CN106582578A - High-pressure-resistant affinity chromatography medium - Google Patents

High-pressure-resistant affinity chromatography medium Download PDF

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Publication number
CN106582578A
CN106582578A CN201611201279.5A CN201611201279A CN106582578A CN 106582578 A CN106582578 A CN 106582578A CN 201611201279 A CN201611201279 A CN 201611201279A CN 106582578 A CN106582578 A CN 106582578A
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China
Prior art keywords
high pressure
pressure resistant
chromatography medium
affinity chromatography
pressure
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CN201611201279.5A
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CN106582578B (en
Inventor
瞿欢欢
朱至放
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Suzhou Bo Bo Biotechnology Co Ltd
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Suzhou Bo Bo Biotechnology Co Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/52—Sorbents specially adapted for preparative chromatography

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

The invention relates to a high-pressure-resistant affinity chromatography medium. Polyacrylate microspheres serve as cores, and agarose molecules are bonded on the surfaces of the polyacrylate microspheres. One part of hydroxyl ends in the agarose molecules are connected with high-pressure-resistant groups, and the other part of the hydroxyl ends are connected with ligands, wherein the high-pressure-resistant groups are CH2Ph-p-CH3 or CH2Ph-p-OCH3. The design of surface modification groups of the affinity chromatography medium is improved, rigid large-steric-hindrance groups are introduced to the surface of the medium, the mechanical strength of the affinity chromatography medium is improved, therefore, the separation efficiency of the chromatography medium at high pressure is improved, and the reuse frequency of the chromatography medium at high pressure is increased. Meanwhile, the improvement method is simple, the production cost is low, and large-scale application and popularization are promoted.

Description

High pressure resistant affinity chromatography medium
Technical field
The present invention relates to a kind of chromatography media, and in particular to a kind of high pressure resistant affinity chromatography medium.
Background technology
At present, in pharmaceutical field, the purity and biological safety of medicine how is improved, is the biologies such as recombiant protein, antibody Medicine is researched and developed and the final steps in production process.Medicine enterprise of China relies primarily on import for the acquisition of separating medium, therefore, state Inside need the dependence broken away to import separating medium badly, reduce the production cost of medicine.
Agar sugar product, but this kind of product mechanical strength are mainly to the affinity chromatography medium of protein drug purifies and separates Relatively low, so as to cause it after filling chromatographic column, proof pressure is relatively low, limits the room for promotion of its separation efficiency, while Inhibit the reuse number of times of agarose.
The content of the invention
For the deficiencies in the prior art part, it is an object of the invention to provide a kind of high pressure resistant affinity chromatography medium.
Technical scheme is summarized as follows：
A kind of high pressure resistant affinity chromatography medium, it is with polyacrylic acid ester microsphere as kernel and micro- in the polyacrylate Ball surface is bonded with agarose molecules；The high pressure resistant group of hydroxyl terminal part connection in the agarose molecules, remainder Connection aglucon；
Wherein, the high pressure resistant group is CH2Ph-p-CH3Or CH2Ph-p-OCH3.(p represents methyl in phenyl ring para-position)
Preferably, described high pressure resistant affinity chromatography medium, wherein, the high pressure resistant group and the aglucon mole Than for 5-15: 100.
Preferably, described high pressure resistant affinity chromatography medium, wherein, the high pressure resistant group and the aglucon mole Than for 8-10: 100.
Preferably, described high pressure resistant affinity chromatography medium, wherein, the high pressure resistant group is CH2Ph-p-CH3。
Preferably, described high pressure resistant affinity chromatography medium, wherein, the high pressure resistant group is CH2Ph-p-OCH3。
Preferably, described high pressure resistant affinity chromatography medium, wherein, the particle diameter of the polyacrylic acid ester microsphere is 30- 100μm。
The invention has the beneficial effects as follows：This case is improved by the design to affinity chromatography medium surface modification group, is being situated between Matter surface introduces the big steric group of rigidity to improve the mechanical strength of affinity chromatography medium, so as to improve chromatography media in height Separation efficiency and recycling number of times under pressure；Meanwhile, this improved method is simple, low production cost, beneficial to pushing away on a large scale Wide application.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to description Word can be implemented according to this.
This case provides the high pressure resistant affinity chromatography medium of an embodiment, and it is gathering with polyacrylic acid ester microsphere as kernel Acrylate microspheres surface bond has agarose molecules；The high pressure resistant group of hydroxyl terminal part connection in agarose molecules, remains Remaining part point connection aglucon；
Wherein, high pressure resistant group is CH2Ph-p-CH3Or CH2Ph-p-OCH3。
Wherein, high pressure resistant group and the mol ratio of aglucon are 5-15: 100；Preferably 8-10: 100.
Wherein, the particle diameter of polyacrylic acid ester microsphere is preferably 30-100 μm.
Polyacrylic acid ester microsphere is a kind of conventional commercial commodity, can be bought.Using agarose gel as medium carrier Chromatographic column pressure voltage is generally 0.3-0.5MPa, and has the polyacrylic acid ester microsphere of agarose molecules as medium using surface bond The chromatographic column pressure voltage of carrier is generally 10-12MPa, and it is 100 times to reuse number of times.
Aglucon (L：Ligand species) is not limited, can be according to the species of material to be adsorbed depending on.For example when need system During standby protein A affinity chromatography medium, aglucon just selects ProA；When needing to prepare protein g affinity chromatography medium, aglucon is just selected ProG.Whether the selection of aglucon is high pressure resistant to the medium without substantial effect.
Embodiment 1
Preparation process is as follows：
1) BrCH is added for 5-15: 100 ratio in the mol ratio of high pressure resistant group and aglucon in 1mol carriers2Ph- p-CH3, 8-10mol toluene and 0.3-0.4mol KOH, stirring reaction 1-1.5h at 70-75 DEG C；Filter, wash three times, do It is dry；Carrier has the polyacrylic acid ester microsphere of agarose molecules for surface bond；
2) 3-4mol acetone, 1.1-1.2mol epoxychloropropane and 1.5-2mol NaOH are added, is stirred at 30-40 DEG C 1-2h, after reaction terminates, filters, and washs drying；
3) NaOH of the aglucon, the ethanol of 5-7mol and 2-3mol of 1.2mol, stirring reaction 12- at 55-60 DEG C are added 15h, reaction is filtered to remove solvent after terminating, wash three times, is drying to obtain chromatography media.
Embodiment 2
Preparation process is as follows：
1) CH is added for 5-15: 100 ratio in the mol ratio of high pressure resistant group and aglucon in 1mol carriers2Ph-p- OCH3, 8-10mol toluene and 0.3-0.4mol KOH, stirring reaction 1-1.5h at 70-75 DEG C；Filter, wash three times, do It is dry；Carrier has the polyacrylic acid ester microsphere of agarose molecules for surface bond；
2) 3-4mol acetone, 1.1-1.2mol epoxychloropropane and 1.5-2mol NaOH are added, is stirred at 30-40 DEG C 1-2h, after reaction terminates, filters, and washs drying；
3) NaOH of the aglucon, the ethanol of 5-7mol and 2-3mol of 1.2mol, stirring reaction 12- at 55-60 DEG C are added 15h, reaction is filtered to remove solvent after terminating, wash three times, is drying to obtain chromatography media.
Following table lists the test result of embodiment 1 and embodiment 2：
Embodiment 1 Embodiment 2
Chromatographic column pressure voltage 18MPa 19MPa
Reuse number of times 200 times 200 times
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in description and embodiment With, it can be applied to completely various suitable the field of the invention, for those skilled in the art, can be easily Other modification is realized, therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited In specific details.

Claims (6)

1. a kind of high pressure resistant affinity chromatography medium, its with polyacrylic acid ester microsphere as kernel, and in the polyacrylic acid ester microsphere Surface bond has agarose molecules；The high pressure resistant group of hydroxyl terminal part connection in the agarose molecules, remainder connects Connect aglucon；
Wherein, the high pressure resistant group is CH2Ph-p-CH3Or CH2Ph-p-OCH3。
2. high pressure resistant affinity chromatography medium according to claim 1, it is characterised in that the high pressure resistant group is matched somebody with somebody with described The mol ratio of base is 5-15: 100.
3. high pressure resistant affinity chromatography medium according to claim 2, it is characterised in that the high pressure resistant group is matched somebody with somebody with described The mol ratio of base is 8-10: 100.
4. high pressure resistant affinity chromatography medium according to claim 1, it is characterised in that the high pressure resistant group is CH2Ph- p-CH3。
5. high pressure resistant affinity chromatography medium according to claim 1, it is characterised in that the high pressure resistant group is CH2Ph- p-OCH3。
6. high pressure resistant affinity chromatography medium according to claim 1, it is characterised in that the grain of the polyacrylic acid ester microsphere Footpath is 30-100 μm.
CN201611201279.5A 2016-12-22 2016-12-22 High pressure resistant affinity chromatography medium Active CN106582578B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107961768A (en) * 2017-12-06 2018-04-27 苏州博进生物技术有限公司 Chromatography media using indoles -3- acethydrazides as functional ligand
CN114054006A (en) * 2021-11-17 2022-02-18 苏州纳微科技股份有限公司 Oligo-dT affinity chromatography filler

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4973683A (en) * 1985-05-23 1990-11-27 Pharmacia Ab Method of cross-linking a porous polysaccharide gel
CN101259406A (en) * 2007-12-17 2008-09-10 南京工业大学 Preparation method of bonding-affinity compound polysaccharide chiral stationary phase
CN102617869A (en) * 2012-03-30 2012-08-01 中国科学院过程工程研究所 Polyacrylic ester and polyacrylic ester copolymer material hydrophilic modification products and hydrophilic modification method of polyacrylic ester
CN103586008A (en) * 2013-10-24 2014-02-19 中国科学院过程工程研究所 Affinity chromatography medium and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4973683A (en) * 1985-05-23 1990-11-27 Pharmacia Ab Method of cross-linking a porous polysaccharide gel
CN101259406A (en) * 2007-12-17 2008-09-10 南京工业大学 Preparation method of bonding-affinity compound polysaccharide chiral stationary phase
CN102617869A (en) * 2012-03-30 2012-08-01 中国科学院过程工程研究所 Polyacrylic ester and polyacrylic ester copolymer material hydrophilic modification products and hydrophilic modification method of polyacrylic ester
CN103586008A (en) * 2013-10-24 2014-02-19 中国科学院过程工程研究所 Affinity chromatography medium and preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107961768A (en) * 2017-12-06 2018-04-27 苏州博进生物技术有限公司 Chromatography media using indoles -3- acethydrazides as functional ligand
CN114054006A (en) * 2021-11-17 2022-02-18 苏州纳微科技股份有限公司 Oligo-dT affinity chromatography filler

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