CN101456903B - Separation and purification method of vancocin - Google Patents
Separation and purification method of vancocin Download PDFInfo
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- CN101456903B CN101456903B CN 200710198599 CN200710198599A CN101456903B CN 101456903 B CN101456903 B CN 101456903B CN 200710198599 CN200710198599 CN 200710198599 CN 200710198599 A CN200710198599 A CN 200710198599A CN 101456903 B CN101456903 B CN 101456903B
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- vancomycin
- aqueous solution
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Abstract
The invention relates to a method for separating vancomycin. The method comprises the following steps: (1) carrying out the column chromatography elution on the vancomycin fermentation liquor in a hydrochloric acid aqueous solution mobile phase macropore absorptive resin chromatographic column containing alcohol so as to obtain vancomycin aqueous solution; (2) adding NH4HCO3 into the hydrochloricacid vancomycin aqueous solution to form precipitate, adjusting the pH value to between 7.5 and 8.5 through ammonia water, and stirring and standing the solution; and (3) obtaining a vancomycin crudeproduct with the content of vancomycin chromatographic purity not less than 80 percent through filtration separation and alcohol top washing. The method is simple and has high yield. The content of the vancomycin chromatographic purity is not less than 80 percent; and the titer of a vancomycin wet product at least reaches 500 mu g/mg.
Description
Technical field
The present invention relates to a kind of separation method of vancomycin.Specifically, at first the method carries out the column chromatography wash-out with the vancomycin fermented liquid in the macroporous adsorption resin chromatography post of the aqueous hydrochloric acid moving phase that contains ethanol, obtain the vancomycin hydrochloride aqueous solution through decolouring and nanofiltration again, at a certain temperature, add NH to this vancomycin hydrochloride aqueous solution
4HCO
3Form throw out, regulate to obtain the vancomycin chromatographic purity with ammoniacal liquor and be not less than 80% vancomycin crude product.
Background technology
Vancomycin is mainly used in treating the severe infections that the aureus strains by anti-β-Nei lactamase causes.To the susceptible patient of penicillin anaphylaxis, vancomycin is a kind of alternative microbiotic, and vancomycin also can be treated the hand post-operation diarrhea that the intractable intestinal tract disease causes.
The business preparation of vancomycin needs multi-level procedure, and this program is expensive and in each stage, the product loss is arranged.Because antibiotic demand increases day by day, need constantly to seek simpler and effective separation method.
The method that reclaims vancomycin comprises: according to United States Patent (USP) 4,440, then the method product separation shown in 753 is precipitated out it from the purification of fermentation substratum that contains water mixing non-solvent such as Virahol, ethanol or acetone; With isolated in form product such as the United States Patent (USP) 4,868 of mantoquita, 285 is described; The imidazoles that the filtration collection forms/vancomycin mixture is as United States Patent (USP) 4,868, shown in 285.
No matter utilize mantoquita still to utilize imidazoles/vancomycin mixture in sepn process, its subsequently resolvent all can make product contamination.And utilize Virahol, ethanol or acetone separation vancomycin can cause vancomycin to present molten pulpous state and be difficult to very much filter.
Therefore, when reclaiming vancomycin from the fermention medium that produces vancomycin, a target is exactly to utilize minimum step to obtain the vancomycin of maximum.And the invention provides a kind of separation method of vancomycin, can obtain the vancomycin chromatographic purity and be not less than 80% vancomycin.
Summary of the invention
The invention provides a kind of separation method of vancomycin, described separation method comprises the steps:
(1) the vancomycin fermented liquid carries out the column chromatography wash-out in the macroporous adsorption resin chromatography post of the aqueous hydrochloric acid moving phase that contains ethanol, obtains the vancomycin hydrochloride aqueous solution;
(2) at 10～20 ℃ of temperature, add NH to this vancomycin hydrochloride aqueous solution
4HCO
3Form throw out, regulate pH=7.5～8.5 with ammoniacal liquor, and stirring, standing;
(3) by filtering separation, and push up with ethanol and wash, obtain the vancomycin chromatographic purity and be not less than 80% vancomycin crude product.
Wherein, before resin absorption, by ceramic membrane filter, pore size filter is 0.02～0.5 μ m to the vancomycin fermented liquid in alkaline condition pH=8.5～10.5 time.
Wherein, described moving phase comprises the ethanol that accounts for moving phase consumption 30～50% and the 1mol/L aqueous hydrochloric acid that accounts for moving phase consumption 50～70%.
Wherein, described macroporous adsorbent resin comprises XAD-1600.
Wherein, the described vancomycin hydrochloride aqueous solution is through activated carbon decolorizing.
Wherein, the concentration of the described vancomycin hydrochloride aqueous solution is greatly about 100mg/mL～120mg/mL.
Wherein, add NH to the described vancomycin hydrochloride aqueous solution
4HCO
3Weight be 6%～10% (W/V) of described vancomycin hydrochloride aqueous solution volume.
Wherein, ammoniacal liquor used is strong aqua.
Wherein, churning time is 45～60 minutes.
Wherein, time of repose is 14～18 hours.
Technical process provided by the present invention specifically describes as follows.
The vancomycin fermented liquid is transferred pH to 8.5～10.5 with sodium hydroxide solution, then be 0.02～0.5 μ m ceramic membrane filter with aperture, the clear filtrate that obtains, pass through macroporous adsorbent resin, vancomycin is attracted to resin, after the resin washing, with the acidic aqueous solution wash-out that contains solvent, typical elutriant is the aqueous hydrochloric acid that contains ethanol.Vancomycin is eluted, and the elutriant that contains vancomycin adds activated carbon decolorizing, filters filtrate the concentrating that obtains and obtains the vancomycin hydrochloride aqueous solution, and the concentration of the aqueous solution is greatly about 100mg/mL.After concentrated the end, then carry out precipitation operation.
Add NH in containing the aqueous solution of vancomycin hydrochloride
4HCO
3(quantity is 6%～10% (W/V) of concentrated solution volume) then regulates vancomycin concentrated solution pH to 7.5～8.5 with ammoniacal liquor, stirs 45～60 minutes, and it is mixed, and a large amount of vancomycins are precipitated out from solution.The temperature of precipitation process must be controlled at 10～20 ℃, after standing 16 ± 2 hours, the mixed solution solid-liquid separation, the vancomycin precipitation is separated from solution obtained the solid Vancomycin, then push up with ethanol and wash, debris in solid is gone on the top, obtains the vancomycin crude product after separation.In the Vancomycin that obtains, the vancomycin chromatographic purity is not less than 80% (HPLC).
Method of the present invention is simple, productive rate is high, and the vancomycin chromatographic purity is not less than 80%, and tiring of vancomycin wet product reaches 500 μ g/mg at least.
Embodiment
By following instance, the present invention is further described in detail, but is not limited in process parameters range in following examples and embodiment.
Embodiment 1: the preparation of the vancomycin hydrochloride aqueous solution
The vancomycin fermented liquid that 200L contains vancomycin concentration 6.8mg/ml time is used 2m in alkaline condition pH=8.5～10.5
2The ceramic membrane filter of 0.02～0.5 μ m, the clear filtrate 500L that obtains is by 40L macroporous adsorbent resin AMBERLITE XAD-1600, vancomycin is attracted to resin, resin contains 40% ethanol and 60%1mol/L aqueous hydrochloric acid wash-out with the washing of 200L purifying is rear with 10L, vancomycin is eluted, obtain containing the elutriant 8.5L of vancomycin, vancomycin concentration is 18.2mg/ml.Add the 85g activated carbon decolorizing in elutriant, filter filtrate the concentrating that obtains and obtain vancomycin hydrochloride aqueous solution 1.05L, wherein vancomycin concentration is 118mg/ml, and the vancomycin chromatographic purity is 80.4%.
Embodiment 2: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 16.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 8.0, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 50 minutes, and it is mixed, stopped stirring standing 18 hours, process temperature is controlled at 19～20 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 38.2g, the 564ug/mg that tires, yield 91.3%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 88.6%.
Embodiment 3: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 16.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 7.5, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 45 minutes, and it is mixed, stopped stirring standing 18 hours, process temperature is controlled at 14.5～15.5 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 43.3g, the 505ug/mg that tires, yield 92.7%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 86.2%.
Embodiment 4: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 16.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 8.5, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 60 minutes, and it is mixed, stopped stirring standing 18 hours, process temperature is controlled at 19～20 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 35.1g, the 604ug/mg that tires, yield 89.8%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 89.7%.
Embodiment 5: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 12.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 8.5, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 50 minutes, and it is mixed, stopped stirring standing 14 hours, process temperature is controlled at 19～20 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 34.2g, the 593ug/mg that tires, yield 85.9%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 90.3%.
Embodiment 6: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 20.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 7.5, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 50 minutes, and it is mixed, stopped stirring standing 18 hours, process temperature is controlled at 10～11 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 41.5g, the 535ug/mg that tires, yield 94.1%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 85.9%.
Embodiment 7: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 20.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 7.5, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 50 minutes, and it is mixed, stopped stirring standing 16 hours, process temperature is controlled at 10～11 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 41.8g, the 544ug/mg that tires, yield 96.4%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 86.1%.
Embodiment 8: the preparation of vancomycin crude product
Get vancomycin hydrochloride volume of water 200mL in embodiment 1, contain vancomycin chromatographic purity 80.4% (HPLC), pH value of solution is 3.0, adds while stirring NH
4HCO
3Be total to 16.0g, in the salt adding process, solution constantly has Bubble formation, vancomycin is therefrom separated out, and has formed soup compound, then adds strong aqua and regulates pH to 8.0, this moment, vancomycin was separated out more, soup compound is denseer, continues to stir 50 minutes, and it is mixed, stopped stirring standing 18 hours, process temperature is controlled at 10～11 ℃.Mixture pushes up by filtering separation and with 50mL ethanol and washes, and has obtained Vancomycin wet product 39.6g, the 564 μ g/mg that tire, yield 94.6%.Vancomycin HPLC detects and contains vancomycin chromatographic purity 88.6%.
Can see from above invention example, the content of the vancomycin chromatographic purity that present method obtains is minimum is 85.9%, vancomycin to tire minimum be 505 μ g/mg, minimum yield is 85.9%.
The present invention is illustrated by top embodiment, still, should be appreciated that the present invention is not limited to particular example as described herein and embodiment.The purpose that comprises these particular example and embodiment here is to help those of skill in the art to put into practice the present invention.Any those of skill in the art are easy to be further improved without departing from the spirit and scope of the present invention and perfect, therefore the present invention only is subject to the restriction of content and the scope of claim of the present invention, and its intention contains all and is included in alternatives and equivalent in the spirit and scope of the invention that is limited by appendix claim.
Claims (4)
1. method of separating vancomycin, described method comprises the steps:
(1) the vancomycin fermented liquid in alkaline condition pH=8.5～10.5 time by the ceramic membrane filter of 0.02～0.5 μ m, the clear filtrate that obtains is by macroporous adsorbent resin AMBERLITEXAD-1600, vancomycin is attracted to macroporous adsorbent resin, macroporous adsorbent resin is used the ethanol that accounts for moving phase consumption 30～50% and the aqueous hydrochloric acid wash-out that accounts for moving phase consumption 50～70% after washing with purifying again, vancomycin is eluted, obtain containing the elutriant of vancomycin, add activated carbon decolorizing in elutriant, the concentrated vancomycin hydrochloride aqueous solution that obtains of the filtrate that filtration obtains,
(2) at 10～20 ℃ of temperature, add NH to this vancomycin hydrochloride aqueous solution
4HCO
3Form throw out, regulate pH=7.5～8.5 with ammoniacal liquor, and stirring, standing;
(3) by filtering separation, and push up with ethanol and wash, obtain the vancomycin chromatographic purity and be not less than 80% vancomycin crude product;
Wherein, the concentration of the described vancomycin hydrochloride aqueous solution is greatly about 100mg/mL～150mg/mL; Add NH to the described vancomycin hydrochloride aqueous solution
4HCO
3Weight be 6%～10% (W/V) of described vancomycin hydrochloride aqueous solution volume.
2. the method for claim 1, wherein described ammoniacal liquor is strong aqua.
3. the method for claim 1, wherein churning time is 45～60 minutes.
4. the method for claim 1, wherein time of repose is 14～18 hours.
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| CN 200710198599 CN101456903B (en) | 2007-12-14 | 2007-12-14 | Separation and purification method of vancocin |
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| CN 200710198599 CN101456903B (en) | 2007-12-14 | 2007-12-14 | Separation and purification method of vancocin |
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| CN101456903B true CN101456903B (en) | 2013-06-05 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9428291B2 (en) | 2013-03-15 | 2016-08-30 | Choon Teo | Method and system for producing high purity vancomycin hydrochloride |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102050866B (en) * | 2009-11-04 | 2015-02-18 | 上海来益生物药物研究开发中心有限责任公司 | Method for purifying glycopeptide compound |
| CN102351930B (en) * | 2011-11-02 | 2015-04-08 | 福建和泉生物科技有限公司 | Method for separating spiramycin fermentation liquid by ceramic membrane microfiltration separation technique |
| CN103408639B (en) * | 2013-07-23 | 2015-04-22 | 丽珠集团福州福兴医药有限公司 | Preparation method of vancomycin with high purity |
| CN104610434B (en) * | 2013-11-01 | 2019-06-07 | 浙江医药股份有限公司新昌制药厂 | A kind of isolation and purification method of vancomycin hydrochloride |
| CN103641895B (en) * | 2013-11-18 | 2015-06-17 | 宁夏泰瑞制药股份有限公司 | Method for producing vancomycin hydrochloride by utilizing vancomycin fermentation broth |
| CN105272992A (en) * | 2014-07-26 | 2016-01-27 | 海正药业(杭州)有限公司 | Method for extracting nemadectin from fermentation liquor |
| CN106518985A (en) * | 2015-09-15 | 2017-03-22 | 江苏海阔生物医药有限公司 | Vancomycin precipitation method |
| WO2020182203A1 (en) * | 2019-03-14 | 2020-09-17 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method for vancomycin analogue |
| CN115010792A (en) * | 2022-06-23 | 2022-09-06 | 丽珠集团新北江制药股份有限公司 | Purification method of vancomycin hydrochloride |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033997A (en) * | 1987-12-28 | 1989-07-19 | 伊莱利利公司 | Improved vancomycin precipitation process |
| CN1616651A (en) * | 2004-10-15 | 2005-05-18 | 上海医药工业研究院 | Vancomycine producing fungus and its use |
-
2007
- 2007-12-14 CN CN 200710198599 patent/CN101456903B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033997A (en) * | 1987-12-28 | 1989-07-19 | 伊莱利利公司 | Improved vancomycin precipitation process |
| CN1616651A (en) * | 2004-10-15 | 2005-05-18 | 上海医药工业研究院 | Vancomycine producing fungus and its use |
Non-Patent Citations (1)
| Title |
|---|
| Cheng-Kang Lee et al.Vancomycin Partitioning in Aqueous Two-Phase Systems:Effects of pH,Salts,and an Affinity Ligand.《Biotechnology and Bioengineering》.1990,第35卷(第4期),第408-416页. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9428291B2 (en) | 2013-03-15 | 2016-08-30 | Choon Teo | Method and system for producing high purity vancomycin hydrochloride |
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| CN101456903A (en) | 2009-06-17 |
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