CN102492024A - Method for extracting daptomycin from fermentation broth - Google Patents
Method for extracting daptomycin from fermentation broth Download PDFInfo
- Publication number
- CN102492024A CN102492024A CN2011104093393A CN201110409339A CN102492024A CN 102492024 A CN102492024 A CN 102492024A CN 2011104093393 A CN2011104093393 A CN 2011104093393A CN 201110409339 A CN201110409339 A CN 201110409339A CN 102492024 A CN102492024 A CN 102492024A
- Authority
- CN
- China
- Prior art keywords
- daptomycin
- fermented liquid
- extracting
- ion
- hydrophobic interaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
A method for extracting daptomycin from fermentation broth relates to fermentation broth. Supernatant liquor is obtained after the fermentation broth is centrifuged, the daptomycin enters an organic phase after the supernatant liquor is extracted by an organic solvent, sodium acetate-acetic acid buffer solution is used to perform re-extraction to reach a water phase so as to obtain strip-extraction liquid, the strip-extraction liquid is purified through hydrophobic interaction chromatography, isopropanol-sodium acetate-acetic acid buffer solution with constant intensity is used for elution to obtain hydrophobic interaction chromatography eluent, the eluent is separated and purified through ion-exchange column chromatography to obtain ion-exchange eluent, the sodium acetate-acetic acid buffer solution is used as basic liquid, and neutral salt is added for elution so as to complete extraction of the daptomycin from the fermentation broth. Organic solvent extraction and resin chromatography technology is applied comprehensively, and the daptomycin with purity more than 85% can be separated and purified from the fermentation broth by using the method. Compared with other separating and purifying technology, the method is moderate in operating condition, low in cost and capable of effectively reducing acidolysis of the daptomycin. The daptomycin serving as novel lipopeptide antibiotics has good medical market prospect.
Description
Technical field
The present invention relates to a kind of fermented liquid, especially relate to a kind of method of from fermented liquid, extracting daptomycin.
Background technology
Bacterial drug resistance is one of severe challenge of facing of the current whole world.Defeat the approach of bacterial drug resistance, the one, prevent abuse of antibiotics and cause the quick increase of resistant organism and spread unchecked; The 2nd, produce chemical sproof mechanism of action according to bacterium, constantly research and development can suppress the new antimicrobial agent of bacterium; The 3rd, research and develop and can not make bacterium produce chemical sproof newtype drug.Only in this way could control serious day by day drug-fast bacteria infection problem effectively.
Daptomycin (Daptomycin) is a kind of novel lipopeptide microbiotic that is obtained through fermented extracted by Streptomyces roseosporus; Its chemical structure and mechanism of action are different from all existing microbiotic, are acknowledged as the vancomyein new antibiotic of optimal inhibition gram-positive microorganism afterwards.Daptomycin is to be formed by connecting a decane side chain and the terminal tryptophane of ring-type beta amino acids peptide chain N-, and structural formula is following:
Hydrophilic amino acid ring and lipophilic aliphatic chain have determined it to have good antibacterial activity in the daptomycin structure.
Experiment in vitro shows; Daptomycin can kill most of gram-positive microorganism fast; And, all present excellent activity to the faecalis (VRE) of vancomycin resistance, the streptococcus aureus (MRSA) of methicillin-resistance, streptococcus aureus (GISA), coagulase positive streptococcus aureus (CNS) and penicillin-fast streptococcus pneumoniae (PRSP) etc. to the medium sensitivity of glycopeptide antibiotic.The antibacterial mechanisms of daptomycin is different with most of antibacterials, and it passes through to upset cytolemma to amino acid whose transhipment, thereby hinders the biosynthesizing of bacteria cell wall Polysaccharides, peptide complexes, changes the character of cytoplasmic membrane; It can also be through destroying the cytolemma of bacterium, and its Dissolve things inside is leaked and reach germ-resistant purpose, so daptomycin few resistance strain isolated that produces in clinical experiment.
In November, 1997, Lilly drugmaker produces the exclusive exploitation in the daptomycin whole world and sell and accomplished by Cubist drugmaker.As the focus development kind, the said firm also is that the whole world is unique with the business-like drugmaker of daptomycin at present with this medicine in Cubist company.Year in September, 2003, daptomycin became first by the first lipopeptide antibiotic of drugs approved by FDA listing.In January, 2006, daptomycin has obtained the authentication of European medicine evaluation administration (EMEA) in whole Europe once more.At present, daptomycin is mainly used in the skin infections that the treatment gram-positive microorganism causes.
The complex structure of daptomycin, the complete synthesis difficulty of chemistry, it is present unique method that microbial fermentation is produced daptomycin.Its less stable of chemical structure decision that daptomycin is special can be developed into dehydration daptomycin and β-plurality of impurities such as isomery daptomycin in the fermented extracted process, so be difficult for adopting strong acid, strong basic ion exchange resin in the leaching process.The fermentation unit of Streptomyces roseosporus fermentative prodn daptomycin is low, by product is many, extraction step is various, product yield is low, has restricted the suitability for industrialized production of this product and extensively popularization.At present; Report about high purity daptomycin method of purification mainly contains: the USP 6699412 of Cubist company application; The utilization anion-exchange chromatography; Hydrophobic interaction chromatography, the daptomycin in the combination process separation and purification fermented liquid of anion-exchange chromatography, obtaining purity is the daptomycin more than 98%.USP 228006 adopts anion-exchange chromatography, and the two-stage reversed phase column chromatography obtains purity and is higher than 95% daptomycin.Domestic aspect, North China drugmaker adopts macroporous adsorbent resin separation and purification daptomycin, and the content of daptomycin is 83% in the elutriant, and extraction yield is 90%.Chinese patent 101899094 has proposed the two-stage membrane sepn, cation-exchange chromatography, and the combination process of anion-exchange chromatography separates daptomycin.In addition, Chinese patent 101830970 has proposed reversed-phase silica gel column chromatography and has prepared the high purity daptomycin.The purification techniques that these patents are mentioned; Some adopts acetonitrile as eluent, and bigger to the pollution of environment, some adopts Zeo-karb; Wash-out under strong acidic condition; Be prone to cause the daptomycin acidolysis, some adopts comparatively expensive ion-exchange packing and reversed phase column chromatography filler, has increased the cost of separation and purification greatly.
Summary of the invention
The object of the present invention is to provide a kind of technology simply from fermented liquid, to extract the method for daptomycin.
The present invention includes following steps:
1) fermented liquid obtains supernatant after centrifugal, and behind organic solvent extraction, daptomycin gets into organic phase, strips to water with sodium-acetate-acetate buffer solution again, must anti-stripping agent;
2) with anti-stripping agent through the hydrophobic interaction chromatography purifying, with Virahol-sodium-acetate-acetate buffer solution constant density wash-out, the hydrophobic interaction chromatography elutriant;
3) with the hydrophobic interaction chromatography elutriant through the ion exchange chromatography separation and purification, medium is an ion-exchange cellulose or sepharose ion exchange resin plasma exchange filler, ion-exchanging eluent;
4) ion-exchanging eluent is a basal liquid with sodium-acetate-acetate buffer solution, adds the neutral salt wash-out, accomplishes and from fermented liquid, extracts daptomycin.
In step 1), it is 4～5 that said supernatant usable acid transfers to pH; Said organic solvent can be selected from carbonatoms more than or equal to 4 alcohol, or carbonatoms is more than or equal to 4 ester, and said carbonatoms can be selected from propyl carbinol or Pentyl alcohol etc. more than or equal to 4 alcohol, is preferably propyl carbinol; Said carbonatoms can be selected from ETHYLE ACETATE or butylacetate etc. more than or equal to 4 ester.
In step 2) in, the medium of said hydrophobic interaction chromatography can be selected from HP20 or macroporous adsorbent resins such as HZ818 or HZ816 or X-5 or AB-8, is preferably the HZ816 macroporous adsorbent resin; Said hydrophobic interaction chromatography elutriant can be selected from a kind of in acetonitrile, ethanol, the Virahol etc., and the concentration of volume percent of said hydrophobic interaction chromatography elutriant can be 10%～60%.
In step 3), the pH of said hydrophobic interaction chromatography elutriant transfers to 6～8; Said ion-exchange packing can be selected from anionite-exchange resin, and said anionite-exchange resin can be selected from a kind of among DEAE 52 ion-exchange celluloses, DEAE Sepharose FF, the QAE Sepharose FF etc.
In step 4), said ion-exchanging eluent is that sodium-acetate-acetate buffer solution of 7.0 is a basal liquid with pH preferably; Said neutral salt can be selected from monovalent salt or divalent salts, and said monovalent salt can be selected from NaCl or KCl etc., and said divalent salts can be selected from CaCl
2Or MgCl
2Deng; The volumetric molar concentration of said neutral salt can be 0～1.0M; Said wash-out can adopt the linear elution mode.
Daptomycin Determination on content method can adopt HPLC, and its detection method is: Agilent HC C18 post; With 0.1% trifluoroacetic acid aqueous solution/0.1% trifluoroacetic acid acetonitrile solution (55: 45) is moving phase, and flow velocity 1mL/min detects wavelength 218nm.
Integrated use organic solvent extraction of the present invention and resin chromatographic technique provide a kind of method for preparing the high purity daptomycin.Use this method can be from fermented liquid separation of pure dissolve purity and be higher than 85% daptomycin.With respect to other separation purifying technique, operational condition of the present invention is gentle, and cost is low, can effectively reduce the daptomycin acidolysis.Daptomycin has good medical market outlook as a kind of novel lipopeptide antibiotic, and therefore separation purification method of the present invention has remarkable economical and social value.
Description of drawings
Fig. 1 is embodiment of the invention daptomycin fermented liquid HPLC figure (RT 7.137min is a daptomycin).In Fig. 1, X-coordinate is time (min), and ordinate zou is peak intensity (mAU).
Fig. 2 is the ion-exchanging eluent HPLC figure (RT 7.446min is a daptomycin) of the embodiment of the invention.In Fig. 2, X-coordinate is time (min), and ordinate zou is peak intensity (mAU).
Embodiment
Through embodiment the present invention is elaborated below.
Embodiment 1
1) the daptomycin fermented liquid is removed mycelium with the centrifugal 15min of 4800rpm and is obtained fermented supernatant fluid (referring to Fig. 1).PH transfers to 5.0 with supernatant, adds the propyl carbinol of 1/4 times of volume, extracts 3 times, and 95% daptomycin gets into organic phase in the supernatant, and 3 times of volume pH 7.0 sodium-acetates-acetate buffer solutions are stripped into aqueous phase with daptomycin.
2) anti-stripping agent pH transfers to 5.0, is splined on the chromatography column flow velocity 1.0mL/min that is filled with the HP20 macroporous adsorbent resin, behind the end of the sample, with pH 7.0,10%, 30%, 50% Virahol-sodium-acetate-acetate buffer solution gradient elution, collects the macroporous absorption elutriant.
3) the macroporous absorption elutriant is splined on the DEAE Sepharose FF ion exchange column of using sodium-acetate-acetate buffer solution balance good in advance; Flow velocity 2mL/min is behind the end of the sample, earlier with 50mM pH 7.0 sodium-acetates-acetate buffer solution thorough washing; With 50mM pH 7.0 sodium-acetates-acetate buffer solution is basal liquid; Add NaCl and carry out linear elution, NaCl concentration is 0～0.1M, collects elution peak.Detect through HPLC, daptomycin content is more than 75% in the elutriant.
Embodiment 2
1) the daptomycin fermented liquid is removed mycelium with the centrifugal 15min of 4800rpm and is obtained fermented supernatant fluid.PH transfers to 4.5 with supernatant, adds the Pentyl alcohol of 1/4 times of volume, extracts 4 times, and 3 times of volume pH 7.5 sodium-acetates-acetate buffer solutions are stripped into aqueous phase with daptomycin.
2) anti-stripping agent pH transfers to 4.5, is splined on the chromatography column that is filled with the HZ816 macroporous adsorbent resin, and aspect ratio is 20; Flow velocity 1.0mL/min is behind the end of the sample, with pH 7.0; 10%, 20%, 40% Virahol-sodium-acetate-acetate buffer solution gradient elution, collect the macroporous absorption elutriant.
3) the macroporous absorption elutriant is splined on the DEAE52 ion exchange column of using sodium-acetate-acetate buffer solution balance good in advance; Flow velocity 2mL/min is behind the end of the sample, earlier with 50mM pH 7.0 sodium-acetates-acetate buffer solution thorough washing; With 50mM pH7.0 sodium-acetate-acetate buffer solution is basal liquid; Add NaCl and carry out linear elution, NaCl concentration is 0.05～0.15M, collects elution peak.Detect through HPLC, daptomycin content is at (referring to Fig. 2) more than 85% in the elutriant.
Claims (10)
1. from fermented liquid, extract the method for daptomycin, it is characterized in that may further comprise the steps:
1) fermented liquid obtains supernatant after centrifugal, and behind organic solvent extraction, daptomycin gets into organic phase, strips to water with sodium-acetate-acetate buffer solution again, must anti-stripping agent;
2) with anti-stripping agent through the hydrophobic interaction chromatography purifying, with Virahol-sodium-acetate-acetate buffer solution constant density wash-out, the hydrophobic interaction chromatography elutriant;
3) with the hydrophobic interaction chromatography elutriant through the ion exchange chromatography separation and purification, medium is an ion-exchange cellulose or sepharose ion exchange resin plasma exchange filler, ion-exchanging eluent;
4) ion-exchanging eluent is a basal liquid with sodium-acetate-acetate buffer solution, adds the neutral salt wash-out, accomplishes and from fermented liquid, extracts daptomycin.
2. the method for from fermented liquid, extracting daptomycin as claimed in claim 1 is characterized in that in step 1), and it is 4～5 that said supernatant uses acid to transfer to pH.
3. the method for from fermented liquid, extracting daptomycin as claimed in claim 1 is characterized in that in step 1), and said organic solvent is selected from carbonatoms more than or equal to 4 alcohol, or carbonatoms is more than or equal to 4 ester.
4. the method for from fermented liquid, extracting daptomycin as claimed in claim 3 is characterized in that said carbonatoms is selected from propyl carbinol or Pentyl alcohol more than or equal to 4 alcohol, is preferably propyl carbinol; Said carbonatoms is selected from ETHYLE ACETATE or butylacetate more than or equal to 4 ester.
5. the method for from fermented liquid, extracting daptomycin as claimed in claim 1; It is characterized in that in step 2) in; The medium of said hydrophobic interaction chromatography is selected from HP20 or HZ818 or HZ816 or X-5 or AB-8 macroporous adsorbent resin, is preferably the HZ816 macroporous adsorbent resin.
6. the method for from fermented liquid, extracting daptomycin as claimed in claim 1; It is characterized in that in step 2) in; Said hydrophobic interaction chromatography elutriant is selected from a kind of in acetonitrile, ethanol, the Virahol, and the concentration of volume percent of said hydrophobic interaction chromatography elutriant can be 10%～60%.
7. the method for from fermented liquid, extracting daptomycin as claimed in claim 1 is characterized in that in step 3) the pH of said hydrophobic interaction chromatography elutriant transfers to 6～8; Said ion-exchange packing is selected from anionite-exchange resin, and said anionite-exchange resin is selected from a kind of among DEAE 52 ion-exchange celluloses, DEAE Sepharose FF, the QAE Sepharose FF.
8. the method for from fermented liquid, extracting daptomycin as claimed in claim 1 is characterized in that in step 4), and said ion-exchanging eluent is that sodium-acetate-acetate buffer solution of 7.0 is a basal liquid with pH.
9. the method for from fermented liquid, extracting daptomycin as claimed in claim 1 is characterized in that in step 4) said neutral salt is selected from monovalent salt or divalent salts, and said monovalent salt is selected from NaCl or KCl; Said divalent salts is selected from CaCl
2Or MgCl
2
10. the method for from fermented liquid, extracting daptomycin as claimed in claim 1 is characterized in that in step 4) the volumetric molar concentration of said neutral salt is 0～1.0M; Said wash-out can adopt the linear elution mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104093393A CN102492024A (en) | 2011-12-09 | 2011-12-09 | Method for extracting daptomycin from fermentation broth |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104093393A CN102492024A (en) | 2011-12-09 | 2011-12-09 | Method for extracting daptomycin from fermentation broth |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102492024A true CN102492024A (en) | 2012-06-13 |
Family
ID=46183891
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011104093393A Pending CN102492024A (en) | 2011-12-09 | 2011-12-09 | Method for extracting daptomycin from fermentation broth |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102492024A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001305A (en) * | 2015-04-29 | 2015-10-28 | 利穗科技(苏州)有限公司 | Method for extracting high-purity daptomycin by utilizing chromatographic technique |
| CN106866789A (en) * | 2015-12-11 | 2017-06-20 | 北大方正集团有限公司 | A kind of method for isolating and purifying Daptomycin RS-8 impurity |
| 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 |
| CN107868120A (en) * | 2017-12-22 | 2018-04-03 | 苏州纳微科技有限公司 | A kind of purification process of Daptomycin |
| CN111103373A (en) * | 2020-01-03 | 2020-05-05 | 丽珠集团福州福兴医药有限公司 | Daptomycin detection method |
| CN113717253A (en) * | 2021-09-15 | 2021-11-30 | 丽珠集团福州福兴医药有限公司 | Purification method of daptomycin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001053330A2 (en) * | 2000-01-20 | 2001-07-26 | Cubist Pharmaceuticals, Inc. | High purity lipopeptides, lipopeptide micelles, processes for preparing same and pharmaceutical compositions containing them |
| 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 |
-
2011
- 2011-12-09 CN CN2011104093393A patent/CN102492024A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001053330A2 (en) * | 2000-01-20 | 2001-07-26 | Cubist Pharmaceuticals, Inc. | High purity lipopeptides, lipopeptide micelles, processes for preparing same and pharmaceutical compositions containing them |
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| 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》, vol. 19, no. 4, 31 August 2011 (2011-08-31), pages 318 - 321 * |
| 石磊等: "达托霉素发酵液大孔树脂吸附分离的研究", 《中国抗生素杂志》, vol. 33, no. 2, 29 February 2008 (2008-02-29), pages 84 - 87 * |
| 覃尚忠等: "达托霉素研究进展", 《中国药事》, vol. 20, no. 6, 30 June 2006 (2006-06-30), pages 375 - 377 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001305A (en) * | 2015-04-29 | 2015-10-28 | 利穗科技(苏州)有限公司 | Method for extracting high-purity daptomycin by utilizing chromatographic technique |
| CN106866789A (en) * | 2015-12-11 | 2017-06-20 | 北大方正集团有限公司 | A kind of method for isolating and purifying Daptomycin RS-8 impurity |
| 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 |
| CN107868120A (en) * | 2017-12-22 | 2018-04-03 | 苏州纳微科技有限公司 | A kind of purification process of Daptomycin |
| CN107868120B (en) * | 2017-12-22 | 2021-06-01 | 苏州纳微科技股份有限公司 | Daptomycin purification method |
| CN111103373A (en) * | 2020-01-03 | 2020-05-05 | 丽珠集团福州福兴医药有限公司 | Daptomycin detection method |
| CN111103373B (en) * | 2020-01-03 | 2022-04-19 | 丽珠集团福州福兴医药有限公司 | Daptomycin detection method |
| CN113717253A (en) * | 2021-09-15 | 2021-11-30 | 丽珠集团福州福兴医药有限公司 | Purification method of daptomycin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102492024A (en) | Method for extracting daptomycin from fermentation broth | |
| EP3064214B1 (en) | Separation and purification method for vancomycin hydrochloride of high purity | |
| CN101830970B (en) | Purification and preparation method of high-purity Daptomycin | |
| CN102718843B (en) | Preparation method of single teicoplanin components | |
| EP2208732B1 (en) | Deshydroxy vancomycin, the preparation, pharmaceutical composition and the use | |
| CN102827258A (en) | Method for purifying Enfuvirtide | |
| CN103724400A (en) | Method for separating and purifying dehydrated daptomycin | |
| CN104774182A (en) | Extraction and purification method of ergothioneine | |
| CN102952178A (en) | Preparation method for high-purity echinocandin compound | |
| CN102924572B (en) | Method for preparing high-purity daptomycin | |
| US20180179244A1 (en) | Fidaxomicin purification method | |
| CN106866790A (en) | The preparation method of Daptomycin RS-5/6, RS-7 and RS-7a/7b impurity | |
| CN100463912C (en) | Membrane separating and purifying process for aminoglycoside antibiotics | |
| CN102690333B (en) | Preparation method of high-purity teicoplanin | |
| CN103012519B (en) | The method of a kind of veterinary antibiotic avilamycin separation and purification | |
| CN102558302A (en) | Method for separating and purifying pristinamycin | |
| CN101838315B (en) | Method for separating Ramoplanin | |
| CN102477067B (en) | 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine and preparation method and application thereof | |
| CN105648001A (en) | Method for extracting mycosubtilin through separation and purification of myxococcus fulvus JCH-04 secondary metabolite | |
| CN106866789A (en) | A kind of method for isolating and purifying Daptomycin RS-8 impurity | |
| CN102351882B (en) | Flucloxacillin sodium compound and preparation method thereof | |
| CN106866791A (en) | A kind of preparation method of high-purity daptomycin lactone hydrolysate | |
| CN102967683B (en) | High-performance liquid chromatography method for methyl(3R)-3-(tert-butyldimethylsilyloxy)-5-oxo-6-triphenylphos phoranylidenehexanoate | |
| CN106565448A (en) | Method for separating and purifying 7-hydroxyl tropolone from bacteria supernatant | |
| CN105669789A (en) | Preparation method for norvancomycin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120613 |