CN105001305A - Method for extracting high-purity daptomycin by utilizing chromatographic technique - Google Patents
Method for extracting high-purity daptomycin by utilizing chromatographic technique Download PDFInfo
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- CN105001305A CN105001305A CN201510212580.5A CN201510212580A CN105001305A CN 105001305 A CN105001305 A CN 105001305A CN 201510212580 A CN201510212580 A CN 201510212580A CN 105001305 A CN105001305 A CN 105001305A
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Abstract
The invention discloses a method for extracting high-purity daptomycin by utilizing a chromatographic technique. The method comprises the following steps that firstly, pre-processing is carried out: filtering is carried out on fermentation liquor containing with the daptomycin, and the turbidity of the filtered daptomycin fermentation liquor is lower than a pre-set value; secondly, extracting is carried out: the fermentation liquor which is subjected to the processing in the first step is injected into a chromatographic column filled with first filter to be subjected to absorbing, the first filter is anion exchanging gel filter, wherein glucan or agarose serves as the framework of the anion exchanging gel filter, after the absorbing is carried out, eluent is added, and the daptomycin is eluted; thirdly, refining is carried out; obtained eluting liquid which is obtained in the second step is injected into a chromatographic column filled with second filter to be subjected to the absorbing, polystyrene-divinyl benzene or bonded silica gel serves as the framework of the second filter, after the absorbing is carried out, buffer salt eluting solvent is added, eluting is carried out, and therefore the high-purity daptomycin is obtained. According to the method, the technology is simple, the operating cost is low, and a final daptomycin product with the purity above 98% can be obtained.
Description
Technical field
The present invention relates to a kind of method utilizing chromatographic technique to extract high-purity daptomycin, particularly a kind of method utilizing reversed phase chromatography and anion-exchange chromatography technology to extract high-purity daptomycin, belongs to medicine industry liquid waste disposal technique field.
Background technology
The cyclic lipopeptide medicine that daptomycin is developed as the success of first, the whole world, used good effect at resisting gram-positive bacteria, due to its complex structure, chemical complete synthesis difficulty is more, and current daptomycin all adopts the method for fermentable to produce.Due to the chemical structure that it is special, in the process of broth extraction, daptomycin can develop into the impurity such as dehydration daptomycin and β-isomery daptomycin.The purification process of current industrial employing is mainly the method for macroporous resin adsorption and butanols lixiviate or ion exchange resin extraction, is difficult to prepare highly purified daptomycin product.
The purifying process of current daptomycin extracts by organic phase, after repeatedly extracting merging, then regulates aqueous pH values to strip after being mainly the pH value regulating fermented liquid, and aqueous phase is carried out adsorbing through macroporous resin or ion exchange resin, wash-out.Due to the chemical structure that daptomycin is special, in the purge process of multi-step, easily cause degraded, develop into the impurity such as dehydration daptomycin and β-isomery daptomycin.Therefore purity is difficult to improve, and is only about 89%.The purifying process of multi-step and the use of a large amount of organic reagent, not only add production cost, also cause pollution to environment, three industrial wastes are difficult to get rid of.
Summary of the invention
The present invention seeks to: for the problems referred to above, provide a kind of method utilizing chromatographic technique to extract high-purity daptomycin, the method technique is simple, and running cost is low, can obtain the daptomycin finished product that purity reaches 98%.
Technical scheme of the present invention is: the described chromatographic technique that utilizes extracts the method for high-purity daptomycin, it is characterized in that the method comprises the following steps:
1) pre-treatment: filter the fermented liquid containing daptomycin, makes the turbidity of the daptomycin fermented liquid after filtration lower than preset value;
2) extracting: by through step 1) fermented liquid after process injects the chromatography column that the first filler is housed and adsorbs, the anion-exchange gel filler that described first filler is is skeleton with dextran or agarose, adds eluent and is eluted by daptomycin after absorption;
3) refining: by step 2) elutriant that obtains injects the chromatography column that the second filler is housed and adsorbs, the filler that described second filler is is skeleton with polystyrene-divinylbenzene or bonded silica gel, add buffering salt eluting solvent after absorption and carry out wash-out, thus obtain highly purified daptomycin.
The present invention, on the basis of technique scheme, also comprises following preferred version:
In described step 1) in, be that employing 0.45 micron membrane filter carries out filtration treatment to the fermented liquid containing daptomycin.
The particle diameter of described first filler is 30 microns ~ 500 microns.
The particle diameter of described second filler is 30 microns ~ 300 microns.
Described step 2) in eluent be ammonium acetate-sodium chloride buffer.
In described step 2) in, first rinse with the Ammonium Acetate aqueous solution, then with described ammonium acetate-sodium chloride buffer, daptomycin is eluted.
Described step 3) in buffering salt eluting solvent be PBS-acetonitrile solution.
The method is further comprising the steps of:
4) concentrated: to adopt membrane filtration technique, concentrating, by step 3 through elutriant) high-purity daptomycin that obtains concentrates, the part salinity in removing high-purity daptomycin;
5) crystallization: by the solution freeze-drying after concentrated desalination, obtains dry pulverulent solids.
Described step 4) in membrane filtration technique adopt be through the nanofiltration membrane that molecular weight is 150.
Described step 5) in by the process of the solution freeze-drying after concentrated desalination be: by step 4) to put into 40 degrees below zero freezing for solution after nanofiltration, then puts into Freeze Drying Equipment freeze-drying.
Advantage of the present invention is: the present invention is after screening is groped, adopt novel preparation technology, by means of only twice purification step, the daptomycin finished product that purity reaches 98% can be obtained, technological process significantly reduces the use of organic solvent, reduce further production and cost recovery.At a lower cost, significantly improve purity and the rate of recovery of daptomycin finished product, thus improve the competitiveness of product in market.
1. compared with traditional technology, the present invention only adopts twice purification step, significantly reduces the use of organic reagent in purifying process, reduces the cost of separation and purification.
2. adopt daptomycin finished product prepared by present invention process, purity can reach 98%, and comparatively traditional technology improves a lot in end product purity.
3. present invention process environmental protection, waste liquid is only the salts solution of lower concentration, there is not the problem of three-waste pollution and industrial waste discharge.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme of the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 be in the embodiment of the present invention daptomycin in step 2) in extraction collection of illustrative plates, according to whole components at information gathering climax.In this figure, horizontal axis representing time, its unit is min; Left vertical represents uv-absorbing height, and its unit is ultraviolet absorption value mAu; Right vertical represents percentage of solvents, and its unit is the percentage of B phase moving phase; In figure, black line is the change curve of B phase moving phase percentage, and blue line is the ultraviolet absorption curve of different time.This width figure indicates the uv-absorbing peak shape that crude product goes out when preparative chromatography.
Fig. 2 be in the embodiment of the present invention daptomycin in step 3) in refining collection of illustrative plates, according to whole components at information gathering climax.In this figure, horizontal axis representing time, its unit is min; Left vertical represents uv-absorbing height, and its unit is ultraviolet absorption value mAu; Right vertical represents percentage of solvents, and its unit is the percentage of B phase moving phase; In figure, black line represents the change curve of B phase moving phase percentage, and blue line represents the ultraviolet absorption curve of different time, and the line segment shown by 47min to 59min is the sample collecting the collection of line correspondence, conveniently sees the sample of publishing picture corresponding to medium ultraviolet absorption value.The uv-absorbing peak shape that this width figure goes out when indicating secondary preparative chromatography.
Fig. 3 be in the embodiment of the present invention daptomycin through step 3) analysis chart after refinement treatment.In this figure, horizontal axis representing time, its unit is min; The longitudinal axis represents uv-absorbing height, and its unit is ultraviolet absorption value mAu; Curve is uv-absorbing wavelength curve, above the data mainly percentage composition of each component in the retention time of show sample and sample.This width figure indicates the purity finally making sample, can find out that the purity of daptomycin is more than 98% in figure.
Embodiment
Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiments are not limited to for illustration of the present invention limit the scope of the invention.The implementation condition adopted in embodiment can do further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in normal experiment.
This method utilizing chromatographic technique to extract high-purity daptomycin disclosed in this enforcement, comprises the following steps:
1) pre-treatment: filter the fermented liquid containing daptomycin, makes the turbidity of the daptomycin fermented liquid after filtration lower than preset value.
In order to obtain preferably filter effect, general 0.45 micron membrane filter that adopts carries out filtration treatment to the fermented liquid containing daptomycin.Containing the transparent state of fermented liquid of daptomycin after filtering, without solid impurity.
2) extracting: by through step 1) fermented liquid after process injects the chromatography column that the first filler is housed and adsorbs, the anion-exchange gel filler that described first filler is is skeleton with dextran or agarose, adds eluent and is eluted by daptomycin after absorption.
This step 2) the chromatography extracting method applied belongs to ion exchange chromatography.
In order to obtain preferably chromatographic effect and elute effect, the particle diameter of the first filler described in this example is between 30 microns ~ 500 microns, and described eluent adopts ammonium acetate-sodium chloride buffer.
In order to improve elute effect further, this example is in this step 2) in, first rinse with the Ammonium Acetate aqueous solution, then with described ammonium acetate-sodium chloride buffer, daptomycin is eluted.
3) refining: by step 2) elutriant that obtains injects the chromatography column that the second filler is housed and adsorbs, the filler that described second filler is is skeleton with polystyrene-divinylbenzene or bonded silica gel, add buffering salt eluting solvent after absorption and carry out wash-out, thus obtain highly purified daptomycin.
This step 3) the chromatography extracting method applied belongs to reverse chromatography.
In order to obtain preferably chromatography and elute effect, the particle diameter of the second filler described in this example is between 30 microns ~ 300 microns, and described buffering salt eluting solvent adopts PBS-acetonitrile solution.
As can be seen from Figure 3, the daptomycin purity that this method of the present embodiment extracts reaches more than 98%.
Conveniently the high-purity daptomycin extracted saved, this routine embodiment has also carried out following step 4) and step 5) operation:
4) concentrated: to adopt membrane filtration technique, concentrating, by step 3 through elutriant) high-purity daptomycin that obtains concentrates, the most of salinity in removing high-purity daptomycin.This step 4) in membrane filtration technique adopt be through the nanofiltration membrane that molecular weight is 150.
5) crystallization: by the solution freeze-drying after concentrated desalination, obtains dry pulverulent solids.This step 5) in by the process of the solution freeze-drying after concentrated desalination be: by step 4) to put into 40 degrees below zero freezing for solution after nanofiltration, then puts into Freeze Drying Equipment freeze-drying.
Certainly, above-described embodiment, only for technical conceive of the present invention and feature are described, its object is to people can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalent transformations of doing according to the spirit of main technical schemes of the present invention or modification, all should be encompassed within protection scope of the present invention.
Claims (10)
1. utilize chromatographic technique to extract a method for high-purity daptomycin, it is characterized in that the method comprises the following steps:
1) pre-treatment: filter the fermented liquid containing daptomycin, makes the turbidity of the daptomycin fermented liquid after filtration lower than preset value;
2) extracting: by through step 1) fermented liquid after process injects the chromatography column that the first filler is housed and adsorbs, the anion-exchange gel filler that described first filler is is skeleton with dextran or agarose, adds eluent and is eluted by daptomycin after absorption;
3) refining: by step 2) elutriant that obtains injects the chromatography column that the second filler is housed and adsorbs, the filler that described second filler is is skeleton with polystyrene-divinylbenzene or bonded silica gel, add buffering salt eluting solvent after absorption and carry out wash-out, thus obtain highly purified daptomycin.
2. the method utilizing chromatographic technique to extract high-purity daptomycin according to claim 1, is characterized in that: in described step 1) in, be that employing 0.45 micron membrane filter carries out filtration treatment to the fermented liquid containing daptomycin.
3. the method utilizing chromatographic technique to extract high-purity daptomycin according to claim 1, is characterized in that: the particle diameter of described first filler is 30 microns ~ 500 microns.
4. the method utilizing chromatographic technique to extract high-purity daptomycin according to claim 1, is characterized in that: the particle diameter of described second filler is 30 microns ~ 300 microns.
5. the chromatographic technique that utilizes according to claim 1 extracts the method for high-purity daptomycin, it is characterized in that: described step 2) in eluent be ammonium acetate-sodium chloride buffer.
6. the method utilizing chromatographic technique to extract high-purity daptomycin according to claim 5, is characterized in that: in described step 2) in, first rinse with the Ammonium Acetate aqueous solution, then with described ammonium acetate-sodium chloride buffer, daptomycin is eluted.
7. the chromatographic technique that utilizes according to claim 1 extracts the method for high-purity daptomycin, it is characterized in that: described step 3) in buffering salt eluting solvent be PBS-acetonitrile solution.
8. the method utilizing chromatographic technique to extract high-purity daptomycin according to claim 1, is characterized in that the method is further comprising the steps of:
4) concentrated: to adopt membrane filtration technique, concentrating, by step 3 through elutriant) high-purity daptomycin that obtains concentrates, the part salinity in removing high-purity daptomycin;
5) crystallization: by the solution freeze-drying after concentrated desalination, obtains dry pulverulent solids.
9. the chromatographic technique that utilizes according to claim 8 extracts the method for high-purity daptomycin, it is characterized in that: described step 4) in membrane filtration technique adopt be through the nanofiltration membrane that molecular weight is 150.
10. the method utilizing chromatographic technique to extract high-purity daptomycin according to claim 9, it is characterized in that described step 5) in by the process of the solution freeze-drying after concentrated desalination be: by step 4) to put into 40 degrees below zero freezing for solution after nanofiltration, then puts into Freeze Drying Equipment freeze-drying.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107519665A (en) * | 2017-07-28 | 2017-12-29 | 上海理工大学 | A kind of method of tunning crude separation in situ extracting fermentation |
| CN109666065A (en) * | 2018-11-22 | 2019-04-23 | 北大方正集团有限公司 | A kind of method of quick preparation high-purity daptomycin |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004042350A2 (en) * | 2002-10-30 | 2004-05-21 | Merck & Co., Inc. | Using amines or amino acids as mobile phase modifiers in chromatography |
| CN101240013A (en) * | 2000-01-20 | 2008-08-13 | 卡比斯特制药公司 | High-purity lipopeptide, lipopeptide micelles and preparation method thereof |
| CN101830970A (en) * | 2009-03-12 | 2010-09-15 | 成都雅途生物技术有限公司 | Purification and preparation method of high-purity Daptomycin |
| CN102276696A (en) * | 2010-06-09 | 2011-12-14 | 上海来益生物药物研究开发中心有限责任公司 | Method for purifying daptomuycin |
| CN102325785A (en) * | 2009-02-19 | 2012-01-18 | 埃克斯利亚制药有限公司 | Method for purifying lipopeptides |
| CN102492024A (en) * | 2011-12-09 | 2012-06-13 | 厦门大学 | Method for extracting daptomycin from fermentation broth |
| 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 |
| CN102718839A (en) * | 2012-07-05 | 2012-10-10 | 鲁南新时代生物技术有限公司 | Method for separating and purifying 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 |
| CN102924572A (en) * | 2012-11-12 | 2013-02-13 | 华北制药集团新药研究开发有限责任公司 | Method for preparing high-purity daptomycin |
| CN102965304A (en) * | 2011-10-27 | 2013-03-13 | 四川大学 | Daptomycin high-producing strain and preparation method thereof |
| CN103159829A (en) * | 2011-12-08 | 2013-06-19 | 北大方正集团有限公司 | Extraction method for daptomycin |
| CN103224547A (en) * | 2013-04-28 | 2013-07-31 | 华北制药集团新药研究开发有限责任公司 | Daptomycin separation and purification method |
| CN103724400A (en) * | 2012-10-10 | 2014-04-16 | 北大方正集团有限公司 | Method for separating and purifying dehydrated daptomycin |
-
2015
- 2015-04-29 CN CN201510212580.5A patent/CN105001305A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240013A (en) * | 2000-01-20 | 2008-08-13 | 卡比斯特制药公司 | High-purity lipopeptide, lipopeptide micelles and preparation method thereof |
| WO2004042350A2 (en) * | 2002-10-30 | 2004-05-21 | Merck & Co., Inc. | Using amines or amino acids as mobile phase modifiers in chromatography |
| CN102325785A (en) * | 2009-02-19 | 2012-01-18 | 埃克斯利亚制药有限公司 | Method for purifying lipopeptides |
| CN101830970A (en) * | 2009-03-12 | 2010-09-15 | 成都雅途生物技术有限公司 | Purification and preparation method of high-purity Daptomycin |
| CN102276696A (en) * | 2010-06-09 | 2011-12-14 | 上海来益生物药物研究开发中心有限责任公司 | Method for purifying daptomuycin |
| CN102875652A (en) * | 2011-07-13 | 2013-01-16 | 北大方正集团有限公司 | Method for separating and purifying daptomycin |
| CN102965304A (en) * | 2011-10-27 | 2013-03-13 | 四川大学 | Daptomycin high-producing strain and preparation method thereof |
| CN103159829A (en) * | 2011-12-08 | 2013-06-19 | 北大方正集团有限公司 | Extraction method for daptomycin |
| CN102492024A (en) * | 2011-12-09 | 2012-06-13 | 厦门大学 | Method for extracting daptomycin from fermentation broth |
| 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 |
| CN102718839A (en) * | 2012-07-05 | 2012-10-10 | 鲁南新时代生物技术有限公司 | Method for separating and purifying daptomycin |
| CN103724400A (en) * | 2012-10-10 | 2014-04-16 | 北大方正集团有限公司 | Method for separating and purifying dehydrated daptomycin |
| CN102924572A (en) * | 2012-11-12 | 2013-02-13 | 华北制药集团新药研究开发有限责任公司 | Method for preparing high-purity daptomycin |
| CN103224547A (en) * | 2013-04-28 | 2013-07-31 | 华北制药集团新药研究开发有限责任公司 | Daptomycin separation and purification method |
Non-Patent Citations (5)
| Title |
|---|
| SHARMA 等: "Purification and characterization of a novel lipopeptide from Streptomyces amritsarensis sp. nov. active against methicillin-resistant Staphylococcus aureus", 《AMB EXPRESS》 * |
| 仉文生 等: "《药物化学》", 31 August 2000, 高等教育出版社 * |
| 夏兴 等: "利用高速逆流色谱分离制备达托霉素", 《中国抗生素杂质》 * |
| 熊宗贵: "《生物技术制药》", 31 August 2010, 高等教育出版社 * |
| 王泽根 等: "阴离子交换树脂对Streptomyces roseosporus发酵液中达托霉素的分离纯化研究", 《药学与临床研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107519665A (en) * | 2017-07-28 | 2017-12-29 | 上海理工大学 | A kind of method of tunning crude separation in situ extracting fermentation |
| CN109666065A (en) * | 2018-11-22 | 2019-04-23 | 北大方正集团有限公司 | A kind of method of quick preparation high-purity daptomycin |
| CN109666065B (en) * | 2018-11-22 | 2022-02-25 | 北大方正集团有限公司 | Method for rapidly preparing high-purity daptomycin |
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