CN102875652A - Method for separating and purifying daptomycin - Google Patents
Method for separating and purifying daptomycin Download PDFInfo
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- CN102875652A CN102875652A CN2011101947433A CN201110194743A CN102875652A CN 102875652 A CN102875652 A CN 102875652A CN 2011101947433 A CN2011101947433 A CN 2011101947433A CN 201110194743 A CN201110194743 A CN 201110194743A CN 102875652 A CN102875652 A CN 102875652A
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Abstract
The invention discloses a method for separating and purifying daptomycin. The method comprises the steps of allowing daptomycin to form micelles, filtering by a ceramic membrane system, performing separation and purification and discoloring treatment by using a weak-base anion exchange resin separating system and a macroporous resin separating system, crystallizing, and thus a daptomycin solid with a chromatographic purity higher than 98% is obtained. The method is simple and practicable, is low in cost, and is suitable for industrialized production.
Description
Technical field
The present invention relates to the preparation method of daptomycin, relate to specifically a kind of method that adopts ceramic membrane filter, resin chromatography and crystallization technique to prepare the high purity daptomycin.
Background technology
Along with antibiotic development and antibiotic abuse, pathogenic bacteria is the severe challenge that society faces to Antibiotic resistance.Except the control abuse of antibiotics, seek at present a kind of effective microbiotic to antimicrobial agent and become the optimal path that addresses this problem, vancomycin once was considered to the last line of defense to resisting gram-positive bacteria, had found increasing anti-this medicine bacterium but now the whole world is clinical.
Daptomycin (Daptomycin) is by Lilly (gift comes) company's original research, a cyclic lipopeptide microbiotic of Cubist drugmaker exploitation.Answer patient to the antibiotic active demand of novel resistance, the end of the year 2003, FDA (FDA) is used for the treatment of the concurrency skin and the skin texture that are caused by some Gram-positive sensitive strains through quick trial program approval injection daptomycin (Daptomycin) (trade(brand)name cubicin) and infects, and infects and skin ulcer such as abscess, operative incision.The mechanism of action of daptomycin is different from other microbiotic, and it passes through to upset cytolemma to amino acid whose transhipment, thereby hinders the biosynthesizing of bacteria cell wall peptidoglycan, changes the character of cytoplasmic membrane; In addition, it can also be by destroying the cytolemma of bacterium, its content is leaked and reach the purpose of sterilization, so bacterium produces relatively difficulty of resistance to daptomycin.
Although daptomycin is realized suitability for industrialized production in the U.S., in China and unrealized scale production, its major cause is not have to realize industrialized extractive technique.
Existing daptomycin extracting method, Chinese patent application 200910058577.7 described daptomycin extracting method for example, only adopt macroporous resin separation and purification daptomycin, the daptomycin chromatographic purity of gained only is 80%-90%, not only the pharmacy demand can not be satisfied, and suitability for industrialized production can't be realized because it is with high costs.Although Chinese patent application 200910085837.X adopts the ion exchange resin method to obtain highly purified daptomycin, can't be realized the purpose of separation and purification daptomycin by the problem of havoc because there being daptomycin.
Summary of the invention
The purpose of this invention is to provide the preparation method that a kind of while easy and simple to handle, with low cost can make again the high purity daptomycin.It comprises the steps:
1) with behind the daptomycin fermented liquid adjusting pH2.0-4.5, be that the ceramic membrane of 0.01 μ m-0.1 μ m filters with membrane pore size, then use the acidic aqueous solution circulation cleaning ceramic membrane of pH2.0-4.0, abandon filtrate, use again the alkaline aqueous solution circulation cleaning ceramic membrane of pH8-10, collect daptomycin filtrate;
2) with step 1) gained daptomycin filtrate adjusting pH4.5-7.0, behind weak base anion-exchange resin absorption, wash-out, obtain the daptomycin stripping liquid;
3) with step 2) gained daptomycin stripping liquid after absorption with macroporous adsorbent resin, desorb decolouring the daptomycin destainer;
4) with step 3) gained daptomycin destainer is that the nanofiltration membrane system of 50-500 is concentrated through molecular weight cut-off, obtains the daptomycin concentrated solution;
5) with step 4) gained daptomycin concentrated solution adjusting pH5.0-7.0, then add inorganic calcium salt and alcoholic solvent and carry out crystallization, obtain the solid daptomycin behind the crystal filtration drying.
Above-mentioned steps 1) in, at first fermented liquid pH is transferred to 2.0-4.5, because the daptomycin iso-electric point is PI3.5, under the condition of pH2.0-4.5, daptomycin forms the very large micella of molecular weight, can permeation ceramic membrane.Wash used pH2.0-4.0 acidic aqueous solution and can adopt phosphoric acid solution, its consumption normally fermentating liquid volume 1-2 doubly; Described alkaline aqueous solution can be sodium hydroxide or the potassium hydroxide solution of pH8-10, and its consumption doubly is advisable with the 4-5 of fermentating liquid volume.After having washed, the yield of daptomycin is more than 92%.
Above-mentioned steps 2) adopt weak base anion-exchange resin separation system separation and purification daptomycin, preferred D301 resin, the D301 resin price is cheap, and cost is low, and it is stronger to the selectivity of daptomycin.Filling internal diameter and the aspect ratio of D301 resin be generally 1: 5～and 1: 7, be preferably 1: 6.Elutriant can adopt the NaCl solution of pH5.5-6.0.
Above-mentioned steps 3) adopt the further separation and purification daptomycin of macroporous resin separation system, be preferably the HZ20 resin, this resin price is cheap, and particle is thicker, is difficult for resulting in blockage.Filling internal diameter and the aspect ratio of HZ20 resin be generally 1: 5～and 1: 7, be preferably 1: 6.Behind the macroporous resin adsorption daptomycin, can clean macroporous resin with the 15%-20% ethanolic soln first, the ethanolic soln with 30%-35% elutes daptomycin again.
Above-mentioned steps 4) molecular weight cut-off of nanofiltration membrane is preferably 100-200.
Above-mentioned steps 5) add-on of inorganic calcium salt is advisable with 30%～50% of contained daptomycin quality in the concentrated solution, and described inorganic calcium salt is calcium chloride, lime acetate for example.Described alcoholic solvent is commonly used ethanol and a Virahol, its add-on normally the concentrated solution volume 3-6 doubly.
Above steps, general, adopt phosphoric acid, acetic acid and sodium hydroxide to come the pH of regulator solution.
In the methods of the invention, after daptomycin fermented liquid process ceramic membrane system, weak base anion-exchange resin separation system and the separation and purification of macroporous resin separation system and decolouring are processed, collect purity 85% above component and be concentrated into concentration about 20% through the nanofiltration membrane system again, add the alcoholic solvent crystallization, its chromatographic purity of daptomycin solid that obtains behind the crystal filtration drying is more than 98%.The method is simple, and is with low cost, is fit to carry out suitability for industrialized production.
Embodiment
Further describe by the following examples the present invention, but the scope that does not limit the present invention in any way.
The used experiment material of following embodiment is the daptomycin fermented liquid, its be about pH7.0 than viscous feed liquid.
Embodiment 1
After the daptomycin fermented liquid is adjusted to pH3.0 with phosphoric acid, remove the macromolecular substance such as water-soluble protein in the daptomycin fermented liquid, pigment through the ceramic membrane filter of aperture 0.05 μ m, then use the phosphate aqueous solution circulation cleaning ceramic membrane (filtrate discards) of the pH3.0 of stock liquid 1-2 times volume, sodium hydroxide solution circulation cleaning ceramic membrane with the pH9.0 of stock liquid 4-5 times volume obtains ceramic membrane filtrate again, gained filtrate clear, and color is reddish yellow.
Filtrate is regulated pH5.5 with acetic acid,diluted cross the D301 resin, washing D301 resin is then at the NaCl eluant solution of pH5.5-6.0 with 300mM.
The gained eluate is crossed HZ20 resin (macroporous adsorbent resin that Hua Zhen company in Shanghai produces) after regulating pH4.0-4.5 with acetic acid,diluted and adding the ethanol of material liquid volume 10%; (annotate: the percentage concentration of ethanolic soln refers to concentration expressed in percentage by volume with the 15%-20% ethanolic soln; lower same) clean the HZ20 resin, use again the ethanolic soln wash-out of 30%-35%.
Eluate process molecular weight cut-off is that the nanofiltration membrane nanofiltration of 100-200 is concentrated, and concentrated rear daptomycin concentration is at 20% (w/w).
The gained concentrated solution is regulated pH6.0 with acetic acid,diluted or sodium hydroxide, add the lime acetate of daptomycin mass content about 40% in this concentrated solution, add 95% ethanol of 4 times of volumes of concentrated solution, refrigerator is placed 15h, filtration drying, the solid daptomycin that obtains.Adopt high performance liquid chromatography (testing conditions is identical with European patent " PROCESS FOR THE PURIFICATION OFDAPTOMYCIN " (EP 1 586 580 A2) disclosed method) to detect daptomycin purity, its purity is more than 99%.
Embodiment 2
After the daptomycin fermented liquid is adjusted to pH3.5 with phosphoric acid, remove the macromolecular substance such as water-soluble protein in the daptomycin fermented liquid, pigment through the ceramic membrane filter of aperture 0.05 μ m, then use the phosphate aqueous solution circulation cleaning ceramic membrane (filtrate discards) of the pH3.0 of stock liquid 1-2 times volume, sodium hydroxide solution circulation cleaning ceramic membrane with the pH10 of stock liquid 4-5 times volume obtains ceramic membrane filtrate again, gained filtrate clear, and color is reddish yellow.
Filtrate is regulated pH5.5 with acetic acid,diluted cross the D301 resin, washing D301 resin is then at the NaCl eluant solution of pH5.5-6.0 with 300mM.
The gained eluate is crossed the HZ20 resin after regulating pH4.0-4.5 with acetic acid,diluted and adding the ethanol of 0.1 times of volume of feed liquid, cleans the HZ20 resin with the 15%-20% ethanolic soln, uses the ethanolic soln wash-out of 30%-35% again.
Eluate process molecular weight cut-off is that the nanofiltration membrane nanofiltration of 100-200 is concentrated, and concentrated rear daptomycin concentration is at 20% (w/w).
The gained concentrated solution is regulated pH6.0 with acetic acid,diluted or sodium hydroxide, the lime acetate that adds daptomycin mass content about 40% in this concentrated solution, 95% ethanol that adds 4 times of volumes of concentrated solution, refrigerator is placed 15h, filtration drying, the solid daptomycin that obtains detects through high performance liquid chromatography, and its purity is more than 99%.
Embodiment 3
After the daptomycin fermented liquid is adjusted to pH3.0 with phosphoric acid, remove the macromolecular substance such as water-soluble protein in the daptomycin fermented liquid, pigment through the ceramic membrane filter of aperture 0.05 μ m, then use the phosphate aqueous solution circulation cleaning ceramic membrane (filtrate discards) of the pH3.0 of stock liquid 0.5-1 times volume, sodium hydroxide solution circulation cleaning ceramic membrane with the pH10.0 of stock liquid 2-3 times volume obtains ceramic membrane filtrate again, gained filtrate clear, and color is reddish yellow.
Filtrate is regulated pH6.0-7.0 with acetic acid,diluted cross the D301 resin, washing D301 resin is then at the NaCl eluant solution of pH6.0-7.0 with 300mM.
The gained eluate is crossed the HZ20 resin after regulating pH4.0-4.5 with acetic acid,diluted and adding the ethanol of 0.1 times of volume of feed liquid, cleans the HZ20 resin with the 15%-20% ethanolic soln, uses the ethanolic soln wash-out of 30%-35% again.
Eluate process molecular weight cut-off is that the nanofiltration membrane nanofiltration of 100-200 is concentrated, and concentrated rear daptomycin concentration is about 20% (w/w).
The gained concentrated solution is regulated pH6.5-7.0 with acetic acid,diluted or sodium hydroxide, the lime acetate that adds daptomycin mass content about 40% in this concentrated solution, 95% ethanol that adds 5 times of volumes of concentrated solution, refrigerator is placed 15h, filtration drying, the solid daptomycin that obtains detects through high performance liquid chromatography, and its purity is more than 98%.
The used film of the present invention all will clean with cleaning agents of membrane after use, soaks with 0.5% sodium sulfite solution in case of necessity and preserves, to reach the recycling of film.
The present invention comprehensively adopts film, chromatographic technique and crystallization means that a kind of Technology of producing preparation high purity daptomycin feasible, with low cost is provided.Although this paper has carried out detailed description to this invention; but be appreciated that; on the basis of the present invention's spirit and essence, those skilled in the art can make some modifications or change, and these modifications or change are all within the scope of protection of present invention.
Claims (10)
1. the method for a separation and purification daptomycin comprises the steps:
1) with behind the daptomycin fermented liquid adjusting pH2.0-4.5, be that the ceramic membrane of 0.01 μ m-0.1 μ m filters with membrane pore size, then use the acidic aqueous solution circulation cleaning ceramic membrane of pH2.0-4.0, abandon filtrate, use again the alkaline aqueous solution circulation cleaning ceramic membrane of pH8-10, collect daptomycin filtrate;
2) with step 1) gained daptomycin filtrate adjusting pH4.5-7.0, behind weak base anion-exchange resin absorption, wash-out, obtain the daptomycin stripping liquid;
3) with step 2) gained daptomycin stripping liquid after macroporous resin adsorption, desorb decolouring the daptomycin destainer;
4) with step 3) gained daptomycin destainer is that the nanofiltration membrane system of 50-500 is concentrated through molecular weight cut-off, obtains the daptomycin concentrated solution;
5) with step 4) gained daptomycin concentrated solution adjusting pH5.0-7.0, then add inorganic calcium salt and alcoholic solvent and carry out crystallization, obtain the solid daptomycin behind the crystal filtration drying.
2. the method for claim 1 is characterized in that step 1) the used pH2.0-4.0 acidic aqueous solution employing phosphoric acid solution of middle washing, its consumption is 1-2 times of fermentating liquid volume.
3. the method for claim 1 is characterized in that step 1) described in alkaline aqueous solution be sodium hydroxide or potassium hydroxide solution, its consumption be fermentating liquid volume 4-5 doubly.
4. the method for claim 1 is characterized in that step 2) described weak base anion-exchange resin is the D301 resin, its filling internal diameter and aspect ratio are 1: 5～1: 7.
5. the method for claim 1 is characterized in that step 2) adopt the NaCl solution of pH5.5-6.0 that daptomycin is eluted from weak base anion-exchange resin.
6. the method for claim 1 is characterized in that step 3) described macroporous resin is the HZ20 resin, its filling internal diameter and aspect ratio are 1: 5～1: 7.
7. the method for claim 1 is characterized in that step 3) with behind the macroporous resin adsorption daptomycin, clean macroporous resin with the 15%-20% ethanolic soln first, the ethanolic soln with 30%-35% elutes daptomycin again.
8. the method for claim 1 is characterized in that step 4) molecular weight cut-off of used nanofiltration membrane is 100-200.
9. the method for claim 1 is characterized in that step 5) in the add-on of inorganic calcium salt be in the concentrated solution contained daptomycin quality 30%～50%; The add-on of alcoholic solvent is 3-6 times of concentrated solution volume.
10. method as claimed in claim 9 is characterized in that step 5) described in inorganic calcium salt be in the following calcium salt one or more: lime acetate and calcium chloride; Described alcoholic solvent is ethanol or Virahol.
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Cited By (9)
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| CN104650189A (en) * | 2013-11-19 | 2015-05-27 | 北大方正集团有限公司 | Preparation method of isomeric daptomycin |
| 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 |
| CN105111285A (en) * | 2015-10-17 | 2015-12-02 | 霍进铭 | Daptomycin extraction method |
| CN106518985A (en) * | 2015-09-15 | 2017-03-22 | 江苏海阔生物医药有限公司 | Vancomycin precipitation method |
| CN106589065A (en) * | 2015-10-16 | 2017-04-26 | 江苏恒瑞医药股份有限公司 | Daptomycin purifying method |
| CN110117310A (en) * | 2019-04-17 | 2019-08-13 | 华北制药华胜有限公司 | A kind of purification process of Daptomycin |
| CN110698472A (en) * | 2019-09-11 | 2020-01-17 | 北大方正集团有限公司 | Purification method of pyrroloquinoline quinone |
| CN114344447A (en) * | 2021-12-16 | 2022-04-15 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN115417822A (en) * | 2022-08-17 | 2022-12-02 | 山东福瑞达生物科技有限公司 | Extraction and purification process of tetrahydropyrimidine |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104650189A (en) * | 2013-11-19 | 2015-05-27 | 北大方正集团有限公司 | Preparation method of isomeric daptomycin |
| 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 |
| CN106518985A (en) * | 2015-09-15 | 2017-03-22 | 江苏海阔生物医药有限公司 | Vancomycin precipitation method |
| CN106589065A (en) * | 2015-10-16 | 2017-04-26 | 江苏恒瑞医药股份有限公司 | Daptomycin purifying method |
| CN106589065B (en) * | 2015-10-16 | 2020-10-20 | 江苏恒瑞医药股份有限公司 | Daptomycin purification method |
| CN105111285A (en) * | 2015-10-17 | 2015-12-02 | 霍进铭 | Daptomycin extraction method |
| CN110117310A (en) * | 2019-04-17 | 2019-08-13 | 华北制药华胜有限公司 | A kind of purification process of Daptomycin |
| CN110698472A (en) * | 2019-09-11 | 2020-01-17 | 北大方正集团有限公司 | Purification method of pyrroloquinoline quinone |
| CN114344447A (en) * | 2021-12-16 | 2022-04-15 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN114344447B (en) * | 2021-12-16 | 2023-08-25 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN115417822A (en) * | 2022-08-17 | 2022-12-02 | 山东福瑞达生物科技有限公司 | Extraction and purification process of tetrahydropyrimidine |
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Application publication date: 20130116 |