CN103130874B - A kind of preparation method of high purity ramoplanin - Google Patents
A kind of preparation method of high purity ramoplanin Download PDFInfo
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- CN103130874B CN103130874B CN201110394932.5A CN201110394932A CN103130874B CN 103130874 B CN103130874 B CN 103130874B CN 201110394932 A CN201110394932 A CN 201110394932A CN 103130874 B CN103130874 B CN 103130874B
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Abstract
The invention discloses a kind of preparation method of high purity ramoplanin, specifically comprise and add flocculating aids by Ramoplanin fermentation liquor, filter after stirring, obtain mycelium, polar solvent is added in mycelium, adjust pH to 3.0-3.5, agitation and filtration obtains ramoplanin polar solvent vat liquor, filter after polar solvent vat liquor is concentrated, concentrated solution dilute with water after filtering, filtrate after dilution is adsorbed by polyamide column, use the mixed solution wash-out separation and purification of polar solvent and water, obtain the ramoplanin fine powder of high-content, purity is greater than 95.0%, total recovery is greater than 85%.Present invention process is easy, is suitable for commercial scale production high purity ramoplanin.
Description
Technical field
The invention belongs to industrial microbial technology field, be specifically related to a kind of method preparing high purity ramoplanin from fermenting culture.
Background technology
Glycopeptide antibiotics is the choice drug for the treatment of methicillin-resistant staphylococcus aureus (staphylococcus aureus, MRSA).From nineteen fifty-nine vancomycin be used for clinical since, it is one for the treatment of gram-positive microorganism the most effective medicine causing severe infections always.The overwhelming majority causes the gram-positive microorganism of clinical infection, all to vancomycin sensitive.But along with vancomycin widely uses, and the un-reasonable phenomenon in clinical application, result in resistance problems and occur gradually and have popular possibility, make exploitation vancomycin substitute extremely urgent.Ramoplanin (Ramoplanin, A-16686, MDL62) is wherein one of kind having Development volue most, 1984, the people such as Italian Bruno actinoplanes (
actinoplanessp.) be separated first in and obtain a kind of new antibiotic---the glycopeptide antibiotics ramoplanin of resisting gram-positive bacteria.Within 1989, complete structural confirmation, after carry out commercial development by companies such as Italian Biosearch, Genome Therapeutics.Mode due to ramoplanin attack bacteria allows bacterium cannot produce resistivity easily, and the possibility therefore producing drug resistance strain is lower, and its prospect is very good by external pharmacy expert.
The antibiotic cocktail that ramoplanin is made up of A1, A2, A3, A1 ＇, A2 ＇, A3 ＇, Ramoplanose 7 components, by cyclic polypeptide, join sugar and fatty acid chain forms, wherein, A2 component is the main component of ramoplanin, accounts for more than 75%.Ramoplanin 7 components all have 1 identical parent nucleus, are the cyclic polypeptide of 17 Amino acid profiles, according to joining the difference that sugar is trisaccharide, disaccharide or monose and fatty acid chain, form ramoplanin 7 components.The space structure of ramoplanin is by 2 antiparallel β
2chain is formed, and its intramolecular 2 ~ 7 and 10 ~ 14 amino acids residue is each formation 1 β respectively
2chain, β 2 corner that 2 antiparallel chain structures are formed by the threonine residues of 8 and the phenylalanine residue of 9 links up.The topological framework of whole molecule is U type, defines 1 Loop ring structure at the opening part of U-shaped molecule by the amino-acid residue of 15 ~ 17.Ramoplanin structure is as figure below:
The biosynthesizing of peptidoglycan on ramoplanin T suppression cell wall, its antimicrobial mechanism is different from the antimicrobial mechanism of vancomycin and teicoplanin.On the extension that vancomycin and teicoplanin mainly occur in peptidoglycan chain to the restraining effect of bacteria cell wall and cross-linking level, namely the peptidoglycan biosynthetic later stage is acted on, competitive binding peptidoglycan precursor or generation specific inhibitory effect, and before the restraining effect of ramoplanin occurs in transpeptidation reaction, be the inhibition that a kind of film reacts, what have suppression UDP-N-acetyl-D-Glucose amine mixes effect.This new mode of action just people, to the interested reason of ramoplanin, because only have the microbiotic of the different step suppressing same reaction, just likely becomes the new antibiotic suppressing pathogenic bacteria resistance problems.
In vivo, in external test, ramoplanin all shows good broad spectrum antibiotic activity, most of pathogenic bacteria (Gram-positive) to ramoplanin susceptibility all higher than vancomycin and teicoplanin.In addition, the cytotoxicity of ramoplanin is less than other glycopeptide antibiotics and without obvious adverse reaction and renal toxicity.At present, hospital's available microbiotic when treating the clinical infection that gram-positive microorganism (particularly staphylococcus and faecalis) causes is very limited, ramoplanin, because of its special antimicrobial mechanism and good fungistatic effect, has good prospect being used for the treatment of in the infectious diseases that gram-positive microorganism causes.
Chinese patent CN 101024662A discloses a kind of purification process of ramoplanin, after ramoplanin crude extract is mixed with silica gel, be placed on silicagel column, with methyl alcohol and sour water as moving phase wash-out, because silica gel is positive phase filling, the long-time aqueous mobile phase that uses can cause silica gel inactivation, causes chromatographic separation ability to decline, often need change silica gel, and the method products obtained therefrom purity lower (about 85%).Chinese patent CN 101838315A discloses a kind of separation method of ramoplanin, after adopting macroporous ion exchange resin absorption, inhales, can reach good separating effect with acidic methanol hydrolysis, but fails to carry out adjusting to each component ratio of ramoplanin and control.All in fermented liquid, regulate pH in above two patents, because ramoplanin solubleness in sour environment is very large, effective constituent can be caused to be lost in filtrate.
Summary of the invention
The object of the invention is to overcome aforesaid method weak point, obtain highly purified ramoplanin preparation technology.The present invention adds flocculating aids before filtering fermentation liquor, effectively purify mycelium, pH is regulated at leaching process, the effective constituent preventing fermented liquid directly to adjust pH to cause is lost, the feature many according to hydroxyl in ramoplanin chemical structure, adopts polyamide separation and purification ramoplanin, achieve the high efficiency separation of each component of ramoplanin and impurity, can obtain the ramoplanin fine powder of high-content, purity is greater than 95.0%, and total recovery is greater than 85%.Present invention process is easy, is suitable for commercial scale production high purity ramoplanin.
In the methods of the invention, flocculating aids is added in Ramoplanin fermentation liquor, filter to obtain mycelium, with polar acidic extraction mycelium, concentrated vat liquor, filter to obtain crude extract, after the mixed solution of crude extract polar solvent and water dilutes, with polyamide column absorption, then use the mixed solution wash-out of polar solvent and water, concentrated, filter, dry, obtain high purity ramoplanin fine powder.
Gained ramoplanin of the present invention can for medicine, and purity can reach more than 95.0%.Particularly, the present invention relates to a kind of preparation method of high purity ramoplanin, comprise the steps:
1) add flocculating aids in Ramoplanin fermentation liquor, filter after stirring, obtain mycelium;
2) add polar solvent in mycelium, adjust pH to 3.0-3.5, stir 0.5-1.0 hour, filter, obtain ramoplanin polar solvent vat liquor;
3) concentrated ramoplanin polar solvent vat liquor, obtains concentrated solution;
4) by concentrated solution filtering insolubles, dilute with water after filtrate is obtained;
5) filtrate after dilution is adsorbed by polyamide column;
6) the mixed solution wash-out of polar solvent and water is used, fraction collection elutriant;
7) concentrating under reduced pressure object elutriant, obtains ramoplanin fine powder.
Flocculating aids wherein described in step 1) is diatomite or perlite, and the volume ratio of flocculating aids add-on and fermented liquid is 10-30g/L.
Wherein step 2) described in polar solvent be methyl alcohol or ethanol, each add-on be the 0.1-0.5 of fermentating liquid volume doubly, extracting times is 2-3 time.
20% of wherein step 3) concentrated solution volume≤polar solvent vat liquor volume.
Wherein step 4) dilute with water filtrate volume to fermentating liquid volume 0.3-0.5 doubly.
Polyamide column dress column volume wherein described in step 5) is 1/10000-1/6000 mL/μ g with the ratio of ramoplanin weight.
Polar solvent wherein described in step 6) is methyl alcohol or ethanol, and the concentration of polar solvent is 75-85%.
The present invention has the following advantages: 1. in Ramoplanin fermentation liquor, add flocculating aids, greatly improves filtration velocity, has purified mycelium.2. regulate pH again during lixiviate, the effective constituent avoided when mycelia is filtered is lost.3. polymeric amide good separating effect, product purity is high.4. simple process, is suitable for commercial scale production high purity ramoplanin, for production pharmaceutical grade raw material provides safer technical guarantee.
Accompanying drawing explanation
Fig. 1: ramoplanin fine powder high-efficient liquid phase chromatogram prepared by embodiment 1.
Specific embodiment
Following embodiment only realizes method of the present invention for setting forth, and should not be construed as limitation of the present invention.
Ramoplanin fermentation liquor used in the present invention is that North China Pharmacuetical Group New Drug Research & Development Co., Ltd's fermentation culture obtains.Polymeric amide is 300-400 order, Cangzhou Bao En sorbing material Science and Technology Ltd..The reagent such as ethanol, methyl alcohol is commercially available.The high performance liquid chromatograph that the present invention uses is 996 type detectors, 515 pumps (Waters company).
Embodiment 1
Get Ramoplanin fermentation liquor 1000mL, include ramoplanin 957 μ g/mL, add diatomite 10g, filter after stirring, obtain mycelium, 300mL ethanol is added in mycelium, pH to 3.5 is adjusted with 2mol/L hydrochloric acid, stir 0.5 hour, lixiviate, filter, obtain first time vat liquor, then in mycelium, ethanol 100 mL is added, stirring and leaching, filter, obtain second time vat liquor, continue in mycelium, add ethanol 100 mL, stirring and leaching, filter, obtain third time vat liquor, merge three vat liquors, 15% of vat liquor volume is evaporated at 40 DEG C, insolubles in filtering concentrated solution, filtrate is diluted with water to 500mL, diluent is adsorbed by 160mL polyamide column, use 75% ethanol elution, fraction collection, high performance liquid chromatography (HPLC) detects elutriant, merge ramoplanin content and be greater than 85% part, be evaporated to dry at 40 DEG C, obtain ramoplanin 0.88g, purity 96.1%, total recovery 88.4%, HPLC area normalization A
1ratio 12.5%, A
2ratio 79.8%, A
3ratio 4.6%, A
otherratio 1.3% (high-efficient liquid phase chromatogram is shown in Fig. 1).
Embodiment 2
Get Ramoplanin fermentation liquor 10L, include ramoplanin 982 μ g/mL, add perlite 300g, filter after stirring, obtain mycelium, 3L methyl alcohol is added in mycelium, pH to 3.3 is adjusted with 2mol/L hydrochloric acid, stir 1 hour, lixiviate, filter, obtain first time vat liquor, 1L methyl alcohol is added again in mycelium, stirring and leaching, filter, obtain secondary vat liquor, merge vat liquor, be evaporated to 20% of vat liquor volume, insolubles in filtering concentrated solution, filtrate is diluted with water to 3L, diluent is adsorbed by 1L polyamide column, use 80% methanol-eluted fractions, fraction collection, HPLC detects elutriant, merge ramoplanin content and be greater than 85% part, be evaporated to dry, obtain ramoplanin 8.86g, purity 95.9%, total recovery 86.5%, HPLC area normalization A
1ratio 10.5%, A
2ratio 82.2%, A
3ratio 4.2%, A
otherratio 1.4%(high-efficient liquid phase chromatogram is similar to Fig. 1).
Embodiment 3
Get Ramoplanin fermentation liquor 8L, include ramoplanin 1003 μ g/mL, add perlite 120g, filter after stirring, obtain mycelium, 4L methyl alcohol is added in mycelium, pH to 3. is adjusted with 2mol/L hydrochloric acid, 0, stir 40 minutes, lixiviate, filter, obtain first time vat liquor, 1L methyl alcohol is added again in mycelium, stirring and leaching, filter, obtain second time vat liquor, merge vat liquor, be evaporated to 18% of vat liquor volume, insolubles in filtering concentrated solution, filtrate is diluted with water to 3.2L, diluent is adsorbed by 1L polyamide column, use 85% methanol-eluted fractions, fraction collection, HPLC detects elutriant, merge ramoplanin content and be greater than 85% part, be evaporated to dry, obtain ramoplanin 7.31g, purity 95.4%, total recovery 86.9%, HPLC area normalization A
1ratio 12.2%, A
2ratio 81.2%, A
3ratio 4.1%, A
otherratio 1.3%(high-efficient liquid phase chromatogram is similar to Fig. 1).
Claims (7)
1. a preparation method for high purity ramoplanin, comprises the steps:
1) add flocculating aids in Ramoplanin fermentation liquor, filter after stirring, obtain mycelium;
2) add polar solvent in mycelium, adjust pH to 3.0-3.5, stir, filter, obtain ramoplanin polar solvent vat liquor;
3) concentrated ramoplanin polar solvent vat liquor, obtains concentrated solution;
4) by concentrated solution filtering insolubles, dilute with water after filtrate is obtained;
5) filtrate after dilution is adsorbed by polyamide column;
6) the mixed solution wash-out of polar solvent and water is used;
7) concentrate eluant, obtains ramoplanin fine powder.
2. method according to claim 1, the flocculating aids wherein described in step 1) is diatomite or perlite, and the volume ratio of flocculating aids add-on and fermented liquid is 10-30g/L.
3. method according to claim 1, wherein step 2) described in polar solvent be methyl alcohol or ethanol.
4. method according to claim 1,20% of wherein step 3) concentrated solution volume≤polar solvent vat liquor volume.
5. method according to claim 1, wherein step 4) dilute with water filtrate volume to fermentating liquid volume 0.3-0.5 doubly.
6. method according to claim 1, the polyamide column dress column volume wherein described in step 5) is 1/10000-1/6000 mL/μ g with the ratio of ramoplanin weight.
7. method according to claim 1, the polar solvent wherein described in step 6) is methyl alcohol or ethanol, and the concentration of polar solvent is 75-85%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838316A (en) * | 2009-03-17 | 2010-09-22 | 上海医药工业研究院 | Method for preprocessing Ramoplanin fermentation liquor |
| CN102040638A (en) * | 2009-10-20 | 2011-05-04 | 华北制药集团新药研究开发有限责任公司 | Method for preparing nonsolvent of high-purity natamycin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838316A (en) * | 2009-03-17 | 2010-09-22 | 上海医药工业研究院 | Method for preprocessing Ramoplanin fermentation liquor |
| CN102040638A (en) * | 2009-10-20 | 2011-05-04 | 华北制药集团新药研究开发有限责任公司 | Method for preparing nonsolvent of high-purity natamycin |
Non-Patent Citations (1)
| Title |
|---|
| 糖肽类抗生素提取分离的研究概况;邓小宽等;《中国医药工业杂志》;20061231;第37卷(第11期);1,2.1,2.2.3 * |
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