CN105111285A - Daptomycin extraction method - Google Patents
Daptomycin extraction method Download PDFInfo
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- CN105111285A CN105111285A CN201510668192.8A CN201510668192A CN105111285A CN 105111285 A CN105111285 A CN 105111285A CN 201510668192 A CN201510668192 A CN 201510668192A CN 105111285 A CN105111285 A CN 105111285A
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- daptomycin
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Abstract
The invention discloses a daptomycin extraction method. Firstly, daptomycin in a fermentation broth is gathered, the daptomycin obtained through gathering is roughly extracted, rough extraction is performed through acidic column chromatography and neutral column chromatography, and a daptomycin fermentation broth is obtained after chromatography. Clear filtrate is obtained after the daptomycin fermentation broth is filtered through a polyethylene film and passes through a macroporous adsorption chromatographic resin column, daptomycin is adsorbed on the macroporous adsorption chromatographic resin column, the settling process is easy to control, the mixed solution is easy to filter, and the obtained daptomycin has light color and is loosely powdery and easy to dissolve. The extraction method has the advantages that a settling method is simple and convenient and the technological process is applicable to mass production.
Description
Technical field
The present invention relates to daptomycin intermediate processing technical field, particularly relate to a kind of extracting method of daptomycin.
Background technology
Daptomycin is first product that a class is called cyclic ester Tai Lei new antibiotic family.It is from streptomyces parvus (
streptomycesparvus) the central Cyclic lipopeptide antibiotic extracting the novel structure of 13 the amino acid compositions obtained of fermented liquid, wherein ten Amino acid profile ring texturees, in the outer capric acid of ring and tryptophane generation esterification.It not only has novel chemical structure, and its binding mode is also from arbitrary to have got permission microbiotic different.It can destroy bacterial cell membrane function in many aspects, kills gram positive organism rapidly thus.Daptomycin decapacitation acts on outside most of clinical relevant gram positive organism, the more important thing is in vitro in X-1497 (methicillin), the resistance isolated strains such as vancomycin and Linezolid still has a strong active.Summary of the invention
Daptomycin is tunning, the ferment filtrate obtained by fermentation culture, a large amount of pigments can be produced in fermenting process and with the akin by product of daptomycin structure as the daptomycin that dewaters, therefore the extracting and purifying method of daptomycin is more complicated.General daptomycin producing strains, throughput is unstable, and output is lower, fermentation byproduct is more, and impurity is more, causes rear extraction work comparatively complicated, substantially increase the purification difficulty of postorder, be difficult to obtain highly purified final product, thus cannot industrialization produce.
Had many reported in literature to the extracting and purifying method of daptomycin postorder, roughly technique is: fermentation liquor ultrafiltration, nanofiltration, resin cation (R.C.) absorption pickling, resin anion(R.A) neutralization, nanofiltration is concentrated, crystallization.Membrane filtration yield reaches 98-99%, and product purity after crystallization also can obtain more than 98.5%.But daptomycin is an amphiprotic substance, iso-electric point is about pH4-5, under different pH conditions, its solvability is different, especially daptomycin has a large amount of homologues, isomer, and these impurity are very similar to daptomycin in nature, therefore, by so simple technique, industrially want separation and purification even crystallization daptomycin be very difficult.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of extracting method of daptomycin is provided.
Technical scheme of the present invention is as follows: the extracting method that the invention provides a kind of daptomycin, comprises the following steps:
A, the daptomycin obtained first by daptomycin enrichment in fermented liquid, then by enrichment slightly extract, and wherein thick extraction adopts acid column chromatography, then uses neutral column chromatography, obtains daptomycin fermented liquid after chromatography.
B, daptomycin fermented liquid to be regulated after pH2.0-4.5, filter with the ceramic membrane that membrane pore size is 0.01 μm-0.1 μm, then the acidic aqueous solution circulation cleaning ceramic membrane of pH2.0-4.0 is used, abandon filtrate, use the alkaline aqueous solution circulation cleaning ceramic membrane of pH8-10 again, collect daptomycin filtrate;
C, in precipitation vessel the daptomycin of gained in settling step b, obtain crystal, and stir this crystal slurry, pH value is adjusted to required degree continuously, until precipitate completely, obtain crystal precipitation;
D, prepare liquid through lyophilize daptomycin and obtain daptomycin.
The present invention is preferred, and according in step c, by carrying out crystallization containing adding the organic solvent dissolved each other with water in clean antibiotic activity elutriant, before crystallization, in solution, daptomycin content is 5.0% ~ 30.0%.
The present invention is preferred, and described machine solvent is methyl alcohol, ethanol, Virahol or acetone.
The present invention is preferred, and described machine solvent adding amount is: methyl alcohol is 2 ~ 6 times of crystal solution volumes; Ethanol is 1.5 ~ 5.5 times of crystal solution volumes; Virahol is 1.2 ~ 5 times of crystal solution volumes; Acetone is 1.0 ~ 4.5 times of crystal solution volumes.
The present invention is preferred, and according in step c, crystallisation process starting temperature is 25 ~ 60 DEG C; Crystallisation process terminal temperature is 0 ~ 30 DEG C; Crystallisation process cooling rate is 0.5 ~ 5 DEG C/min.
Beneficial effect of the present invention is as follows:
Adopt such scheme, the present invention is filtered by polyethylene film, and the clear filtrate obtained is by macroporous absorption chromatography resin post, daptomycin is attracted on macroporous absorption chromatography resin post, and precipitation process is easy to control, and mixed solution easily filters, the daptomycin alkali obtained is of light color, and pulverulent bulk is soluble; The present invention has the advantage that the easy and technical process of intermediate processing is applicable to produce in enormous quantities.
Accompanying drawing explanation
Fig. 1 is the schema of the extracting method of a kind of daptomycin of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Refer to Fig. 1, the invention provides a kind of extracting method of daptomycin, comprise the following steps:
A, the daptomycin obtained first by daptomycin enrichment in fermented liquid, then by enrichment slightly extract, and wherein thick extraction adopts acid column chromatography, then uses neutral column chromatography, obtains daptomycin fermented liquid after chromatography.
B, daptomycin fermented liquid to be regulated after pH2.0-4.5, filter with the ceramic membrane that membrane pore size is 0.01 μm-0.1 μm, then the acidic aqueous solution circulation cleaning ceramic membrane of pH2.0-4.0 is used, abandon filtrate, use the alkaline aqueous solution circulation cleaning ceramic membrane of pH8-10 again, collect daptomycin filtrate;
C, the daptomycin of gained in settling step a in precipitation vessel, obtain crystal, and stir this crystal slurry, pH value is adjusted to required degree continuously, until precipitate completely, obtain crystal precipitation, by carrying out crystallization containing adding the organic solvent dissolved each other with water in clean antibiotic activity elutriant, before crystallization, in solution, daptomycin content is 5.0% ~ 30.0%, by carrying out crystallization containing adding the organic solvent dissolved each other with water in clean antibiotic activity elutriant, before crystallization, in solution, daptomycin content is 5.0% ~ 30.0%, described machine solvent adding amount is: methyl alcohol is 2 ~ 6 times of crystal solution volumes, ethanol is 1.5 ~ 5.5 times of crystal solution volumes, Virahol is 1.2 ~ 5 times of crystal solution volumes, acetone is 1.0 ~ 4.5 times of crystal solution volumes, and crystallisation process starting temperature is 25 ~ 60 DEG C, crystallisation process terminal temperature is 0 ~ 30 DEG C, crystallisation process cooling rate is 0.5 ~ 5 DEG C/min,
D, prepare liquid through lyophilize daptomycin and obtain daptomycin.。
embodiment one:
The present invention includes following steps:
A, the daptomycin obtained first by daptomycin enrichment in fermented liquid, then by enrichment slightly extract, and wherein thick extraction adopts acid column chromatography, then uses neutral column chromatography, obtains daptomycin fermented liquid after chromatography
B, daptomycin fermented liquid to be regulated after pH3.0, filter with the ceramic membrane that membrane pore size is 0.05 μm, then use the acidic aqueous solution circulation cleaning ceramic membrane of pH3.0, abandon filtrate, use the alkaline aqueous solution circulation cleaning ceramic membrane of pH9 again, collect daptomycin filtrate;
C, the daptomycin of gained in settling step a in precipitation vessel, obtain crystal, and stir this crystal slurry, pH value is adjusted to required degree continuously, until precipitate completely, obtain crystal precipitation, by carrying out crystallization containing adding the organic solvent dissolved each other with water in clean antibiotic activity elutriant, before crystallization, in solution, daptomycin content is 21.0%, by carrying out crystallization containing adding the organic solvent dissolved each other with water in clean antibiotic activity elutriant, before crystallization, in solution, daptomycin content is 18.0%, described machine solvent adding amount is: methyl alcohol is 4 times of crystal solution volumes, ethanol is 3.5 times of crystal solution volumes, Virahol is 2.3 times of crystal solution volumes, acetone is 3.3 times of crystal solution volumes, and crystallisation process starting temperature is 50 DEG C, crystallisation process terminal temperature is 10 DEG C, crystallisation process cooling rate is 5 DEG C/min,
D, prepare liquid through lyophilize daptomycin and obtain daptomycin.
In sum, adopt such scheme, the present invention is filtered by polyethylene film, the clear filtrate obtained is by macroporous absorption chromatography resin post, and daptomycin is attracted on macroporous absorption chromatography resin post, and precipitation process is easy to control, mixed solution easily filters, the daptomycin alkali obtained is of light color, and pulverulent bulk is soluble; The present invention has the advantage that the easy and technical process of intermediate processing is applicable to produce in enormous quantities.
These are only preferred embodiment of the present invention, be not limited to the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. an extracting method for daptomycin, is characterized in that, comprises the following steps:
A, the daptomycin obtained first by daptomycin enrichment in fermented liquid, then by enrichment slightly extract, and wherein thick extraction adopts acid column chromatography, then uses neutral column chromatography, obtains daptomycin fermented liquid after chromatography.
2.b, daptomycin fermented liquid to be regulated after pH2.0-4.5, filter with the ceramic membrane that membrane pore size is 0.01 μm-0.1 μm, then the acidic aqueous solution circulation cleaning ceramic membrane of pH2.0-4.0 is used, abandon filtrate, use the alkaline aqueous solution circulation cleaning ceramic membrane of pH8-10 again, collect daptomycin filtrate;
C, in precipitation vessel the daptomycin of gained in settling step b, obtain crystal, and stir this crystal slurry, pH value is adjusted to required degree continuously, until precipitate completely, obtain crystal precipitation;
D, prepare liquid through lyophilize daptomycin and obtain daptomycin.
3. the extracting method of a kind of daptomycin according to claim 1, it is characterized in that, according in step c, by carrying out crystallization containing adding the organic solvent dissolved each other with water in clean antibiotic activity elutriant, before crystallization, in solution, daptomycin content is being 5.0% ~ 30.0%.
4. the extracting method of a kind of daptomycin according to claim 2, is characterized in that, described machine solvent is methyl alcohol, ethanol, Virahol or acetone.
5. the extracting method of a kind of daptomycin according to claim 3, is characterized in that, described machine solvent adding amount is: methyl alcohol is 2 ~ 6 times of crystal solution volumes; Ethanol is 1.5 ~ 5.5 times of crystal solution volumes; Virahol is 1.2 ~ 5 times of crystal solution volumes; Acetone is 1.0 ~ 4.5 times of crystal solution volumes.
6. the extracting method of a kind of daptomycin according to claim 1, is characterized in that, according in step b, crystallisation process starting temperature is 25 ~ 60 DEG C; Crystallisation process terminal temperature is 0 ~ 30 DEG C; Crystallisation process cooling rate is 0.5 ~ 5 DEG C/min.
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| CN201510668192.8A CN105111285A (en) | 2015-10-17 | 2015-10-17 | Daptomycin extraction method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106518985A (en) * | 2015-09-15 | 2017-03-22 | 江苏海阔生物医药有限公司 | Vancomycin precipitation method |
| CN106866791A (en) * | 2015-12-11 | 2017-06-20 | 北大方正集团有限公司 | A kind of preparation method of high-purity daptomycin lactone hydrolysate |
| CN108250270A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | A kind of method of the enrichment extraction Daptomycin from zymotic fluid |
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| CN102675426A (en) * | 2012-04-26 | 2012-09-19 | 杭州华东医药集团生物工程研究所有限公司 | Extraction and purification method of daptomycin |
| CN102875652A (en) * | 2011-07-13 | 2013-01-16 | 北大方正集团有限公司 | Method for separating and purifying daptomycin |
| CN104672310A (en) * | 2013-11-27 | 2015-06-03 | 北大医药重庆大新药业股份有限公司 | Preparation method of vancomycin hydrochloride |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875652A (en) * | 2011-07-13 | 2013-01-16 | 北大方正集团有限公司 | Method for separating and purifying daptomycin |
| CN102675426A (en) * | 2012-04-26 | 2012-09-19 | 杭州华东医药集团生物工程研究所有限公司 | Extraction and purification method of daptomycin |
| CN104672310A (en) * | 2013-11-27 | 2015-06-03 | 北大医药重庆大新药业股份有限公司 | Preparation method of vancomycin hydrochloride |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106518985A (en) * | 2015-09-15 | 2017-03-22 | 江苏海阔生物医药有限公司 | Vancomycin precipitation method |
| CN106866791A (en) * | 2015-12-11 | 2017-06-20 | 北大方正集团有限公司 | A kind of preparation method of high-purity daptomycin lactone hydrolysate |
| CN108250270A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | A kind of method of the enrichment extraction Daptomycin from zymotic fluid |
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