CN101659692B - Method for preparing echinocandin B - Google Patents
Method for preparing echinocandin B Download PDFInfo
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- CN101659692B CN101659692B CN2009101331176A CN200910133117A CN101659692B CN 101659692 B CN101659692 B CN 101659692B CN 2009101331176 A CN2009101331176 A CN 2009101331176A CN 200910133117 A CN200910133117 A CN 200910133117A CN 101659692 B CN101659692 B CN 101659692B
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Abstract
The invention discloses a method for preparing echinocandin B. The method comprises the following steps of: a) centrifuging the fermentation liquid of echinocandin B, taking mycelium, leaching the echinocandin B in the mycelium with a first solvent and filtering and removing the mycelium; b) distilling the solvent in the first solvent leaching solution of echinocandin B to dryness, soaking with a second solvent and filtering and removing the insoluble substances; c) distilling the second solvent soak solution of echinocandin B to dryness and then dissolving in the first solvent, utilizing macroporous absorption resin, performing eluting with the first solvent and collecting the part with higher purity; d) distilling the collected solution of echinocandin B to dryness and then dissolving in the second solvent, utilizing an alumina column, performing eluting with the first solvent and collecting eluent; e) distilling the eluent of echinocandin B to dryness and then dissolving in the first solvent, utilizing inverse resin, performing eluting with the first solvent and then collecting the part with higher purity; and f) distilling the collected solution of echinocandin B to dryness and then dissolving in the first solvent, adding in a small amount of water by dripping to achieve the purpose of separation by crystallization after supersaturation, and preparing the echinocandin B. The method for preparing the echinocandin B not only can well remove the pigment, but also is suitable for the large-scale industrial production and has good commercial value; and the method leads the final purity of the echinocandin B to be improved by more than 95 percent.
Description
Technical field
The invention belongs to the pharmaceutical chemistry field, be specifically related to a kind of method for preparing the precursor echinocandin B of anidulafungin.
Background technology
Since the seventies in 20th century; In treatment clinical course; Fungi infestation disease and the mortality ratio that causes owing to uncontrollable fungi infestation improve gradually, and this has direct relation with widely-used to the prejudicial medicine of human immune system and a large amount of extensive pedigree antibiotics that use.In addition, relevant with the infection of clinical use implants in vivo and chronic immunosuppressant virus as AIDS.Therefore, safe, the novel and effective antifungal drug of research and development seems extremely important.As everyone knows, the fungal cell has cell walls, and mammalian cell does not have cell walls.Therefore, should be with the cell walls of fungi action target spot as the novel anti fungi-medicine.The echinocandin class promptly is the medicine that acts on fungal cell wall.
Echinocandin (Echinocandins) is one type of natural product finding the seventies in 20th century, has the activity that noncompetitive ground suppresses β-1,3 glucan synthase.This title of echinocandin refers to one type of natural antimycotic product of ring-type lipopeptid class with identical ring type polypeptide core and different fatty acids side chain at first, and echinocandin family then comprises members such as echinocandin, cilofungin, pneumocandins, aculeucins, mulundocandin and W F11899 now.Echinocandin mainly contains three types: B, C and D, and wherein echinocandin B is topmost type, it is produced by A spergilusnid ulans and A.rugulosus.A Nifen is quiet to be the semi-synthetic verivate of echinocandin B, is in the clinical study stage three phases at present.
Domestic research for echinocandin B preparation technology is not almost reported; External do not have any new progress to the preparation technology of echinocandin B these years yet; The achievement in research of the comparison system that can know is at present mainly come company from gift scientific research personnel; But their technology can only small-scale preparation in laboratory scope, is difficult to realize suitability for industrialized production.
Introduced gift in the USP 4288549 in detail and come the research of the scientific research personnel of company, this process using silicagel column, method purifying echinocandin Bs such as extraction the separation-extraction technology of echinocandin B.Its main drawback was the hypertonia of post, and preparation technology is difficult to amplify; And can not well remove the pigment in the fermented liquid of the upper reaches.
Summary of the invention
For solving the technical problem of still not having the method for effective separation and Extraction echinocandin B in the prior art; The applicant has invented a kind of simple method for preparing echinocandin B; This method not only can well be removed the pigment in the echinocandin B; And be fit to large-scale industrial production, have good commercial value.This method can be brought up to the final purity of echinocandin B more than 95%.
Therefore, the present invention provides a kind of method for preparing echinocandin B, and this method comprises the following steps:
A) fermented liquid of centrifugal echinocandin B is got mycelium, with echinocandin B in first kind of solvent leaching mycelium, removes by filter mycelium;
B) with the solvent evaporate to dryness in first kind of solvent leaching solution of echinocandin B,, remove by filter insolubles with second kind of solvent soaking;
C) with being dissolved in first kind of solvent behind second kind of solvent soaking liquid of echinocandin B evaporate to dryness, last macroporous adsorbent resin carries out wash-out with first kind of solvent, collects elutriant;
D) be dissolved in second kind of solvent after echinocandin B is collected the liquid evaporate to dryness, last alumina column carries out wash-out with first kind of solvent, collects elutriant;
E) echinocandin B elutriant evaporate to dryness is dissolved in first kind of solvent, last reversed-phase resin carries out wash-out with first kind of solvent, collects elutriant;
F) echinocandin B is collected behind the liquid evaporate to dryness with first kind of dissolution with solvents, dripped less water its supersaturation post crystallization is separated out, make echinocandin B.
First kind of solvent in the inventive method is methyl alcohol or ethanol, particular methanol; Second kind of solvent is propyl carbinol.
With oxalic acid the pH value of echinocandin B fermented liquid in a) step is adjusted to 2～4 in the inventive method, preferred pH value is adjusted to 3.
The inventive method a) in the step the used volume of methyl alcohol be preferably mycelial 1.5～2.5 times, preferred 1.5 times.
The inventive method is step and b a)) concentration of first kind of solvent in the step is preferably greater than 90%.
The inventive method b) concentration of second kind of solvent in the step is preferably 100%.
The inventive method c) concentration of first kind of solvent is 60%～70% during the upper prop in the step, and preferred concentration is 65%, and first kind of solvent strength is 85%～95% during wash-out, and preferred concentration is 90%.
The inventive method d) concentration of second kind of solvent is 80%～100% during the upper prop in the step, and preferred concentration is 100%, and first kind of solvent strength is 70%～80% during wash-out, and preferred concentration is 75%.
The inventive method e) concentration of first kind of solvent is 60%～70% during the upper prop in the step, and preferred concentration is 65%, and first kind of solvent strength is 85%～95% during wash-out, and preferred concentration is 90%.
The inventive method c) elutriant in the step is a purity greater than 40% part; E) elutriant in the step is a purity greater than 90% part.
Preferably, the inventive method d) macroporous adsorbent resin in the step is the HP20 resin.
Preferably, the inventive method e) reversed-phase resin in the step is a YPR II resin.
The applicant proves through Comparative Examples; Because the pigment in the echinocandin B solution is very many, therefore, through c) polymeric adsorbent of step; The pigment absorption that a lot of polarity are bigger is not got on; And echinocandin B can be good at being adsorbed up, has a lot of pigments can not washed during with first kind of eluate solvent wash-out of 9: 1 on the resin, and therefore the keying action in this step is the removal pigment.
And d) the main effect of step is to have removed the bigger impurity of a lot of polarity; Make the purity of echinocandin B be greatly improved; Echinocandin B can be good at being adsorbed during upper prop, and a lot of polar impurities can not eluted when selecting first kind of solvent elution of 4: 1 for use.Simultaneously, it has also removed the pigment that polymeric adsorbent can not be removed fully.
E of the present invention) the main effect of step is the enrichment echinocandin B; First kind of solvent upper prop echinocandin B of 3: 1 can be by absorption fully; Echinocandin B is concentrated and is eluted during 9: 1 wash-outs; Liquid Detection finds that this resin can be good near the impurity of the RT echinocandin B peak.
With respect to prior art, the invention has the advantages that: whole process flow is simple, and cost is lower, does not have factory's scale-up problem, is easy to realize suitability for industrialized production.And this technology can remove pigment in the fermented liquid and impurity basically, makes the purity of title product reach the ideal level.
Embodiment
Echinocandin B HPLC testing conditions of the present invention is following:
Chromatographic column: ODS-C18 (5 μ);
Detect wavelength: 222nm;
Moving phase: water: methyl alcohol: own nitrile (20: 70: 10);
Flow velocity: 0.800ml/min
Column temperature: 40 ℃
Echinocandin B fermentation condition of the present invention is following:
Bacterial classification: ATCC 58397
Slant culture
Substratum: PDA
Condition: 25 ℃, 10 days
Seed culture
Substratum: glucose 1.0%, glycerine 1.0%, cottonseed meal 2.5%, pH are 6.8-7.0
Condition: 25 ℃, 2 days, shaking speed: 250rpm
Fermentation culture
Substratum:
Glucose 2.0%, sucrose 2.0%, glycerine 2.0%, soya-bean oil 2.0%, cottonseed meal 2.0%, analysis for soybean powder 1.0%, yeast powder 0.5%, KH
2PO
40.5%, proline(Pro) 0.5%, and preceding pH7.0 disappears
121 ℃ of sterilization 20min, inoculum size 10% is shaken bottled amount 100ml/750ml, places the 25 ℃ of rotary shaking table 250rpm of thermostatic chamber shake flask fermentations after 9 days, obtains the echinocandin B fermented liquid.
Embodiment 1,2, and 3 investigate the influence of the fermented liquid of different pH to separating technology
Embodiment 1:
Get echinocandin B fermented liquid 500ml, its purity is 3.1%, and unit is 647ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 2.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.0%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 34.7%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 67.1%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 88.2%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 92.3%, more than the total recovery in several steps be about 36%.
Embodiment 2: best pH
Get echinocandin B fermented liquid 500ml, its purity is 3.1%, and unit is 647ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 39.6%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 70.4%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 91.3%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 95.6%, more than the total recovery in several steps be about 36%.
Embodiment 3:
Get echinocandin B fermented liquid 500ml, its purity is 3.1%, and unit is 647ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 4.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 7.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 34.2%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 65.4%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 88.2%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 91.5%, more than the total recovery in several steps be about 36%.
Embodiment 4～6 investigates the influence of the solvent of different concns proportioning to adsorption and desorption by resin
Embodiment 4:
Get echinocandin B fermented liquid 500ml, its purity is 3.6%, and unit is 698ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution that contains 30% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, again with the methanol solution wash-out pillar that contains 5% water; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 33.5%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 70.4%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution of 30% water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the methanol solution wash-out pillar of 5% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 88.4%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 92.5%, more than the total recovery in several steps be about 36%.
Embodiment 5: best proportioning
Get echinocandin B fermented liquid 500ml, its purity is 3.6%, and unit is 698ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution that contains 35% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, again with the methanol solution wash-out pillar that contains 10% water; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 39.5%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 72.4%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution of 35% water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the methanol solution wash-out pillar of 10% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 93.8%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 95.7%, more than the total recovery in several steps be about 34%.
Embodiment 6:
Get echinocandin B fermented liquid 500ml, its purity is 3.6%, and unit is 698ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution that contains 40% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, again with the methanol solution wash-out pillar that contains 15% water; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 35.2%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 69.3%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, in the methanol solution water that is dissolved in 40% water behind the echinocandin B evaporate to dryness; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the methanol solution wash-out pillar of 15% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 90.1%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 93.9%, more than the total recovery in several steps be about 35%.
Embodiment 7,8, and 9 investigate the influence of the solvent of different concns proportioning to the aluminum oxide wash-out
Embodiment 7
Get echinocandin B fermented liquid 500ml, its purity is 3.9%, and unit is 663ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution that contains 35% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, again with the methanol solution wash-out pillar that contains 10% water; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 38.9%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Echinocandin B is collected behind the liquid evaporate to dryness propyl carbinol dissolving upper prop with 80%; The upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid, use 70% methanol-eluted fractions pillar again; Collect all effluent; This moment, echinocandin B purity was 68.9%, and most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPRII resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution of 35% water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the methanol solution wash-out pillar of 10% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 90.4%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 92.4%, more than the total recovery in several steps be about 30%.
Embodiment 8
Get echinocandin B fermented liquid 500ml, its purity is 3.9%, and unit is 663ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution that contains 35% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, again with the methanol solution wash-out pillar that contains 10% water; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 38.9%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Echinocandin B is collected behind the liquid evaporate to dryness propyl carbinol dissolving upper prop with 100%; The upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid, use 75% methanol-eluted fractions pillar again; Collect all effluent; This moment, echinocandin B purity was 74.5%, and most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution of 35% water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the methanol solution wash-out pillar of 10% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 94.3%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 96.8%, more than the total recovery in several steps be about 30%.
Embodiment 9
Get echinocandin B fermented liquid 500ml, its purity is 3.9%, and unit is 663ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.9%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution that contains 35% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, again with the methanol solution wash-out pillar that contains 10% water; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 38.9%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Echinocandin B is collected behind the liquid evaporate to dryness propyl carbinol dissolving upper prop with 100%; The upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid, use 80% methanol-eluted fractions pillar again; Collect all effluent; This moment, echinocandin B purity was 71.1%, and most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the methanol solution of 35% water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the methanol solution wash-out pillar of 10% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 91.3%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 94.6%, more than the total recovery in several steps be about 30%.
Embodiment 10,11, and 12 investigate the influence of the solvent soaking mycelium of different volumes to separating technology
Embodiment 10
Get echinocandin B fermented liquid 500ml, its purity is 4.2%, and unit is 712ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1 times; Remove by filter mycelium, again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally; Detecting its purity is 8.9%, and yield is 60%.
Embodiment 11
Get echinocandin B fermented liquid 500ml, its purity is 4.2%, and unit is 712ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium, again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally; Detecting its purity is 8.7%, and yield is 85%.
Embodiment 12
Get echinocandin B fermented liquid 500ml, its purity is 4.2%, and unit is 712ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 2 times; Remove by filter mycelium, again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally; Detecting its purity is 8.4%, and yield is 90%.
Conclusion: methyl alcohol and propyl carbinol soaking fermentation liquid with different volumes are little to the influence of fermented liquid purity, but very big to the influence of its yield, and the solvent of two volumes and monoploid be long-pending, and to compare raising be not a lot, in line with the principle of saving, selects 1.5 times for use
Embodiment 13 investigates ethanol as the influence of first kind of solvent to separating technology
Get echinocandin B fermented liquid 500ml, its purity is 3.3%, and unit is 598ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is extracted echinocandin B wherein with mycelium with 1.5 times alcohol immersion; Remove by filter mycelium; Again the propyl carbinol with same volume behind the ethanol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.2%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the ethanolic soln that contains 35% water; Flow velocity with 10ml/min is crossed post, and echinocandin B is not attracted on the post fully, and a spot of leakage is arranged; With the ethanolic soln wash-out pillar that contains 10% water, distribute and collect the higher part of elutriant moderate purity again, detect and find that purity is 33.5%; Contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Echinocandin B is collected behind the liquid evaporate to dryness propyl carbinol dissolving upper prop with 100%; The upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid, use 75% ethanol elution pillar again; Collect all effluent; This moment, echinocandin B purity was 60.2%, and partial pigment is removed, and solution is incarnadine.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in the ethanolic soln of 35% water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses the ethanolic soln wash-out pillar of 10% water again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 89.4%, pigment is removed basically; Solution is faint yellow vitreous state, again the echinocandin B evaporate to dryness is used dissolve with ethanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 91.0%, more than the total recovery in several steps be about 30%.
Comparative Examples 1: investigate step b) without the influence of propyl carbinol leaching to separating technology
Get echinocandin B fermented liquid 500ml; Its purity is 5%, and unit is 677ug/ml, leaves standstill for some time after fermented liquid pH is adjusted to 3.0; Abandoning supernatant behind the high speed centrifugation; With methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times, remove by filter mycelium, this moment, its purity was 4%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 6: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 30.8%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 59.1%; Most of pigment is removed, and it is faint yellow that solution is.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 4: 1 the methanol-water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity, detect and find that purity is 81.4%, pigment is removed basically; Solution is the water white transparency state, again the echinocandin B evaporate to dryness is used dissolve with methanol at last, adds less water its supersaturation post crystallization is separated out; This moment, purity was 85.1%, more than the total recovery in several steps be about 41%.
Conclusion: if there is not propyl carbinol to soak this step in the technology, a lot of water miscible impurity are difficult to remove in the fermented liquid, make the purity of final finished decrease.
Comparative Examples 2: investigation lacks the influence of step c) to separating technology
Get echinocandin B fermented liquid 500ml, its purity is 3.5%, and unit is 612ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.4%.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 55.3%; A lot of pigments are removed, and solution is incarnadine.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 79.2%, most of pigment is removed, and solution is faint yellow state.
Again the echinocandin B evaporate to dryness is used dissolve with methanol at last, add and to put into crystallizer after less water makes its supersaturation, discovery can't crystallization, finally can only obtain purity and be 79.2% faint yellow amorphous substance.
Comparative Examples 3: investigation lacks the influence of step d) to separating technology
Get echinocandin B fermented liquid 500ml, its purity is 3.5%, and unit is 612ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.4%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 39.6%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high about 20cm, diameter is about the YPR II resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 15ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 68.2%, most of pigment is removed, and solution is the incarnadine state.
Again the echinocandin B evaporate to dryness is used dissolve with methanol at last, add and to put into crystallizer after less water makes its supersaturation, discovery can't crystallization, finally can only obtain purity and be 68.2% faint yellow amorphous substance.
Comparative Examples 4: investigation lacks the influence of step e) to separating technology
Get echinocandin B fermented liquid 500ml, its purity is 3.5%, and unit is 612ug/ml; Leave standstill for some time after fermented liquid pH is adjusted to 3.0, abandoning supernatant behind the high speed centrifugation is with methyl alcohol soaking and extracting wherein the echinocandin B of mycelium with 1.5 times; Remove by filter mycelium; Again the propyl carbinol with same volume behind the methyl alcohol evaporate to dryness is leached echinocandin B, remove by filter insolubles wherein equally, detecting its purity is 8.4%.
Adorn a high about 20cm, diameter is about the HP20 resin column of 4cm, with being dissolved in behind the echinocandin B evaporate to dryness in 3: 1 the methanol-water; Flow velocity with 10ml/min is crossed post, and echinocandin B is attracted on the post fully, uses 9: 1 methanol-water wash-out pillar again; Distribute and collect the higher part of elutriant moderate purity; Detect and find that purity is 39.6%, contain comparatively significantly pigment, solution is incarnadine.
Adorn a high 15cm of being about, the alumina column of diameter 3cm.Dissolve upper prop with propyl carbinol after echinocandin B collected the liquid evaporate to dryness, the upper prop flow rate control does not have echinocandin at 12ml/min in the waste liquid; Use 4: 1 methanol-water wash-out pillar again, collect all effluent, this moment, echinocandin B purity was 70.4%; Most of pigment is removed, and solution is almost colourless.
Again the echinocandin B evaporate to dryness is used dissolve with methanol at last, add and to put into crystallizer after less water makes its supersaturation, discovery can't crystallization, finally can only obtain purity and be 70.4% white amorphous substance.
Attach: the used resin model of the present invention
1DIAION?HP20
Manufacturer: MIT
Specific surface area (m2/g): 600
Pore radius (
):＞200
Apparent density (g/L-R): 680
Water-content (%): 55-65
2YPR?II
Manufacturer: Anhui Samsung resin Science and Technology Ltd.
The relative Oxacyclotetradecane,erythromycin deriv u/ml of exchanging equivalent: >=8 ten thousand
Moisture content %:45-55
Granularity (0.315-1.25mm): >=95
Physical strength %: >=90
Claims (15)
1. the method for preparing echinocandin B, this method comprises the following steps:
A) fermented liquid of centrifugal echinocandin B is got mycelium, with echinocandin B in first kind of solvent leaching mycelium, removes by filter mycelium;
B) with the solvent evaporate to dryness in first kind of solvent leaching solution of echinocandin B, soak with propyl carbinol, remove by filter insolubles;
C) with being dissolved in first kind of solvent behind the echinocandin B propyl carbinol soak solution evaporate to dryness; Last macroporous adsorbent resin; Carry out wash-out with first kind of solvent; Collect elutriant, wherein the concentration as first kind of solvent of solvent is 60%～70%, is 85%～95% as the concentration of first kind of solvent of eluent;
D) be dissolved in the propyl carbinol after echinocandin B is collected the liquid evaporate to dryness, last alumina column carries out wash-out with first kind of solvent, collects elutriant, and the concentration of wherein said first kind of solvent is 70%～80%;
E) echinocandin B elutriant evaporate to dryness is dissolved in first kind of solvent; Last reversed-phase resin carries out wash-out with first kind of solvent, collects elutriant; Wherein the concentration as first kind of solvent of solvent is 60%～70%, is 85%～95% as the concentration of first kind of solvent of eluent;
F) echinocandin B is collected behind the liquid evaporate to dryness with first kind of dissolution with solvents, dripped less water its supersaturation post crystallization is separated out, make echinocandin B;
Wherein said first kind of solvent is methyl alcohol or ethanol.
2. method according to claim 1 is characterized in that, the pH value of echinocandin B fermented liquid in a) step is adjusted to 2～4.
3. method according to claim 2 is characterized in that, the pH value of echinocandin B fermented liquid in a) step is adjusted to 3.
4. method according to claim 1 is characterized in that, a) first kind of solvent and b in the step) the used volume of propyl carbinol is mycelial 1.5～2.5 times in the step.
5. method according to claim 4 is characterized in that, a) first kind of solvent and b in the step) the used volume of propyl carbinol is mycelial 1.5 times in the step.
6. method according to claim 1 is characterized in that, b) concentration of the propyl carbinol in the step is 100%.
7. method according to claim 1 is characterized in that, c) and e) concentration as first kind of solvent of solvent in the step is 65%.
8. method according to claim 1 is characterized in that, c) and e) concentration as first kind of solvent of eluent in the step is 90%.
9. method according to claim 1 is characterized in that, d) concentration as the propyl carbinol of solvent is 80%～100% in the step.
10. method according to claim 9 is characterized in that, d) concentration as the propyl carbinol of solvent is 100% in the step.
11. method according to claim 1 is characterized in that, d) concentration as first kind of solvent of eluent is 75% in the step.
12. method according to claim 1 is characterized in that, c) elutriant in the step is a purity greater than 40% part.
13. method according to claim 1 is characterized in that, e) elutriant in the step is a purity greater than 90% part.
14. method according to claim 1 is characterized in that, c) macroporous adsorbent resin in the step is selected from the HP20 resin.
15. method according to claim 1 is characterized in that, e) reversed-phase resin in the step is selected from YPR II resin.
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| CN2009101331176A CN101659692B (en) | 2008-08-27 | 2009-04-01 | Method for preparing echinocandin B |
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| CN103724403B (en) * | 2012-10-12 | 2018-02-16 | 重庆乾泰生物医药有限公司 | The isolation and purification method and purposes of a kind of ECB |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050267019A1 (en) * | 1997-12-10 | 2005-12-01 | Oliver Courtin | Echinocandin derivatives, their method of preparation and their application as anti-fungal agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050267019A1 (en) * | 1997-12-10 | 2005-12-01 | Oliver Courtin | Echinocandin derivatives, their method of preparation and their application as anti-fungal agents |
Non-Patent Citations (2)
| Title |
|---|
| David W Denning.Echinocandin antifungal drugs.《THE LANCET》.2003,第362卷1142-1151. * |
| 张致平.抗真菌药物研究进展.《中国处方药》.2006,(第48期),28-32. * |
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