteXAD-1180N, Amberlite XAD-1600, Amberlite XAD-2, AmberliteXAD-4, Diaion HP-20 or Diaion SP-825.
Preferably, at step b) in, after extraction liquid is through the echinocandin compounds in the described filtrate of macroporous resin adsorption, after the macroporous resin adsorption waste liquid concentrates with nanofiltration membrane first, nanofiltration sees through liquid as extraction agent, and turns back to step and a) again loop extraction.
Preferably, at step b) in, before macroporous resin carries out prewashing, parse operation, wash first resin column with water.
Preferably, at step b) in, when macroporous resin carried out the prewashing operation, pre-washing agent was the mixed solvent of organic solvent and water, wherein the organic solvent volume ratio is controlled at 20%～50%.
Preferably, at step b) in, when macroporous resin carried out parse operation, resolving agent was the mixed solvent of organic solvent and water, wherein the organic solvent volume ratio is controlled at 50%～90%.
Preferably, at step c) in, before described desorbed solution concentrates, perhaps desorbed solution is through partial concentration, but when the echinocandin class compound precipitation is not separated out, add discoloring agent and decolour under 0 ℃～30 ℃ temperature, described discoloring agent is gac, aluminum oxide or decolorizing resin, and the discoloring agent ratio of adding is 0.5～5 times of echinocandin compounds gross weight in the feed liquid to be decoloured.
Preferably, at step c) in, desorbed solution carries out vacuum-concentrcted under 20 ℃～60 ℃ temperature.
Preferably, at step e) in, described solidliquid mixture is directly filtered, obtain echinocandin compounds wet-milling.
Preferably, at step e) in, described echinocandin compounds wet-milling is carried out drying, described dry adopt vacuum decompression heat drying or vacuum decompression lyophilize.
Preferably, at step e) in, described vacuum decompression heat drying carries out drying under 20 ℃～60 ℃ temperature.
Preferably, in steps d) in, described desorbed solution through concentrated remove the second solvent after, add again the third solvent when being water, separate out the echinocandin class compound precipitation after, described solidliquid mixture is directly carried out the vacuum decompression lyophilize.
Method of the present invention is by using membrane technique, the extraction process route is short, the solvent species that uses is also relatively less, and the simple nanofiltration enrichment step of process in the leaching process, extraction agent just can turn back to and continue extraction in the microfiltration membrane, realized that the solvent cycle in leaching process applies mechanically, greatly reduced the usage quantity of solvent, reduced the cost of solvent recuperation; Simultaneously, use the membrane concentration technology, realize cryoconcentration, not only remove small molecular weight impurity, also reduced temperature drift when the vacuum decompression heating is concentrated and produce the problem of new impurity, improved quality product.Technique of the present invention is extracted the product purity that obtains behind the purifying to target compound high, and yield is high, use quantity of solvent few, and the cyclic utilization rate of solvent is high, is fit to large-scale industrial production.
Embodiment
Further specify the present invention below by embodiment, embodiments of the invention are only used for the present invention is described and provide, rather than limitation of the present invention.
Embodiment 1～embodiment 3 illustrates by microbial fermentation, obtains the embodiment that lung is read rhzomorph B0 (Pneumocandin B0), WF11899A (FR901379), three kinds of product fermented liquids of ECB (Echinocandin B).
Embodiment 1: lung is read the preparation of rhzomorph B0 (Pneumocandin B0) fermented liquid
Bacterial strain: Zalerion arboricola (HCCB30111, CGMCC No.5412) (referring to No. 201210041247.9 application of Chinese patent application);
Slant medium: PDA;
The slant culture condition: 28 ℃, 5 days;
Seed culture medium (%): glucose 2.5, KH
2PO
40.9, yeast powder 0.5, medicine matchmaker 1.0,85% lactic acid 0.2ml, micro-0.1ml, pH 6.0;
The seed culture condition: dig the piece inoculation, 25 ℃, 220rpm, 3 days;
Fermention medium (%): sucrose 12.5, Sodium Glutamate 0.8, yeast powder 0.8, proline(Pro) 1.5, KH
2PO
40.15, bitter salt 0.04, micro-1ml, MES buffering salt 1.5, pH 5.3;
Fermentation condition: 10%, 25 ℃ of transferred species amount, 220rpm, fermentation culture 7 days;
Trace element forms (g/L): iron vitriol 1.0, manganese sulfate monohydrate 0.2, Calcium dichloride dihydrate 0.1, boric acid 0.056, copper chloride dihydrate 0.025, four water ammonium molybdate 0.019,12N hydrochloric acid 50ml.
Lung is read the product validation of rhzomorph B0 (Pneumocandin B0): detect through HPLC, to read rhzomorph B0 (Pneumocandin B0) content be 4.5g/L to lung in the fermented liquid.
The preparation of embodiment 2:WF11899A (FR901379) fermented liquid
Bacterial strain: Coleophoma empetri WF11899A (FR901379) (referring to " improvement of FR901379 production by mutant selection andmedium optimization by Munekazu Kanda published in Vol.107 No.5; 530-534; 2009, Journal of Bioscience and Bioengineering ")
Slant medium: PDA;
The slant culture condition: 28 ℃, 7 days;
Seed culture medium (%): sucrose 1.0, medicine matchmaker 2.0, yeast powder 1.0, peptone 1.0, KH
2PO
40.2, CaCO
30.2 Tween 80 0.1, pH 6.5;
The seed culture condition: dig the piece inoculation, 25 ℃, 220rpm, fermentation culture 5 days;
Fermention medium (%): starch 3.0, glucose 1.0, wheatgerm 0.2, medicine matchmaker 2.0, Zein powder 0.5, KH
2PO
42.0, NaHPO
41.5, ZnSO
4.7H
2O 0.001, and pH 6.7;
Fermentation condition: 10%, 25 ℃ of transferred species amount, 220rpm, 7 days.
The product validation of WF11899A (FR901379): detect through HPLC, WF11899A in the fermented liquid (FR901379) content is 1.03g/l.
Embodiment 3: the preparation of ECB (Echinocandin B) fermented liquid
Bacterial strain: Aspergillus rugulovalvus (HCCB30110, CGMCC No.5413) (referring to No. 201210041876.1 application of Chinese patent application);
Slant medium: PDA;
The slant culture condition: 28 ℃, 5 days;
Seed culture medium (%): glucose 1.0, glycerine 1.0, cottonseed meal 2.5, pH 6.3;
The seed culture condition: dig the piece inoculation, 25 ℃, 220rpm, fermentation culture 8 days;
Fermention medium (%): maltose 6.0, dextrin 2.0, cottonseed meal 3.0, yeast powder 1.5, soya-bean oil 0.2, L-glutamic acid 0.3, KH
2PO
40.1 pH 7.0;
Fermentation condition: 10%, 25 ℃ of transferred species amount, 220rpm, 7 days.
The product validation of ECB (Echinocandin B): detect through HPLC, ECB in the fermented liquid (Echinocandin B) content is 0.87g/l.
Embodiment 4～embodiment 27 illustrates respectively in embodiment 1～embodiment 3 fermented liquid that three kinds of microbial fermentations obtain as raw material, extracts the embodiment that lung is read rhzomorph B0 (Pneumocandin B0), WF11899A (FR901379), three kinds of products of ECB (Echinocandin B).
Embodiment 4: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 30L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% phosphorus acid for adjusting pH to 3.0 that mycelium suspended liquid is washed in 0.05 μ m ceramic membrane; After being filtered to 15L, directly extract in ceramic membrane with methyl alcohol (20%) and water mixed extractant, temperature is controlled at 5 ℃, finishes simultaneously extraction-filter operation.After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/5 with the nanofiltration membrane of 200 Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction.The nanofiltration concentrated solution directly adsorbs with the 3LXAD-1180N macroporous resin, and the waste back-cycling after the absorption extracts in ceramic membrane again, and resin column washes with water first, be 20% methyl alcohol prewashing with concentration again after, 60% methyl alcohol is resolved.After the desorbed solution elder generation nanofiltration that contains lung Colistin B 0 is concentrated into original volume 1/10, add the gac of 0.5 times of weight of active principle, temperature remains on 0 ℃ and decolours again; 40 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added entry behind the methyl alcohol and precipitate; Contain the direct vacuum lyophilization of solidliquid mixture of lung Colistin B 0 precipitation, obtain lung Colistin B 0 dry powder, product yield is 84.3%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=78.9% through HPLC.
Embodiment 5: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 20L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% phosphorus acid for adjusting pH to 6.3 that mycelium suspended liquid is washed in 0.5 μ m stainless steel membrane; After being filtered to 10L, the alcohol-water mixed extractant with 90% directly extracts in stainless steel membrane, and temperature is controlled at 50 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/5 with the nanofiltration membrane of 400 Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to alcohol concn is 45%, adsorb with 2.5L XAD-1600 macroporous resin, resin column concentration is after 45% the ethanol prewashing, and 90% ethanol is resolved; After the desorbed solution elder generation nanofiltration that contains sour jujube lung Colistin B 0 is concentrated into original volume 1/15, add the aluminum oxide of 2 times of weight of active principle, temperature remains on 30 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added ethyl acetate behind the ethanol and precipitate; The solidliquid mixture that contains lung Colistin B 0 precipitation filters and obtains wet-milling, and 60 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 82.7%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=79.0% through HPLC.
Embodiment 6: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 21L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% careless acid for adjusting pH to 4.6 that mycelium suspended liquid is washed in molecular weight cut-off is the ultra-filtration membrane of 30K Dalton; After being filtered to 10L, the n-propyl alcohol with 80%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature is controlled at 20 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/4 with the nanofiltration membrane of 800 Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to n-propyl alcohol concentration is 40%, adsorb with 2L XAD-7HP macroporous resin, resin column washes with water first, be 40% n-propyl alcohol prewashing with concentration again after, 80% n-propyl alcohol is resolved; The desorbed solution that contains lung Colistin B 0 adds the decolorizing resin of 3 times of weight of active principle, and temperature remains on 20 ℃ and decolours; After the nanofiltration of feed liquid after decolouring elder generation was concentrated into original volume 1/15,60 ℃ of vacuum concentration were again removed and are added acetone behind the n-propyl alcohol and precipitate; The solidliquid mixture that contains lung Colistin B 0 precipitation filters and obtains wet-milling, and 20 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 85.1%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=80.1% through HPLC.
Embodiment 7: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 25L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 30% careless acid for adjusting pH to 5.8 that mycelium suspended liquid is washed in molecular weight cut-off is the ultra-filtration membrane of 3000KDalton; After being filtered to 12L, the isopropanol-water mixed extractant with 80% directly extracts in ultra-filtration membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/5 with the nanofiltration membrane of 600 Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to isopropyl alcohol concentration is 40%, adsorb with 2L HP-20 macroporous resin, resin column concentration is after 40% the Virahol prewashing, and 80% Virahol is resolved; After the desorbed solution elder generation nanofiltration that contains lung Colistin B 0 is concentrated into original volume 1/10, add the gac of 5 times of weight of active principle, temperature remains on 10 ℃ and decolours again; 60 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added acetonitrile behind the Virahol and precipitate; The solidliquid mixture that contains lung Colistin B 0 precipitation filters and obtains wet-milling, and 30 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 85.9%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=79.5% through HPLC.
Embodiment 8: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 23L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating, the water that utilizes 20% ammoniacal liquor to regulate pH to 8.0 in 0.5 μ m ceramic membrane is washed mycelium suspended liquid; After being filtered to 10L, the acetone-water mixed extractant with 30% directly extracts in ceramic membrane, and temperature is controlled at 25 ℃, finishes simultaneously extraction-filter operation; The extraction liquid that contains lung Colistin B 0 directly adsorbs with 2.5L SP-207 macroporous resin, and resin column concentration is after 30% the acetone prewashing, and 50% acetone is resolved; The desorbed solution that contains lung Colistin B 0 adds the aluminum oxide of 4 times of weight of active principle, and temperature remains on 20 ℃ and decolours; 30 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added isobutyl acetate behind the acetone and precipitate; The solidliquid mixture that contains lung Colistin B 0 precipitation filters and obtains wet-milling, and 30 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 84.9%
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=75.7% through HPLC.
Embodiment 9: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 32L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% careless acid for adjusting pH to 3.6 that mycelium suspended liquid is washed in 0.2 μ m ceramic membrane; After being filtered to 15L, the methanol-water mixed extractant with 30% directly extracts in ceramic membrane, and temperature is controlled at 10 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/7 with the nanofiltration membrane of 400 Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution directly adsorbs with 3L SP-825 macroporous resin, and the absorption waste liquid is concentrated with nanofiltration membrane, and nanofiltration sees through liquid and turns back in the stainless steel membrane and extract, and resin column washes with water first, be 30% methyl alcohol prewashing with concentration again after, 60% methyl alcohol parsing; The desorbed solution that contains lung Colistin B 0 adds the gac of 1 times of weight of active principle, and temperature remains on 10 ℃ and decolours; After the nanofiltration of feed liquid after decolouring elder generation was concentrated into original volume 1/10,50 ℃ of vacuum concentration were again removed and are added n-butyl acetate behind the methyl alcohol and precipitate; Obtain wet-milling after the solidliquid mixture that contains lung Colistin B 0 precipitation filters, 45 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 86.6%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=75.5% through HPLC.
Embodiment 10: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 26L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% salt acid for adjusting pH to 5.0 that mycelium suspended liquid is washed in 0.1 μ m stainless steel membrane; After being filtered to 13L, the alcohol-water mixed extractant with 60% directly extracts in stainless steel membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/4 with the nanofiltration membrane of 600 Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to alcohol concn is 30%, adsorb with 2.5L XAD-2 macroporous resin, resin column concentration is after 30% the ethanol prewashing, and 70% ethanol is resolved; After the desorbed solution elder generation nanofiltration that contains lung Colistin B 0 is concentrated into original volume 1/15, add the aluminum oxide of 2.5 times of weight of active principle, temperature remains on 20 ℃ and decolours again; 40 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added ether behind the ethanol and precipitate; The solidliquid mixture that contains lung Colistin B 0 precipitation filters and obtains wet-milling, and 35 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 86.3%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=78.7% through HPLC.
Embodiment 11: the extraction of lung Colistin B 0 (Pneumocandin B0)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 27L that is made by embodiment 1 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating is that the water that utilizes 10% ammoniacal liquor to regulate pH to 7.5 in the ultra-filtration membrane of 500K Dalton is washed mycelium suspended liquid at molecular weight cut-off; After being filtered to 15L, feed liquid is put into the ceramic membrane of 0.1um, directly extracts in the n-propyl alcohol with 50%-water mixed extractant, and temperature is controlled at 30 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains lung Colistin B 0 was concentrated into original volume 1/4 with the nanofiltration membrane of 200Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution adsorbs with 3L XAD-4 macroporous resin, and resin column concentration is after 50% the n-propyl alcohol prewashing, and 90% n-propyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains lung Colistin B 0 is concentrated into original volume 1/15, add the gac of 3 times of weight of active principle, temperature remains on 15 ℃ and decolours again; 60 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added hexane behind the n-propyl alcohol and precipitate; The solidliquid mixture that contains lung Colistin B 0 precipitation filters and obtains wet-milling, and 40 ℃ of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 85.1%.
The HPLC testing conditions of lung Colistin B 0 (Pneumocandin B0) is as follows:
Chromatographic column: Lichrocart Silica 5 μ m
250 * 4.6mm
Detect wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of Pneumocandin B0 (lung is read rhzomorph B0): detect the content of lung Colistin B 0 in the dry powder (Pneumocandin B0)=75.9% through HPLC.
The extraction of embodiment 12:WF11899A (FR901379)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 20L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% salt acid for adjusting pH to 4.8 that mycelium suspended liquid is washed in molecular weight cut-off is the ultra-filtration membrane of 1000KDalton; After being filtered to I0L, feed liquid is put into the stainless steel membrane of 0.2um, and the isopropanol-water mixed extractant with 90% directly extracts, and temperature is controlled at 30 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/5 with the nanofiltration membrane of 600 Dalton, nanofiltration saw through liquid and turns back in the stainless steel membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to isopropyl alcohol concentration is 45%, adsorb with 2L HP-20 macroporous resin, resin column washes with water first, be 45% Virahol prewashing with concentration again after, 80% Virahol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/15, add the gac of 1.5 times of weight of active principle, temperature remains on 20 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added acetonitrile behind the Virahol and precipitate; The solidliquid mixture that contains the WF11899A precipitation filters and obtains wet-milling, and 30 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 87.9%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=73.0% through HPLC.
The extraction of embodiment 13:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 25L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% careless acid for adjusting pH to 4.0 that mycelium suspended liquid is washed in 0.2 μ m ceramic membrane; After being filtered to 12L, the methanol-water mixed extractant with 30% directly extracts in ceramic membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/5 with the nanofiltration membrane of 200Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution directly adsorbs with the 2.5LXAD-1600 macroporous resin, and the absorption waste liquid is concentrated with nanofiltration membrane, and nanofiltration sees through liquid and turns back in the ceramic membrane and extract, and resin column concentration is after 30% the methyl alcohol prewashing, 60% methyl alcohol parsing; The desorbed solution that contains WF11899A adds first the aluminum oxide of 0.5 times of weight of active principle, and temperature remains on 0 ℃ and decolours; After feed liquid nanofiltration after the decolouring is concentrated into original volume 1/10,40 ℃ of vacuum concentration again, remove methyl alcohol after.Adding n-butyl acetate precipitates; The solidliquid mixture that contains the WF11899A precipitation filters and obtains wet-milling, and 50 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 83.5%
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=74.6% through HPLC.
The extraction of embodiment 14:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 23L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 30% phosphorus acid for adjusting pH to 5.3 that mycelium suspended liquid is washed in 0.2 μ m stainless steel membrane; After being filtered to 10L, the alcohol-water mixed extractant with 80% directly extracts in stainless steel membrane, and temperature is controlled at 50 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/5 with the nanofiltration membrane of 200Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to alcohol concn is 40%, adsorb with 2L XAD-1180N macroporous resin, resin column washes with water first, be 40% ethanol prewashing with concentration again after, 80% ethanol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/15, add the gac of 2 times of weight of active principle, temperature remains on 25 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added entry behind the ethanol and precipitate; Contain the direct vacuum lyophilization of solidliquid mixture of WF11899A precipitation, obtain WF11899A dry powder, product yield is 81.3%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=76.7% through HPLC.
The extraction of embodiment 15:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 28L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% salt acid for adjusting pH to 3.5 that mycelium suspended liquid is washed in 0.3 μ m stainless steel membrane; After being filtered to 15L, the alcohol-water mixed extractant with 50% directly extracts in stainless steel membrane, and temperature is controlled at 30 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/4 with the nanofiltration membrane of 600Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution adsorbs with 3L XAD-2 macroporous resin, and resin column concentration is after 50% the ethanol prewashing, and 80% ethanol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/10, add the decolorizing resin of 2.5 times of weight of active principle, temperature remains on 20 ℃ and decolours again; 40 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added hexane behind the ethanol and precipitate; The solidliquid mixture that contains the WF11899A precipitation filters and obtains wet-milling, and 30 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 87.1%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=76.3% through HPLC.
The extraction of embodiment 16:WF11899A (FR901379)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 20L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 25% careless acid for adjusting pH to 5.1 that mycelium suspended liquid is washed in molecular weight cut-off is the ultra-filtration membrane of 300K Dalton; After being filtered to 10L, the n-propyl alcohol with 90%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/4 with the nanofiltration membrane of 600Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to n-propyl alcohol concentration is 45%, adsorb with 2L XAD-7HP macroporous resin, resin column concentration is after 45% the n-propyl alcohol prewashing, and 80% n-propyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/15, add the aluminum oxide of 3 times of weight of active principle, temperature remains on 25 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added acetone behind the n-propyl alcohol and precipitate; The solidliquid mixture that contains the WF11899A precipitation filters and obtains wet-milling, and 25 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 85.6%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=80.6% through HPLC.
The extraction of embodiment 17:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 30L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% phosphorus acid for adjusting pH to 4.9 that mycelium suspended liquid is washed in 0.3 μ m ceramic membrane; After being filtered to 15L, the methanol-water mixed extractant with 40% directly extracts in ceramic membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/6 with the nanofiltration membrane of 800Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution directly adsorbs with 3L SP-825 macroporous resin, and resin column concentration is after 40% the methyl alcohol prewashing, and 60% methyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/15, add the gac of 2 times of weight of active principle, temperature remains on 20 ℃ and decolours again; 45 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added isobutyl acetate behind the methyl alcohol and precipitate; Obtain wet-milling after the solidliquid mixture that contains WF11899A precipitation filters, 45 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 83.6%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=79.5% through HPLC.
The extraction of embodiment 18:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 25L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% careless acid for adjusting pH to 5.3 that mycelium suspended liquid is washed in 0.05 μ m stainless steel membrane; After being filtered to 10L, the alcohol-water mixed extractant with 80% directly extracts in stainless steel membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/5 with the nanofiltration membrane of 400Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to alcohol concn is 40%, adsorb with 2.5L XAD-2 macroporous resin, resin column concentration is after 40% the ethanol prewashing, and 85% ethanol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/15, add the gac of 2.5 times of weight of active principle, temperature remains on 30 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added ethyl acetate behind the ethanol and precipitate; The solidliquid mixture that contains the WF11899A precipitation filters and obtains wet-milling, and 60 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 83.7%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=77.3% through HPLC.
The extraction of embodiment 19:WF11899A (FR901379)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 24L that is made by embodiment 2 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating is that the water that utilizes 20% ammoniacal liquor to regulate pH to 7.4 in the ultra-filtration membrane of 100K Dalton is washed mycelium suspended liquid at molecular weight cut-off; After being filtered to 10L, the n-propyl alcohol with 45%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains WF11899A was concentrated into original volume 1/5 with the nanofiltration membrane of 200Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution adsorbs with 2.5L XAD-4 macroporous resin, and resin column concentration is after 45% the n-propyl alcohol prewashing, and 90% n-propyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains WF11899A is concentrated into original volume 1/15, add the gac of 1.5 times of weight of active principle, temperature remains on 15 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added ether behind the n-propyl alcohol and precipitate; The solidliquid mixture that contains the WF11899A precipitation filters and obtains wet-milling, and 40 ℃ of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 86.1%.
The HPLC testing conditions of WF11899A is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of WF11899A: detect the content of WF11899A in the dry powder (FR901379)=76.9% through HPLC.
Embodiment 20: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 27L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% phosphorus acid for adjusting pH to 3.3 that mycelium suspended liquid is washed in 0.1 μ m stainless steel membrane; After being filtered to 15L, the methanol-water mixed extractant with 20% directly extracts in stainless steel membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/6 with the nanofiltration membrane of 400Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution adsorbs with the 2.5LXAD-1600 macroporous resin, and the absorption waste back-cycling extracts in stainless steel membrane, and resin column washes with water first, be 20% methyl alcohol prewashing with concentration again after, 75% methyl alcohol parsing; After the desorbed solution elder generation nanofiltration that contains ECB is concentrated into original volume 1/10, add the decolorizing resin of 1.5 times of weight of active principle, temperature remains on 30 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added n-butyl acetate behind the methyl alcohol and precipitate; The solidliquid mixture that contains the ECB precipitation filters and obtains wet-milling, and 50 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 83.9%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=64.1% through HPLC.
Embodiment 21: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 30L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 25% salt acid for adjusting pH to 5.8 that fermented liquid is washed in molecular weight cut-off is the ultra-filtration membrane of 2000KDalton; After being filtered to 15L, the isopropanol-water mixed extractant with 80% directly extracts in ultra-filtration membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/5 with the nanofiltration membrane of 600Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to n-propyl alcohol concentration is 40%, adsorb with 2.5L XAD-4 macroporous resin, resin column washes with water first, be 40% n-propyl alcohol prewashing with concentration again after, 80% n-propyl alcohol is resolved; The desorbed solution that contains ECB adds first the gac of 3 times of weight of active principle, and temperature remains on 15 ℃ and decolours; After feed liquid nanofiltration after the decolouring was concentrated into original volume 1/15,50 ℃ of vacuum concentration were again removed and are added acetone behind the Virahol and precipitate; The solidliquid mixture that contains the ECB precipitation filters and obtains wet-milling, and 35 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 82.6%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=68.3% through HPLC.
Embodiment 22: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 28L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% careless acid for adjusting pH to 6.3 that mycelium suspended liquid is washed in 0.3 μ m stainless steel membrane; After being filtered to 15L, the alcohol-water mixed extractant with 80% directly extracts in stainless steel membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/5 with the nanofiltration membrane of 400Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to alcohol concn is 40%, adsorb with 2L XAD-7HP macroporous resin, resin column washes with water first, be 40% ethanol prewashing with concentration again after, 80% ethanol is resolved; After the desorbed solution elder generation nanofiltration that contains ECB is concentrated into original volume 1/15, add the aluminum oxide of 2.5 times of weight of active principle, temperature remains on 25 ℃ and decolours again; 40 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added acetonitrile behind the ethanol and precipitate; The solidliquid mixture that contains the ECB precipitation filters and obtains wet-milling, and 35 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 83.9%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=71.6% through HPLC.
Embodiment 23: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 31L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% careless acid for adjusting pH to 5.2 that mycelium suspended liquid is washed in molecular weight cut-off is the ultra-filtration membrane of 1500KDalton; After being filtered to 15L, the isopropanol-water mixed extractant with 90% directly extracts in ultra-filtration membrane, and temperature is controlled at 30 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/5 with the nanofiltration membrane of 200Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; After nanofiltration concentrated solution thin up to isopropyl alcohol concentration is 45%, adsorb with 3L SP-207 macroporous resin, resin column concentration is after 45% the Virahol prewashing, and 80% Virahol is resolved; After the desorbed solution elder generation nanofiltration that contains ECB is concentrated into original volume 1/10, add the gac of 1.5 times of weight of active principle, temperature remains on 20 ℃ and decolours again; 60 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added entry behind the Virahol and precipitate; Contain the direct vacuum lyophilization of solidliquid mixture of ECB precipitation, obtain ECB dry powder, product yield is 84.9%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=70.1% through HPLC.
Embodiment 24: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 25L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 10% careless acid for adjusting pH to 4.6 that mycelium suspended liquid is washed in 0.2 μ m ceramic membrane; After being filtered to 10L, the methanol-water mixed extractant with 35% directly extracts in ceramic membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/6 with the nanofiltration membrane of 200Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution directly adsorbs with the 2.5LXAD-1180N macroporous resin, and resin column concentration is after 35% the methyl alcohol prewashing, and 60% methyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains ECB is concentrated into original volume 1/10, add the gac of 0.5 times of weight of active principle, temperature remains on 10 ℃ and decolours again; 40 ℃ of vacuum concentration of feed liquid after the decolouring, remove methyl alcohol after, add ether and precipitate; The solidliquid mixture that contains the ECB precipitation filters and obtains wet-milling, and 50 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 83.5%
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=72.0% through HPLC.
Embodiment 25: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) the echinocandin compounds fermented liquid 28L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating is that the water that utilizes 15% ammoniacal liquor to regulate pH to 7.2 in the ultra-filtration membrane of 200K Dalton is washed mycelium suspended liquid at molecular weight cut-off; After being filtered to 15L, the n-propyl alcohol with 50%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/5 with the nanofiltration membrane of 400Dalton, nanofiltration saw through liquid and turns back in the ultra-filtration membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution adsorbs with 3L HP-20 macroporous resin, and resin column concentration is after 50% the n-propyl alcohol prewashing, and 85% n-propyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains ECB is concentrated into original volume 1/15, add the gac of 1 times of weight of active principle, temperature remains on 15 ℃ and decolours again; 50 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added isobutyl acetate behind the n-propyl alcohol and precipitate; The solidliquid mixture that contains the ECB precipitation filters and obtains wet-milling, and 40 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 87.3%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=71.4% through HPLC.
Embodiment 26: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the stainless steel membrane pipe of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 20L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 15% careless acid for adjusting pH to 3.9 that mycelium suspended liquid is washed in 0.3 μ m stainless steel membrane; After being filtered to 10L, the alcohol-water mixed extractant with 45% directly extracts in stainless steel membrane, and temperature is controlled at 35 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/4 with the nanofiltration membrane of 400Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution adsorbs with 2L XAD-2 macroporous resin, and resin column washes with water first, be 45% ethanol prewashing with concentration again after, 70% ethanol is resolved; The desorbed solution that contains ECB adds first the gac of 1.5 times of weight of active principle, and temperature remains on 20 ℃ and decolours; After feed liquid nanofiltration after the decolouring was concentrated into original volume 1/10,40 ℃ of vacuum concentration were again removed and are added hexane behind the ethanol and precipitate; The solidliquid mixture that contains the ECB precipitation filters and obtains wet-milling, and 30 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 85.4%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=66.4% through HPLC.
Embodiment 27: the extraction of ECB (Echinocandin B)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customization, the ceramic-film tube of two φ 3cm * 100cm is housed) the echinocandin compounds fermented liquid 23L that is made by embodiment 3 is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating utilizes the water of 20% salt acid for adjusting pH to 4.3 that mycelium suspended liquid is washed in 0.1 μ m ceramic membrane; After being filtered to 15L, the methanol-water mixed extractant with 40% directly extracts in ceramic membrane, and temperature is controlled at 40 ℃, finishes simultaneously extraction-filter operation; After the extraction liquid that contains ECB was concentrated into original volume 1/5 with the nanofiltration membrane of 800Dalton, nanofiltration saw through liquid and turns back in the ceramic membrane as extraction agent, proceeds extraction; The nanofiltration concentrated solution directly adsorbs with 2L SP-825 macroporous resin, and resin column concentration is after 40% the methyl alcohol prewashing, and 70% methyl alcohol is resolved; After the desorbed solution elder generation nanofiltration that contains ECB is concentrated into original volume 1/10, add the aluminum oxide of 2.5 times of weight of active principle, temperature remains on 20 ℃ and decolours again; 45 ℃ of vacuum concentration of feed liquid after the decolouring are removed and are added ethyl acetate behind the methyl alcohol and precipitate; Obtain wet-milling after the solidliquid mixture that contains ECB precipitation filters, 45 ℃ of vacuum decompression dryings obtain ECB dry powder, and product yield is 84.6%.
The HPLC testing conditions of ECB (Echinocandin B) is as follows:
Chromatographic column: Phenomenex Luna 5 μ m C18 (2)
250 * 4.6mm
Detect wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (formic acid of interpolation 0.3%-ammonium formiate buffering salt)
Flow velocity: 1mL/min
Column temperature: 30 ℃
The product validation of ECB (Echinocandin B): detect the content of ECB in the dry powder (Echinocandin B)=68.2% through HPLC.
What need statement is that foregoing invention content and embodiment are intended to prove the practical application of technical scheme provided by the present invention, should not be construed as the restriction to protection domain of the present invention.Those skilled in the art are in spirit of the present invention and principle, when doing various modifications, being equal to and replacing or improve.
Claims (18)
1. method of from echinocandin compounds fermented liquid, extracting the echinocandin compounds, the method may further comprise the steps:
A) adopt microfiltration equipment that echinocandin compounds fermented liquid is carried out cross flow filter, the mycelium suspended liquid that obtains concentrating, then adding the water that is adjusted to pH=3.0～8.0 cleans mycelium suspended liquid, mycelium suspended liquid after obtaining behind the micro-filtration cleaning, then at adding extraction agent under 5 ℃～50 ℃ temperature the mycelium suspended liquid after cleaning is extracted, obtain containing the filtrate of echinocandin compounds behind the micro-filtration; Wherein, described extraction agent is the mixed solvent of the first solvent and water, and described the first solvent is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone;
B) with the echinocandin compounds in the described filtrate of macroporous resin adsorption, then use the prewashing of the second solvent, parsing, obtain containing the desorbed solution of echinocandin compounds; Wherein, described macroporous adsorbent resin is nonpolar macroporous adsorption resin or low-pole macroporous adsorbent resin, and described the second solvent is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone;
C) first with the concentrated described desorbed solution of nanofiltration, continue concentrating under reduced pressure to remove the second solvent;
D) add after the third solvent separates out the echinocandin compounds, obtain containing the solidliquid mixture of echinocandin compounds; Wherein, described the third solvent is acetone, acetonitrile, ethyl acetate, isobutyl acetate, n-butyl acetate, hexane, ether or water; And
E) described solidliquid mixture is filtered, obtain echinocandin compounds dry powder after the wet-milling drying.
2. method according to claim 1 is characterized in that, described echinocandin compounds comprises that lung reads rhzomorph B0 (Pneumocandin B0), WF11899A (FR901379) and ECB (Echinocandin B).
3. method according to claim 1 is characterized in that, step a) in, use respectively phosphoric acid, oxalic acid, hydrochloric acid or ammoniacal liquor to regulate water to pH=3.0～8.0, described phosphoric acid, oxalic acid, hydrochloric acid or ammonia concn are 10wt.%～30wt.%.
4. method according to claim 1, it is characterized in that, step a) in, described micro-filtration adopts the multiple microfiltration membrane compounding in series in a kind of micro-filtrate membrane filtration in ceramic membrane, stainless steel membrane, the ultra-filtration membrane or ceramic membrane, stainless steel membrane, the ultra-filtration membrane to filter; Described microfiltration membrane is that the aperture is that the ceramic membrane of 0.05～0.5 μ m or stainless steel membrane or molecular weight cut-off are the ultra-filtration membrane of 30KDalton～3000K Dalton.
5. method according to claim 1 is characterized in that, step a) in, the volume ratio of the first solvent described in the described mixed solvent is 20%～90%, and is preferred 40～60%, more preferably 20%～50%.
6. method according to claim 1 is characterized in that, step a) in, the mycelium suspended liquid after cleaning is extracted adding extraction agent under 20～40 ℃ of temperature.
7. method according to claim 1, it is characterized in that, step a) in, before with the echinocandin compounds in the described filtrate of macroporous resin adsorption, the use molecular weight cut-off is that the nanofiltration membrane of 200 Dalton～800 Dalton concentrates, nanofiltration sees through liquid and turns back in the solvent extraction, and the nanofiltration concentrated solution then carries out step b) operation.
8. method according to claim 1 is characterized in that, at step b) in, described low-pole macroporous adsorbent resin is Amberlite XAD-7HP or Diaion SP-207; Described nonpolar macroporous adsorption resin is Amberlite XAD-1180N, AmberliteXAD-1600, Amberlite XAD-2, Amberlite XAD-4, Diaion HP-20 or Diaion SP-825.
9. method according to claim 1, it is characterized in that, at step b) in, after extraction liquid is through the echinocandin compounds in the described filtrate of macroporous resin adsorption, after the macroporous resin adsorption waste liquid concentrates with nanofiltration membrane first, nanofiltration sees through liquid as extraction agent, and turns back to step and a) again loop extraction.
10. method according to claim 1 is characterized in that, at step b) in, before macroporous resin carries out prewashing, parse operation, wash first resin column with water.
11. method according to claim 10 is characterized in that, at step b) in, when macroporous resin carried out the prewashing operation, pre-washing agent was the mixed solvent of organic solvent and water, wherein the organic solvent volume ratio is controlled at 20%～50%.
12. method according to claim 10 is characterized in that, at step b) in, when macroporous resin carried out parse operation, resolving agent was the mixed solvent of organic solvent and water, wherein the organic solvent volume ratio is controlled at 50%～90%.
13. method according to claim 1, it is characterized in that, at step c) in, before described desorbed solution concentrates, perhaps desorbed solution is through partial concentration, but the echinocandin class compound precipitation adds discoloring agent and decolours under 0 ℃～30 ℃ temperature when not separating out, described discoloring agent is gac, aluminum oxide or decolorizing resin, and the discoloring agent ratio of adding is 0.5～5 times of echinocandin compounds gross weight in the feed liquid to be decoloured.
14. method according to claim 13 is characterized in that, at step c) in, desorbed solution carries out vacuum-concentrcted under 20 ℃～60 ℃ temperature.
15. method according to claim 1 is characterized in that, at step e) in, described solidliquid mixture is directly filtered, obtain echinocandin compounds wet-milling.
16. method according to claim 15 is characterized in that, at step e) in, described echinocandin compounds wet-milling is carried out drying, described dry adopt vacuum decompression heat drying or vacuum decompression lyophilize.
17. method according to claim 16 is characterized in that, at step e) in, described vacuum decompression heat drying carries out drying under 20 ℃～60 ℃ temperature.
18. method according to claim 1, it is characterized in that, in steps d) in, described desorbed solution through concentrated remove the second solvent after, add again the third solvent when being water, after separating out the echinocandin class compound precipitation, described solidliquid mixture is directly carried out the vacuum decompression lyophilize.
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| CN106755221A (en) * | 2016-11-28 | 2017-05-31 | 无锡福祈制药有限公司 | A kind of preparation method of MFG parent nucleus FR179642 |
| CN107778359A (en) * | 2016-08-27 | 2018-03-09 | 鲁南制药集团股份有限公司 | A kind of method for purifying ECB parent nucleus |
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| CN105272992A (en) * | 2014-07-26 | 2016-01-27 | 海正药业(杭州)有限公司 | Method for extracting nemadectin from fermentation liquor |
| CN105693824A (en) * | 2015-08-27 | 2016-06-22 | 博瑞生物医药（苏州）股份有限公司 | Method for reducing micafungin sodium raw material drug solvent residual amount |
| CN107778359A (en) * | 2016-08-27 | 2018-03-09 | 鲁南制药集团股份有限公司 | A kind of method for purifying ECB parent nucleus |
| CN107778359B (en) * | 2016-08-27 | 2020-08-28 | 鲁南制药集团股份有限公司 | Method for purifying echinocandin B mother nucleus |
| CN106399431A (en) * | 2016-11-28 | 2017-02-15 | 无锡福祈制药有限公司 | Preparation method of micafungin precursor |
| CN106755221A (en) * | 2016-11-28 | 2017-05-31 | 无锡福祈制药有限公司 | A kind of preparation method of MFG parent nucleus FR179642 |
| CN110655557A (en) * | 2018-06-28 | 2020-01-07 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of pneumocandin B0 serine analogue |
| CN111187339A (en) * | 2018-11-15 | 2020-05-22 | 江苏豪森药业集团有限公司 | Method for extracting FR901379 from fermentation liquor |
| CN111187339B (en) * | 2018-11-15 | 2023-12-01 | 江苏豪森药业集团有限公司 | Method for extracting FR901379 from fermentation broth |
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Effective date of registration: 20180426 Address after: 312366 188 Zhiyuan Avenue, Binhai New Town, Shaoxing, Zhejiang Patentee after: Zhejiang Changhai Pharmaceutical Co., Ltd. Address before: No. 59 around the East Road around Xinchang, Zhejiang, Zhejiang Patentee before: Xinchang Pharmaceutical Factory, Zhejiang Medicine Co., Ltd. |