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CN102276699A - Application of monodisperse polymethacrylate microballoons in column chromatography purification of vancomycin - Google Patents

Application of monodisperse polymethacrylate microballoons in column chromatography purification of vancomycin Download PDF

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Publication number
CN102276699A
CN102276699A CN2011101643353A CN201110164335A CN102276699A CN 102276699 A CN102276699 A CN 102276699A CN 2011101643353 A CN2011101643353 A CN 2011101643353A CN 201110164335 A CN201110164335 A CN 201110164335A CN 102276699 A CN102276699 A CN 102276699A
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Prior art keywords
vancomycin
column chromatography
column
polymethacrylate
volume
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CN2011101643353A
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Chinese (zh)
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江必旺
陈荣姬
吴俊成
林生跃
裘金明
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Suzhou Nano-Micro Bio-Technology Co Ltd
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Suzhou Nano-Micro Bio-Technology Co Ltd
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Abstract

The invention discloses application of monodisperse polymethacrylate microballoons in column chromatography purification of vancomycin. According to the invention, a column filled with monodisperse polymethacrylate microballoons (produced by Suzhou Nanomicro Technology Co., Ltd. and named Uni-PGM-50) is employed for column chromatography. As a column chromatography filling material, Uni-PGM-50 has good hydrophilicity, high mechanical strength, acid and alkali resistance, a good separation effect and the like; therefore, sample separation with an aqueous solution as a mobile phase is benefited, the volume of a filling material bed does not change with salinity of the mobile phase, and vancomycin adsorbed by the filling material can be eluted merely with a low concentration brine solution in purifying and separating vancomycin, which enables high purity of vancomycin and a high recovery rate of the filling material bed to be achieved.

Description

The application of monodispersity polymethacrylate microballoon in the vancomycin column chromatography purification
Technical field
The present invention relates to a kind of application of monodispersity polymethacrylate microballoon, relate in particular to the extraction Application in Purification of monodispersity polymethacrylate microballoon in antibiotic vancomycin.
Background technology
Vancomycin is a kind of glycopeptide antibiotics that is produced under the fermentation condition in control by microorganism, known that can suppress bacteria cell wall with become and change the cell membrane permeability.Its main application is catching of causing of treatment gram-positive microorganism, for the resistance streptococcus aureus tangible curative effect is arranged, and also is used for the treatment of penicillin anaphylaxis or uses penicillin and cynnematin not to have the patient of effect.
In separation and purification microbiotic, amino acid, polypeptide, protein and natural product program, ion exchange chromatography is one of the most frequently used method.Its chromatography media mainly is made up of three parts, and first part is a precursor structure, i.e. carrier; Second section is the charged functional group that is fixed on the precursor structure; Third part is the movable ion with the functional group oppositely charged.Compare with traditional ion exchange resin, be used for the isolating ion-exchange chromatography media of biomacromolecule, also require precursor structure enough hydrophilic, and will have uniform particle diameter except possessing high ion density, having certain withstand voltage, acid-proof alkaline.Be used for the isolating medium great majority of biomacromolecule at present and do not possess all above characteristics, have defective in actual applications.
(analytical chemistry research report, 1999,27 such as Li Rong, 517-522) use silica gel to be matrix, the silane coupling agent of band weak cation on the bonding, synthetic weak cation chromatography media is used for the separation and purification of N,O-Diacetylmuramidase, and it reaches good separating effect to albumen mass-energy.But silica matrix does not have alkali resistance, and this has limited its range of application.
Chinese patent 101440127 discloses a kind of method of using CM Sephadex C-25 (GE Healthcare) the separation and purification vancomycin of dextran matrix, this method is moving phase with the ammonium bicarbonate aqueous solution, can obtain the purity and the 70% above rate of recovery more than 95%.But the separating medium that uses in this method is dextran matrix, and very obvious variation can take place its bed volume under different salt concn, and dextran matrix is softer, not high pressure resistant simultaneously, and this is having this great limitation in separation fast.
(Journal of Separation Science such as Wei, 2006,29,5 – 13) by copolymerization glytidyl methacrylate and ethylene glycol dimethacrylate in stainless steel chromatogram post, through modifying the synthetic weak cation chromatographic column in back, but this separating medium is block, and its specific surface area is less, and groups density is lower.
(chemistry world such as Lu Miaolong, 1984,10, be matrix 369-373) with the polystyrene/divinylbenzene polymkeric substance, synthetic weak cation chromatography media, this medium hydrophobicity is stronger, and chloromethylation step wherein will use the strong carinogenicity reagent of chloromethyl ether, on producing limitation is arranged.
Summary of the invention
The invention provides a kind of monodispersity polymethacrylate microballoon as the application of chromatography media in the vancomycin column chromatography purification.
For achieving the above object, technical scheme of the present invention is in the vancomycin column chromatography purification, adopts Uni-PGM-50(monodispersity polymethacrylate microballoon, Suzhou Nano-Micro Bio-technology Co., Ltd.) carry out column chromatography as the post of filler.
Monodispersity polymethacrylate microballoon Uni-PGM-50 is become stationary phase as the filler chromatographic column of packing into, and the filler chromatographic column method of packing into is a known technology, does not repeat them here.Flow through this chromatographic column containing to be separated or analyzing the mixture solution that contains vancomycin, be adsorbed on the vancomycin of the to be separated or analysis on the Uni-PGM-50 stationary phase then with the aqueous solution of salt as the moving phase wash-out.
The aqueous solution of described salt is NH 4HCO 3The aqueous solution.
The invention has the beneficial effects as follows: Uni-PGM-50 not only is beneficial to the aqueous solution as the column chromatography bed of packings and reaches high purity and high-recovery as moving phase separation and purification vancomycin, and the volume of column chromatography bed of packings is along with the salt concn of moving phase changes, simultaneously high pressure resistant, anti-moving phase potential of hydrogen significantly changes, and the vancomycin that available low concentration salt solution of while will be adsorbed on the filler elutes.
Description of drawings
Fig. 1 is scanning electron microscope (the Scanning Electron Microscopy) photo of the Uni-PGM-50 of Suzhou Nano-Micro Bio-technology Co., Ltd. monodispersity polymethacrylate microballoon of embodiment one use.
Embodiment
Illustrate in greater detail the present invention below by embodiment.But scope of the present invention is not limited to these embodiment.
Embodiment one
The purifying of vancomycin: adopt the adjustable glass column of 10 * 300 mm, one end, Uni-PGM-50 resin (monodispersity polymethacrylate microballoon, 50 microns of sphere diameters, Suzhou Nano-Micro Bio-technology Co., Ltd.) is chromatographic stuffing, dress post height 20.5cm, dress column volume 16mL with the 3%NaCl activation chromatographic column of 3 column volumes (CV), uses the flow velocity balance chromatographic column of 5CV ultrapure water with 1mL/min earlier then.Vancomycin sample (purity 89%) transfers pH to 5.0 after-filtration with the ultrapure water dissolving with dilute hydrochloric acid, concentration 20mg/mL, and last sample volume 16mL, last sample flow velocity 0.4mL/min is with the ultrapure water of 5CV and the 0.2%NH of 10CV 4HCO 3With the flow velocity prewashing of 1mL/min, use 1% NH then 4HCO 3With the flow velocity wash-out of 0.5mL/min, be in charge of collection, be 20.2cm at elution process center pillar bed height, packing volume is 15.9mL, the bed of packings volume does not have change substantially.Collect liquid after testing, purity is 77.7% greater than 95% vancomycin yield.
Embodiment two
The purifying of vancomycin: adopt the adjustable glass column of 10 * 300 mm, one end, CM Sephadex C-25(GE Healthcare, particle size range 40-125 micron) be chromatographic stuffing, dress post height 20.5cm, dress column volume 16mL, the 5CV ultrapure water is with the flow velocity balance chromatographic column of 1mL/min.Vancomycin sample (purity 89%) spends the ultrapure water dissolving, transfers pH to 5.0 after-filtration with dilute hydrochloric acid, concentration 20mg/mL, and last sample volume 16mL, last sample flow velocity 0.4mL/min is with the ultrapure water of 5CV and 0.4% NH of 8CV 4HCO 3With the flow velocity prewashing of 1mL/min, use 4% NH then 4HCO 3With the flow velocity wash-out of 0.5mL/min, be in charge of collection, the bed of packings volume obviously reduces in elution process.Collect liquid after testing, purity is 68% greater than 95% vancomycin yield.
Embodiment three
The purifying of vancomycin: adopt the adjustable glass column of 10 * 300 mm, one end, CM Sephadex C-25(GE Healthcare, particle size range 40-125 micron) is chromatographic stuffing, dress post height 20.5cm, dress column volume 16mL, with the 3%NaCl activation chromatographic column of 3 column volumes (CV), use the flow velocity balance chromatographic column of 5CV ultrapure water then earlier with 1mL/min.Vancomycin sample (purity 89%) transfers pH to 5.0 after-filtration with the ultrapure water dissolving with dilute hydrochloric acid, concentration 20mg/mL, and last sample volume 16mL, last sample flow velocity 0.4mL/min is with the ultrapure water of 5CV and the 0.2%NH of 10CV 4HCO 3With the flow velocity prewashing of 1mL/min, use 1% NH then 4HCO 3With the flow velocity wash-out of 0.5mL/min, wash-out 5CV, the bed of packings volume obviously reduces in elution process.Collect in the liquid and do not detect vancomycin.
Embodiment four
Salts solution influences packing volume: adopt the adjustable glass column of 10 * 300 mm, one end, Uni-PGM-50 (monodispersity polymethacrylate microballoon, Suzhou Nano-Micro Bio-technology Co., Ltd.) is chromatographic stuffing, after finishing, the dress post uses the flow velocity balance chromatographic column of 3CV ultrapure water with 1.5mL/min, the measurement column bed height is 20.5cm, volume 16mL, with the flow velocity flushing chromatographic column of 3CV 3%NaCl solution with 1.5mL/min, it is 20.5cm that flushing finishes back measurement column bed height, volume is 16mL, and volume is compared no change with original volume.
Embodiment five
Salts solution influences packing volume: adopt the adjustable glass column of 10 * 300 mm, one end, CM Sephadex C-25(GE Healthcare, particle size range 40-125 micron) is chromatographic stuffing, with the flow velocity balance chromatographic column of 3CV ultrapure water with 1.5mL/min, the measurement column bed height was 21.5cm, volume 16.9mL after the dress post finished, with the flow velocity flushing chromatographic column of 3CV 1%NaCl solution with 1.5mL/min, the measurement column bed height is 18.5cm, and volume is 14.5mL, and the volume ratio original volume reduces 14%.Continue with the flow velocity flushing chromatographic column of 3CV 2%NaCl solution with 1.5mL/min, it is 17.1cm that flushing finishes back measurement column bed height, and volume is 13.4mL, and the volume ratio original volume reduces 20.5%.With the flow velocity flushing chromatographic column of 3CV 3%NaCl solution with 1.5mL/min, it is 16.0cm that flushing finishes back measurement column bed height at last, and volume is 12.6mL, and the volume ratio original volume reduces 25.6%.
Embodiment six
Pressure is to fillers affect: adopt the adjustable glass column of 10 * 300 mm, one end, Uni-PGM-50 (monodispersity polymethacrylate microballoon, Suzhou Nano-Micro Bio-technology Co., Ltd.) is chromatographic stuffing, after finishing, the dress post uses the flow velocity balance chromatographic column of 3CV ultrapure water with 1.5mL/min, the measurement column bed height is 21.5cm, volume 16.9mL, regulating flow velocity makes increased pressure arrive 43.5psi (pound-force per square inch), continue flushing 3CV ultrapure water, Deng the stable back of post bed measurement column bed height is 21.5cm, volume 16.9mL, volume does not change.
Embodiment seven
Pressure is to fillers affect: adopt the adjustable glass column of 10 * 300 mm, one end, CM Sephadex C-25(GE Healthcare, particle size range 40-125 micron) be chromatographic stuffing, with the flow velocity balance chromatographic column of 3CV ultrapure water with 1.5mL/min, the measurement column bed height was 22.5cm after the dress post finished, volume 17.7mL, regulate flow velocity and make increased pressure arrive 43.5psi, continue flushing 3CV ultrapure water, waiting the stable back of post bed measurement column bed height is 20.1cm, volume 15.8mL, volume reduces 10.7%.
With embodiment one, five, six and result and the performance of embodiment two, three, four, seven compare (wherein " zero " expression "Yes", " * " expression " not being ")
Figure 641110DEST_PATH_IMAGE002

Claims (5)

1. the application of monodispersity polymethacrylate microballoon in the vancomycin column chromatography purification.
2. the application of monodispersity polymethacrylate microballoon in the vancomycin column chromatography purification is characterized in that: employing monodispersity polymethacrylate microballoon is that the post of filler carries out column chromatography.
3. application according to claim 1 and 2 is characterized in that: described monodispersity polymethacrylate microballoon is that Suzhou Nano-Micro Bio-technology Co., Ltd. produces, product type is the monodispersity polymethacrylate microballoon of Uni-PGM-50.
4. application according to claim 3, it is characterized in that: with the stationary phase of monodispersity polymethacrylate microballoon as chromatographic column, to contain on the sample of vancomycin sample on this chromatographic column, be adsorbed on vancomycin on the filler as the moving phase wash-out with the aqueous solution of salt.
5. application according to claim 4 is characterized in that: the aqueous solution of described salt is NH 4HCO 3The aqueous solution.
CN2011101643353A 2011-06-19 2011-06-19 Application of monodisperse polymethacrylate microballoons in column chromatography purification of vancomycin Pending CN102276699A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603872A (en) * 2012-04-11 2012-07-25 苏州纳微生物科技有限公司 Application of monodisperse polymethacrylate mixed type cation exchange chromatography media to vancomycin purification by column chromatography
CN102659872A (en) * 2012-04-11 2012-09-12 苏州纳微生物科技有限公司 Preparation method of high purity scutellarin
CN103408639A (en) * 2013-07-23 2013-11-27 丽珠集团福州福兴医药有限公司 Preparation method of vancomycin with high purity

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1242055C (en) * 2004-10-15 2006-02-15 上海医药工业研究院 Vancomycine producing fungus and its use
CN101031582A (en) * 2004-10-27 2007-09-05 阿尔法马股份公司 Purification of glycopeptides
CN101146609A (en) * 2005-01-25 2008-03-19 马林克罗特贝克公司 Chromatographic media
CN102079696A (en) * 2011-01-15 2011-06-01 深圳职业技术学院 Hypericin purifying method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1242055C (en) * 2004-10-15 2006-02-15 上海医药工业研究院 Vancomycine producing fungus and its use
CN101031582A (en) * 2004-10-27 2007-09-05 阿尔法马股份公司 Purification of glycopeptides
CN101146609A (en) * 2005-01-25 2008-03-19 马林克罗特贝克公司 Chromatographic media
CN102079696A (en) * 2011-01-15 2011-06-01 深圳职业技术学院 Hypericin purifying method

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603872A (en) * 2012-04-11 2012-07-25 苏州纳微生物科技有限公司 Application of monodisperse polymethacrylate mixed type cation exchange chromatography media to vancomycin purification by column chromatography
CN102659872A (en) * 2012-04-11 2012-09-12 苏州纳微生物科技有限公司 Preparation method of high purity scutellarin
CN102659872B (en) * 2012-04-11 2014-11-19 苏州纳微科技有限公司 Preparation method of high purity scutellarin
CN103408639A (en) * 2013-07-23 2013-11-27 丽珠集团福州福兴医药有限公司 Preparation method of vancomycin with high purity
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

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Application publication date: 20111214