CN102603872A - Application of monodisperse polymethacrylate mixed type cation exchange chromatography media to vancomycin purification by column chromatography - Google Patents
Application of monodisperse polymethacrylate mixed type cation exchange chromatography media to vancomycin purification by column chromatography Download PDFInfo
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- CN102603872A CN102603872A CN2012101035973A CN201210103597A CN102603872A CN 102603872 A CN102603872 A CN 102603872A CN 2012101035973 A CN2012101035973 A CN 2012101035973A CN 201210103597 A CN201210103597 A CN 201210103597A CN 102603872 A CN102603872 A CN 102603872A
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Abstract
The invention discloses an application of monodisperse polymethacrylate mixed type cation exchange chromatography media to vancomycin purification by column chromatography. A chromatographic column is used for column chromatography by using the monodisperse polymethacrylate mixed type cation exchange chromatography media as the packing. The crude vancomycin can be separated and purified by using a water solution as a mobile phase to achieve high purity and recycling rate; in addition, column chromatography cannot change along with the changes of salinity and flow velocity, the chromatographic packing can resist high pressure and high flow velocity and has high carrying capacity; and meanwhile, the vancomycin adsorbed on the chromatographic column can be eluted by using low salinity.
Description
Technical field
The present invention relates to the antibiotic purification field, be specifically related to the column chromatography purification field of vancomyein.
Background technology
Vancomyein is a kind of glycopeptide antibiotics that is produced by the actinomyces microorganism strains; It can suppress the synthetic and change cell membrane permeability of bacteria cell wall; To suppressing the disease that gram-positive microorganism such as staphylococcus, suis, clostridial infection cause obvious curative effects is arranged, can be used for treating to penicillin anaphylaxis simultaneously or use penicillium mould or cynnematin not to have the patient of effect.
Ion-exchange chromatography media is one of method the most frequently used in the separation and purification of biological macromolecule, is widely used in the separation and purification of amino acid, polypeptide, protein, microbiotic and natural product.It 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 pair ion with the functional group oppositely charged.Compare with traditional ion exchange resin; Be used for the isolating ion-exchange chromatography media of biomacromolecule except possessing high ion density, having certain withstand voltage, acid-proof alkaline; Also require precursor structure enough hydrophilic; Make it have excellent biological compatibility, and will have uniform particle diameter.Be used for the isolating medium great majority of biomacromolecule at present and do not possess all above characteristics, in practical application, have defective.
(Journal of Chromatography A, 1997,779 113-122) such as Aya Ohkubo use silica gel to be matrix, bonding strong cation group, and synthetic strong cation chromatography media is used for micromolecular separation and purification and can reaches good separating effect.But silica matrix can not alkali resistance, and this has limited its range of application.
USP 7018814 discloses a kind of purification process of vancomycin hydrochloride, and this method is a separating medium with the strong cation-exchanging resin, is that moving phase is slightly carried vancomyein with the ammonium hydroxide aqueous solution, and the recovery is 88%.The ion exchange resin matrix particle diameter that this method is used can not satisfy the polishing purification requirement as the inhomogeneous microballoon of 50-200 μ M.
Chinese patent 200710187300.5 discloses the method for a kind of Sephadex CM-25 with VISOSE matrix (GE Healthcare) separation and purification vancomyein; This method is moving phase with the ammonium bicarbonate aqueous solution; Can obtain the purity more than 95%, the recovery more than 70%.But the separating medium that uses in this method is VISOSE matrix, and very obvious variation can take place its bed volume under different salt concentration, and VISOSE matrix is softer, not high pressure resistant simultaneously, and this is having great limitation in separation fast.
Capto MMC (GE Healthcare) is a kind of compound cation-exchange chromatography medium; Since its surface bond comprise the compound aglucon of hydrophobic grouping, ion-exchange group and hydrogen bond; Therefore it has the selectivity that is different from traditional biological isolating ions displacement chromatography medium, and higher dynamic carrying capacity.But the matrix that this medium uses is agarose, and its high voltage performance is lower, and certain defective is arranged under the operation of high flow rate.
Summary of the invention
The objective of the invention is provides a kind of and can reach high purity and high-recovery to the problems referred to above, and column chromatography can not change along with the variation of salt concn and flow velocity, and chromatographic stuffing can be high pressure resistant and high flow rate and possess the chromatography media of high carrying capacity.
For realizing above-mentioned purpose; Technical scheme provided by the invention is; The application of single dispersion Rohm tech inc mixed type cation-exchange chromatography medium in the vancomyein column chromatography purification, the single dispersion of employing Rohm tech inc mixed type cation-exchange chromatography medium are that the chromatographic column of filler is carried out column chromatography.
Preferably; With said single stationary phase that disperses Rohm tech inc mixed type cation-exchange chromatography medium as chromatographic column; To contain on the sample of vancomyein appearance on this chromatographic column, be adsorbed on the vancomyein on the filler as the moving phase wash-out with the aqueous solution of salt.
Preferably, the aqueous solution of said salt is NH
4HCO
3The aqueous solution.
Preferably, said single dispersion Rohm tech inc matrix mixed type cation-exchange chromatography medium is to be produced by Suzhou Nano-Micro Bio-technology Co., Ltd., and product type is Uni MSP-50S.
Uni MSP-50S is hydrophobic grouping and an ion-exchange group on hydrophilic polymethyl acrylic acid stromal surface bonding; It possesses hydrophobic and dual function IX; Therefore compare conventional ion displacement chromatography medium during as the column chromatography filler and have better choice property, and the volume of bed of packings can not change with moving phase concentration, when purification of vancomycin; Only need the low salt concn salt brine solution just can the vancomyein that be adsorbed on the filler be eluted; Reach high purity and high-recovery, simultaneously, strong anti-back-pressure performance can be operated it under high flow rate.
The invention has the beneficial effects as follows: single dispersion Rohm tech inc mixed type cation-exchange chromatography medium Uni MSP-50S not only is beneficial to the aqueous solution and does moving phase separation and purification vancomyein bullion; Reach high purity and high-recovery; And column chromatography can not change along with the variation of salt concn and flow velocity; Chromatographic stuffing can be high pressure resistant with high flow rate and possess high carrying capacity, the vancomyein that can use low salt concn will be adsorbed on the chromatography column simultaneously elutes.
Description of drawings
Fig. 1 is the single Rohm tech inc mixed type cation-exchange chromatography medium Uni MSP-50S stereoscan photograph that disperses of Suzhou Nano-Micro Bio-technology Co., Ltd. that uses among the embodiment 1.
Fig. 2 is that the HPLC of the vancomyein bullion before purifying among the embodiment 1 analyzes collection of illustrative plates.
Fig. 3 is that the HPLC of the vancomyein after purifying among the embodiment 1 analyzes collection of illustrative plates.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition that after having read content of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the attached claims restricted portion of the application equally.
Embodiment 1,
Adopt 10 * 220mm, the adjustable glass column of an end; Single dispersion Rohm tech inc mixed type cation-exchange chromatography medium Uni MSP-50S that Suzhou Nano-Micro Bio-technology Co., Ltd. produces is a chromatographic stuffing filling chromatography column; Dress post height 19cm; Dress column volume 14.9mL is earlier with the flow velocity balance chromatographic column of 3.0CV ultrapure water with 1.5mL/min.Vancomyein bullion (purity 89%) is with the ultrapure water dissolving, and regulating pH is 3.2, bullion concentration 37.5mg/mL, and last appearance volume 15mL, the chromatographic column carrying capacity is 37.8mg/mL.After the last appearance, wash 3.0BV with ultrapure water earlier, use 0.33% NH then
4HCO
3Wash-out 4.0CV uses 0.45% NH at last to remove impurity
4HCO
3Wash-out 7.0CV also is in charge of the collection elutriant.Collect liquid through detecting, it is about 78% in the recovery more than 95% to obtain chromatographic purity, and chromatographic purity is in the recovery more than 97% about 61.5%.
Embodiment 2,
Adopt 10 * 220mm, the adjustable glass column of an end; Single dispersion Rohm tech inc mixed type cation-exchange chromatography medium Uni MSP-50S that Suzhou Nano-Micro Bio-technology Co., Ltd. produces is a chromatographic stuffing filling chromatography column; Dress post height 19cm; Dress column volume 14.9mL is earlier with the flow velocity balance chromatographic column of 3.0CV ultrapure water with 1.5mL/min.Vancomyein bullion (purity 89%) is with the ultrapure water dissolving, and regulating pH is 3.2, bullion concentration 37.5mg/mL, and last appearance volume 22.0mL, the chromatographic column carrying capacity is 55.4mg/mL.After the last appearance, wash 3.0BV with ultrapure water earlier, use 0.33% NH then
4HCO
3Wash-out 4.0CV uses 0.45% NH at last to remove impurity
4HCO
3Wash-out 7.0CV also is in charge of the collection elutriant.Collect liquid through detecting, it is about 76% to obtain the recovery of chromatographic purity more than 95%, and chromatographic purity is in the recovery more than 97% about 60.1%.
Embodiment 3,
Adopt 10 * 220mm, the adjustable glass column of an end; GE CM-Sephadex C-25 (GE Healthcare; Particle size range 40-120 micron) is chromatographic stuffing filling chromatography column; Dress post height 20.0cm, dress column volume 15.7mL is earlier with the flow velocity balance chromatographic column of 5.0CV ultrapure water with 1.0mL/min.Vancomyein bullion (purity 89%) is with the ultrapure water dissolving, and regulating pH is 3.2, bullion concentration 37.5mg/mL, and last appearance volume 23.2mL, the chromatographic column carrying capacity is 55.4mg/mL.After the last appearance, wash 5.0BV with ultrapure water earlier, use 0.4% NH then
4HCO
3Wash-out 4.0CV uses 4.0% NH at last to remove impurity
4HCO
3Wash-out 6.0CV also is in charge of the collection elutriant.Collect liquid through detecting, it is about 75% to obtain the recovery of chromatographic purity more than 95%, and chromatographic purity is in the recovery more than 97% about 62.5%.
Embodiment 4,
Test the influence of salts solution to packing volume:
Adopt 10 * 250mm, the adjustable glass column of an end, Uni MSP-50S (Suzhou Nano-Micro Bio-technology Co., Ltd.) be a chromatographic stuffing, and the dress post finishes afterwards with the 3CV ultrapure water with 2mL/min flow velocity balance chromatographic column; The measurement column bed height is 21.0cm; Volume is 16.5mL, and with 2mL/min flow velocity flushing chromatographic column, it is 21.0cm that flushing finishes back measurement column bed height with 3CV 3% NaCl solution; Volume is 16.5mL, and volume is compared no change with original volume.
Embodiment 5,
Test the influence of salts solution to packing volume:
Adopt the adjustable glass column of 10 * 250mm, one end, CM Sephadex C-25 (GE Healthcare, particle size range 40-120 micron) is a chromatographic stuffing; The dress post finish the back with the 3CV ultrapure water with 2mL/min flow velocity balance chromatographic column, the measurement column bed height is 22.0cm, volume is 17.3mL; Wash chromatographic column with 3CV 1% NaCl solution with the 2mL/min flow velocity; It is 18.8cm that flushing finishes back measurement column bed height, and volume is 14.8mL, and the volume ratio original volume dwindles 14.5%; Continue to wash chromatographic column with 3CV 2% NaCl solution with the 2mL/min flow velocity, it is 17.3cm that flushing finishes back measurement column bed height, and volume is 13.6mL, and the volume ratio original volume dwindles 21.4%; Continue to wash chromatographic column with 3CV 3% NaCl solution with the 2mL/min flow velocity, it is 16.2cm that flushing finishes back measurement column bed height, and volume is 12.7mL, and the volume ratio original volume dwindles 26.4%.
Embodiment 6,
Test pressure is to the influence of filler:
Adopt the adjustable glass column of 10 * 250mm, one end, Uni MSP-50S (Suzhou Nano-Micro Bio-technology Co., Ltd.) be a chromatographic stuffing, and the dress post finishes afterwards with the 3CV ultrapure water with 2mL/min flow velocity balance chromatographic column; The measurement column bed height is 21.0cm, and volume is 16.5mL, regulates flow velocity and makes increased pressure to 0.3MPa; Continue flushing 3CV ultrapure water; The measurement column bed height is 21.0cm, and volume is 16.5mL, and volume is compared no change with original volume.
Embodiment 7,
Test pressure is to fillers affect:
Adopt the adjustable glass column of 10 * 250mm, one end, CM Sephadex C-25 (GE Healthcare, particle size range 40-120 micron) is a chromatographic stuffing; The dress post finish the back with the 3CV ultrapure water with 2mL/min flow velocity balance chromatographic column, the measurement column bed height is 21.0cm, volume is 16.5mL; Regulate flow velocity and make increased pressure to 0.3MPa, continue flushing 3CV ultrapure water, the measurement column bed height is 18.6cm; Volume is 16.5mL, and volume reduces 11.4%.
Embodiment one, two, four, six and embodiment three, five, seven performances are compared, and comparative result is seen table 1.
Table 1 medium Uni MPS-50S and medium CM Sephadex C-25 comparative result table
| Result and performance | Embodiment one, two, four, six chromatography media Uni MPS-50S | Embodiment three, five, seven chromatography media CM Sephadex C-25 |
| Uniform particle diameter | ○ | × |
| High pressure resistant | ○ | × |
| Volume no change in the salts solution | ○ | × |
| The low salt concn elution samples | ○ | × |
Annotate: wherein " zero " expression is, " * " expression is not.
Visible from Fig. 1, single very homogeneous of Rohm tech inc mixed type cation-exchange chromatography medium Uni MSP-50S particle diameter that disperses.
Fig. 2 is the vancomyein before purifying, and visible assorted peak is many and high, explains that impurity is many and concentration is high, and Fig. 3 is the vancomyein after purifying, and assorted peak is considerably less and low, explains that impurity is considerably less and concentration is low, and the vancomyein purification effect is fine.
The present invention adopts single dispersion Rohm tech inc mixed type cation-exchange chromatography medium Uni MSP-50S not only to be beneficial to the aqueous solution and does moving phase separation and purification vancomyein bullion; Reach high purity and high-recovery; And column chromatography can not change along with the variation of salt concn and flow velocity; Chromatographic stuffing can be high pressure resistant with high flow rate and possess high carrying capacity, the vancomyein that can use low salt concn will be adsorbed on the chromatography column simultaneously elutes.
More than be description,, make this area professional and technical personnel can realize or use the present invention through above-mentioned explanation to the disclosed embodiments to the embodiment of the invention.Multiple modification to these embodiment will be conspicuous concerning those skilled in the art, and defined General Principle can realize under the situation that does not break away from the spirit or scope of the present invention in other embodiments among this paper.Therefore, the present invention will can not be restricted to these embodiment shown in this paper, but will meet and principle disclosed herein and features of novelty the wideest corresponding to scope.
Claims (3)
1. the application of single dispersion Rohm tech inc mixed type cation-exchange chromatography medium in the vancomyein column chromatography purification; It is characterized in that the single dispersion of employing Rohm tech inc mixed type cation-exchange chromatography medium is that the chromatographic column of filler is carried out column chromatography.
2. the single application of Rohm tech inc mixed type cation-exchange chromatography medium in the vancomyein column chromatography purification that disperse as claimed in claim 1; It is characterized in that; With said single stationary phase that disperses Rohm tech inc mixed type cation-exchange chromatography medium as chromatographic column; To contain on the sample of vancomyein appearance on this chromatographic column, be adsorbed on the vancomyein on the filler as the moving phase wash-out with the aqueous solution of salt.
3. the single application of Rohm tech inc mixed type cation-exchange chromatography medium in the vancomyein column chromatography purification that disperse as claimed in claim 2 is characterized in that the aqueous solution of said salt is NH
4HCO
3The aqueous solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102977190A (en) * | 2012-12-17 | 2013-03-20 | 苏州纳微生物科技有限公司 | Application of monodisperse polymethyl acrylate strong cation exchange chromatography medium in purification of thymopentin |
| CN104718450A (en) * | 2012-10-18 | 2015-06-17 | 捷恩智株式会社 | Cation exchange chromatography carrier for refining of antibodies, and method for separation of antibody monomers from polymers thereof produced in antibody drug manufacturing process |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616651A (en) * | 2004-10-15 | 2005-05-18 | 上海医药工业研究院 | Vancomycine producing fungus and its use |
| US7018814B2 (en) * | 2004-06-30 | 2006-03-28 | Cj Corporation | Process of purifying vancomycin hydrochloride |
| CN101146609A (en) * | 2005-01-25 | 2008-03-19 | 马林克罗特贝克公司 | Chromatographic media |
| CN102276699A (en) * | 2011-06-19 | 2011-12-14 | 苏州纳微生物科技有限公司 | Application of monodisperse polymethacrylate microballoons in column chromatography purification of vancomycin |
-
2012
- 2012-04-11 CN CN2012101035973A patent/CN102603872A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7018814B2 (en) * | 2004-06-30 | 2006-03-28 | Cj Corporation | Process of purifying vancomycin hydrochloride |
| CN1616651A (en) * | 2004-10-15 | 2005-05-18 | 上海医药工业研究院 | Vancomycine producing fungus and its use |
| CN101146609A (en) * | 2005-01-25 | 2008-03-19 | 马林克罗特贝克公司 | Chromatographic media |
| CN102276699A (en) * | 2011-06-19 | 2011-12-14 | 苏州纳微生物科技有限公司 | Application of monodisperse polymethacrylate microballoons in column chromatography purification of vancomycin |
Non-Patent Citations (1)
| Title |
|---|
| 纳微: "高效液相色谱填料产品选择指南", 《"匹兹堡分析化学和光谱应用会议暨展览会" 宣传册》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104718450A (en) * | 2012-10-18 | 2015-06-17 | 捷恩智株式会社 | Cation exchange chromatography carrier for refining of antibodies, and method for separation of antibody monomers from polymers thereof produced in antibody drug manufacturing process |
| CN102977190A (en) * | 2012-12-17 | 2013-03-20 | 苏州纳微生物科技有限公司 | Application of monodisperse polymethyl acrylate strong cation exchange chromatography medium in purification of thymopentin |
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Address after: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 No. 218 BioBAY building C1 Applicant after: SUZHOU NANOMICRO TECHNOLOGY COMPANY LIMITED Address before: Suzhou City, Jiangsu Province, Suzhou Industrial Park 215000 Xinghu Street No. 218 BioBAY C1 Dong group A Applicant before: Suzhou Nano-Micro Bio-technology Co., Ltd. |
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Application publication date: 20120725 |