CN103159829B - Extraction method for daptomycin - Google Patents
Extraction method for daptomycin Download PDFInfo
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- CN103159829B CN103159829B CN201110407488.6A CN201110407488A CN103159829B CN 103159829 B CN103159829 B CN 103159829B CN 201110407488 A CN201110407488 A CN 201110407488A CN 103159829 B CN103159829 B CN 103159829B
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Abstract
The invention discloses an extraction method for daptomycin. The method comprises the following steps of: forming micelles from daptomycin in fermentation solution and filtering the micelles via a ceramic membrane system at first; then performing separation and purification by virtue of a multi-section macroporous adsorption resin separation system and a weak-base anion-exchange resin separation system; and finally concentrating and freeze-drying to obtain a daptomycin solid having a chromatographic purity of greater than 96%. The method disclosed by the invention is simple and practicable, low in cost, and 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 chromatographic technique to prepare high purity daptomycin from broth extraction.
Background technology
Along with antibiotic development and antibiotic abuse, pathogenic bacteria is the severe challenge that society faces to antibiotic resistance.Except controlling abuse of antibiotics, searching is a kind of at present has effectively become to the microbiotic of antimicrobial agent the optimal path addressing this problem, vancomycin was once considered to the last line of defense to resisting gram-positive bacteria, had found increasing resistance to 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 through quick trial program approval injection daptomycin (Daptomycin) (trade(brand)name cubicin) the concurrency skin and the skin texture that are caused by some Gram-positive sensitive strains and infects, as abscess, operative incision infect and skin ulcer.The mechanism of action of daptomycin is different from other microbiotic, and it is by upsetting cytolemma to amino acid whose transhipment, thus the biosynthesizing of obstruction bacteria cell wall peptidoglycan, the character of change cytoplasmic membrane; In addition, it can also be by destroying the cytolemma of bacterium, its content leaked and reach the object of sterilization, therefore bacterium daptomycin is produced to resistance may be more difficult.
Although daptomycin is realized suitability for industrialized production in the U.S., in China unrealized scale production, its major cause is not have to realize industrialized extractive technique.
Existing daptomycin extracting method, the described daptomycin extracting method of for example Chinese patent application 200910058577.7, only adopt macroporous resin separation and purification daptomycin, the daptomycin chromatographic purity of gained is only 80%-90%, not only can not meet pharmacy demand, and cannot realize suitability for industrialized production because it is with high costs.Although Chinese patent application 200910085837.X adopts ion exchange resin method to obtain highly purified daptomycin, because existing daptomycin cannot be realized the object of separation and purification daptomycin by the problem of havoc.
Summary of the invention
The object of this invention is to provide a kind of preparation method of Tobramycin easy and simple to handle, with low cost, make high purity daptomycin from daptomycin broth extraction.Technical scheme of the present invention is as follows:
An extracting method for daptomycin, comprises the steps:
1) daptomycin fermented liquid is regulated after pH2.0-4.5, filter with the ceramic membrane that membrane pore size is 0.01 μ m-0.1 μ m, 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) by step 1) gained daptomycin filtrate adjusting pH4.5-7.0, after multistage absorption with macroporous adsorbent resin, wash-out, obtain daptomycin elutriant;
3) by step 2) gained daptomycin elutriant after weak base anion-exchange resin chromatography daptomycin chromatographic solution;
4) by step 3) gained daptomycin chromatographic solution is 50-500 through molecular weight cut-off nanofiltration membrane system is concentrated, obtains daptomycin concentrated solution;
5) by step 4) obtain solid daptomycin after gained daptomycin concentrated solution vacuum-freeze-dry is dry.
Above-mentioned steps 1) in, first fermented liquid pH is adjusted to 2.0-4.5, because 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 pH2.0-4.0 acidic aqueous solution used 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 is doubly advisable with the 4-5 of fermentating liquid volume.After having washed, the yield of daptomycin is more than 92%.
Above-mentioned steps 2) employing macroporous adsorbent resin separation system separation and purification daptomycin, preferably D101 resin.D101 resin price is cheap, and cost is low, and its particle is thicker, is equivalent to play pre-column effect.The macroporous adsorbent resin separation system that this step is used is made up of many segment posts and a large section post, wherein many macroporous adsorbent resin segment posts are connected successively for absorption, the segment post of many series connection when wash-out connect again large section of post of a macroporous adsorbent resin for separating of, make daptomycin just can reach for the first time very high separation and purification effect when chromatography at this.Filling internal diameter and the aspect ratio of D101 resin segment post be generally 1: 7～and 1: 10, be preferably 1: 10, column length 20cm～35cm, its resin demand is 10%～15% of large section of post of D101 resin.Filling internal diameter and the aspect ratio of large section resin column are 1: 5～1: 7, preferably 1: 6, and column length 35cm～52cm.Elutriant can adopt the solution containing 0.06N sodium-acetate and 30%～40% ethanol of pH6.5.
Above-mentioned steps 3) adopt the further separation and purification daptomycin of weak base anion-exchange resin chromatographic system, preferably D301 resin, 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 4) molecular weight cut-off of nanofiltration membrane is preferably 100-200.
Above steps, general, adopt phosphoric acid, acetic acid and sodium hydroxide to carry out the pH of regulator solution.
In the methods of the invention, daptomycin fermented liquid is after ceramic membrane system, the separation and purification of macroporous resin separation system, weak base anion-exchange resin separation system are processed, and its chromatographic purity of the daptomycin obtaining is more than 98%.The method is simple, with low cost, is applicable to carrying out suitability for industrialized production.
Embodiment
Further describe by the following examples the present invention, but the scope not limiting the present invention in any way.
Following embodiment experiment material used is daptomycin fermented liquid, its be about pH7.0 compared with viscous feed liquid.
Embodiment 1
Daptomycin fermented liquid is adjusted to after pH3.0 with phosphoric acid, remove the macromolecular substance such as water-soluble protein, pigment in daptomycin fermented liquid 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, obtain ceramic membrane filtrate with the sodium hydroxide solution circulation cleaning ceramic membrane of the pH9.0 of stock liquid 4-5 times volume again, gained filtrate clear, and color is reddish yellow.
Filtrate is regulated to pH6.0 with acetic acid,diluted, then cross successively the D101 resin column of the series connection of the long 35cm of 3 segment diameter 3.5cm, disconnect series connection, each segment post washes with water respectively to every section of effluent liquid substantially colourless, then after 3 segment D101 resin columns being connected successively, connect with 1 large section of D101 resin of the long 50cm of diameter 5cm again, and with 0.06N sodium acetate, the 30% ethanolic soln wash-out of pH6.5.
Gained eluate regulates the D301 resin column of the upper long 50cm of diameter 5cm of pH5.5 with acetic acid,diluted, wash D301 resin, then uses the sodium chloride solution wash-out of the 300mM of pH5.5～6.0.
The nanofiltration membrane nanofiltration that D301 resin elution thing is 100-200 through molecular weight cut-off is concentrated, and concentrated rear daptomycin concentration is at 20% (w/w).
Concentrated solution vacuum-freeze-dry, gained solid daptomycin adopts high performance liquid chromatography (testing conditions is identical with European patent " PROCESS FOR THE PURIFICATION OF DAPTOMYCIN " (EP 1 586 580 A2) disclosed method) to detect daptomycin purity, and its purity is more than 98%.
Embodiment 2
Daptomycin fermented liquid is adjusted to after pH3.5 with phosphoric acid, remove the macromolecular substance such as water-soluble protein, pigment in daptomycin fermented liquid 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, obtain ceramic membrane filtrate with the sodium hydroxide solution circulation cleaning ceramic membrane of the pH10 of stock liquid 4-5 times volume again, gained filtrate clear, and color is reddish yellow.
Filtrate is regulated to pH6.0 with acetic acid,diluted, cross successively the D101 resin column being cascaded of the long 35cm of 3 segment diameter 3.5cm, disconnect series connection, each segment post washes with water respectively to every section of effluent liquid substantially colourless, then after 3 segment D101 resin columns being connected again, connect with 1 large section of D101 resin of the long 50cm of diameter 5cm of large root again, and with 0.06N sodium acetate, the 30% ethanolic soln wash-out of pH6.5.
Gained eluate regulates the D301 resin column of the upper long 50cm of diameter 5cm of pH5.5 with acetic acid,diluted, wash D301 resin, then uses the sodium chloride solution wash-out of the 300mM of pH5.5～6.0.
The nanofiltration membrane nanofiltration that D301 resin elution thing is 100-200 through molecular weight cut-off is concentrated, and concentrated rear daptomycin concentration is at 20% (w/w).
Concentrated solution vacuum-freeze-dry, gained solid daptomycin adopts high performance liquid chromatography (testing conditions is identical with European patent " PROCESS FOR THE PURIFICATION OF DAPTOMYCIN " (EP 1 586 580 A2) disclosed method) to detect daptomycin purity, and its purity is 96%.
Embodiment 3
Daptomycin fermented liquid is adjusted to after pH3.0 with phosphoric acid, remove the macromolecular substance such as water-soluble protein, pigment in daptomycin fermented liquid 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, obtain ceramic membrane filtrate with the sodium hydroxide solution circulation cleaning ceramic membrane of the pH10.0 of stock liquid 2-3 times volume again, gained filtrate clear, and color is reddish yellow.
Filtrate is regulated to pH6.0 with acetic acid,diluted, cross successively the D101 resin column of the long 35cm series connection of 3 segment diameter 3.5cm, disconnect series connection, it is substantially colourless that each segment post is washed to respectively every section of effluent liquid, then after 3 segment D101 resin columns being connected again, connect with 1 large section of D101 resin of the long 50cm of diameter 5cm of large root again, and with 0.06N sodium acetate, the 30% ethanolic soln wash-out of pH6.5.
Gained eluate regulates the D301 resin column of the upper long 50cm of diameter 5cm of pH5.5 with acetic acid,diluted, wash D301 resin, then uses the sodium chloride solution wash-out of the 300mM of pH5.5～6.0.
The nanofiltration membrane nanofiltration that D301 resin elution thing is 100-200 through molecular weight cut-off is concentrated, and concentrated rear daptomycin concentration is at 20% (w/w).
Concentrated solution vacuum-freeze-dry, gained solid daptomycin adopts high performance liquid chromatography (testing conditions is identical with European patent " PROCESS FOR THE PURIFICATION OF DAPTOMYCIN " (EP 1 586 580 A2) disclosed method) to detect daptomycin purity, and its purity is more than 97%.
The present invention's film used all cleans with cleaning agents of membrane after use, soaks and preserves if desired, to reach the recycling of film with 0.5% sodium sulfite solution.
The present invention comprehensively adopts film, chromatographic technique that a kind of Technology of producing preparation high purity daptomycin feasible, with low cost is provided.Especially fermented liquid is crossed the desorb of ceramic membrane and D101 segmentation absorption string post and is designed the critical process reference mark that especially prepared by high purity daptomycin in micella situation.Although this invention has been carried out to detailed description herein; but be appreciated that; on the basis without prejudice to the present invention's spirit and essence, those skilled in the art can make some amendments or variation, and these amendments or variation are all within the scope of protection of present invention.
Claims (6)
1. an extracting method for daptomycin, comprises the steps:
1) daptomycin fermented liquid is regulated after pH2.0-4.5, filter with the ceramic membrane that membrane pore size is 0.01 μ m-0.1 μ m, 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) by step 1) gained daptomycin filtrate adjusting pH4.5-7.0, adopt many segment posts and a large section post composition multistage D101 resin isolation system, wherein many segment posts are together in series for absorption, when wash-out, by connecting with large section of post again after many segment post series connection, after multistage D101 resin absorption, wash-out, obtain daptomycin elutriant;
3) by step 2) gained daptomycin elutriant after weak base anion-exchange resin chromatography daptomycin chromatographic solution, described weak base anion-exchange resin is D301 resin, its filling internal diameter and aspect ratio are 1: 5～1: 7;
4) by step 3) gained daptomycin chromatographic solution is 100-200 through molecular weight cut-off nanofiltration membrane system is concentrated, obtains daptomycin concentrated solution;
5) by step 4) obtain solid daptomycin after gained daptomycin concentrated solution vacuum-freeze-dry is dry.
2. the method for claim 1, is characterized in that step 1) in washing acidic aqueous solution used be phosphoric acid solution, its consumption be fermentating liquid volume 1-2 doubly.
3. the method for claim 1, is characterized in that step 1) described in alkaline aqueous solution be sodium hydroxide or potassium hydroxide solution, the 4-5 that its consumption is fermentating liquid volume is doubly.
4. the method for claim 1, is characterized in that step 2) described in large section column length 35cm～52cm, filling internal diameter and aspect ratio are 1: 5～1: 7; Described segment column length 20cm～35cm, filling internal diameter and aspect ratio are 1: 7～1: 10, resin demand is 10%～15% of large section post.
5. the method for claim 1, is characterized in that step 2) adopt the solution containing 0.06N sodium-acetate and 30%～40% ethanol of pH6.5 that daptomycin is eluted from D101 resin.
6. the method for claim 1, is characterized in that step 3) adopt the NaCl solution of pH5.5-6.0 that daptomycin is eluted from D301 resin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| CN104387444B (en) * | 2014-11-13 | 2017-12-08 | 北大医药重庆大新药业股份有限公司 | A kind of preparation method of the high-purity samples of Daptomycin impurity RS 2 |
| 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 |
| CN106589065B (en) * | 2015-10-16 | 2020-10-20 | 江苏恒瑞医药股份有限公司 | Daptomycin purification method |
| CN105481950B (en) * | 2016-01-28 | 2019-01-04 | 丽珠集团福州福兴医药有限公司 | A kind of Daptomycin extracting method |
| 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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| 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 |
Citations (4)
| 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 lipopeptides, lipopeptide micelles, processes for preparing same |
| WO2009144739A1 (en) * | 2008-05-26 | 2009-12-03 | Biocon Limited | Amorphous daptomycin and a method of purification thereof |
| WO2010095953A1 (en) * | 2009-02-19 | 2010-08-26 | Axellia Pharmaceuticals Aps | Process for purifying lipopeptides |
| CN101899094A (en) * | 2009-06-01 | 2010-12-01 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of high-purity Daptomycin |
-
2011
- 2011-12-08 CN CN201110407488.6A patent/CN103159829B/en active Active
Patent Citations (4)
| 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 lipopeptides, lipopeptide micelles, processes for preparing same |
| WO2009144739A1 (en) * | 2008-05-26 | 2009-12-03 | Biocon Limited | Amorphous daptomycin and a method of purification thereof |
| WO2010095953A1 (en) * | 2009-02-19 | 2010-08-26 | Axellia Pharmaceuticals Aps | Process for purifying lipopeptides |
| CN101899094A (en) * | 2009-06-01 | 2010-12-01 | 安徽丰原发酵技术工程研究有限公司 | Preparation method of high-purity Daptomycin |
Non-Patent Citations (2)
| Title |
|---|
| 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》.2008,第33卷(第2期),84-89. * |
| 达托霉素发酵液大孔树脂吸附分离的研究;石磊等;《中国抗生素杂志》;20080229;第33卷(第2期);84-89 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Effective date of registration: 20221020 Address after: 3007, Hengqin international financial center building, No. 58, Huajin street, Hengqin new area, Zhuhai, Guangdong 519031 Patentee after: New founder holdings development Co.,Ltd. Patentee after: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee after: Peking University Medical Management Co.,Ltd. Address before: 100871, Beijing, Haidian District Cheng Fu Road 298, founder building, 9 floor Patentee before: PEKING UNIVERSITY FOUNDER GROUP Co.,Ltd. Patentee before: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee before: PKU HEALTHCARE INDUSTRY Group |