CN1354365A - High-effective affinity chromatography filler and its preparation method - Google Patents
High-effective affinity chromatography filler and its preparation method Download PDFInfo
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- CN1354365A CN1354365A CN 00132647 CN00132647A CN1354365A CN 1354365 A CN1354365 A CN 1354365A CN 00132647 CN00132647 CN 00132647 CN 00132647 A CN00132647 A CN 00132647A CN 1354365 A CN1354365 A CN 1354365A
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- heparin
- microballoon
- glycidylmethacrylate
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Abstract
The invention discloses color spectrum filler with high affinity whose matrix material is micro balls made of polymethyl acrylic acid epoxy propyl ester. The filler contains the polymeric unit whose formula is showed on above right, here Hep is heparin, and the structure of P is showed on right below. The invented filler is prepared by linking heparin to micro balls made of polymethyl acrylic acid epoxy propyl ester. The invented color spectrum filler possesses properties of high affinity capacity to atnithrombase III and lower non-specificity adsorption.
Description
The present invention relates to a kind of high-effective affinity chromatography filler and preparation method thereof, being specifically related to the heparin is high-effective affinity chromatography filler of aglucon and preparation method thereof.
Can be divided into two types of flexible glue and ebonites again by the matrix of affine filler.Flexible glue comprises natural polysaccharide and polyacrylamide etc., they have good hydrophilicity, non-specific adsorption to protein is low, under low voltage operated, have good chromatographic separation performance, but their bad mechanical strength, gel particle very easily is out of shape and destroys, on-stream pressure and flow velocity all are very limited, and serviceable life is also short.Macro porous silica gel is the ebonite that is widely used, and they have good mechanical property, can use under high pressure, conditions of high flow rate, but their poor chemical stability is only used under pH2～8 conditions.In addition, the electronegative silicon hydroxyl on silica gel surface has stronger non-specific adsorption to protein, makes the recovery reduce greatly, thereby also is difficult to practical high-effective affinity chromatography.Desirable high-effective affinity chromatography filler should have higher physical strength, better chemical stability, good hydrophilicity and biocompatibility and long serviceable life.
The affine filler that with the heparin is aglucon is the biochemical parting material of a kind of broad-spectrum special type.The affine filler that with the heparin is aglucon has heparin-Sepharose, heparin-polyacrylamide and heparin-silica gel etc.
In work related to the present invention, F.L.Zhuo etc. had reported once that a kind of was high-effective affinity chromatography filler [the Journal ofChromatography of aglucon with the heparin, 476 (1989), 195-203], they are the surfaces that DEAE-agrose are coated on macro porous silica gel, heparin is bonded on the clad, having prepared with the heparin is the high-effective affinity chromatography filler of aglucon again.Characterize through chromatogram, this material has stronger affinity interaction to Antithrombin III, but after the silica gel surface coats natural polysaccharide, can not shield the silicon hydroxyl on silica gel surface fully, and there is stronger non-specific adsorption in protein remains, and mass recovery only is 78%.
Having the object of the present invention is to provide a kind of is the high-effective affinity chromatography filler of aglucon with the heparin, and having overcome in the prior art with the heparin is the low shortcoming of high-effective affinity chromatography filler protein recovery of aglucon.
Another object of the present invention is to provide the preparation method of this filler.
High-effective affinity chromatography filler of the present invention, as matrix, contain the polymerized unit of following formula with the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon in this filler:
Wherein the structure of P is:
Hep is a heparin, and its structure is:
Above-mentioned poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon is the multipolymer of glytidyl methacrylate and methacrylic acid diethylene glycol dilaurate.
The preparation of high-effective affinity chromatography filler of the present invention, adopt following three kinds of methods:
1) with hydrochloric acid heparin is handled earlier, and then utilize amino in the heparin molecule and the epoxy radicals coupling on the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, and obtain filler of the present invention, wherein the heparin treatment step comprises heparin is dissolved in the 0.1M hydrochloric acid, heating joins in the ethanol then.
2) earlier the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon is carried out amination, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, and then with described amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon and heparin in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide, utilize carboxyl and the reaction of the amino on the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon in the heparin molecule, obtain filler of the present invention.
3) earlier the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon is carried out amination, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, and then described amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon and heparin carried out reduction amination in the presence of sodium cyanoborohydride, utilize the aldehyde radical of heparin molecule reducing end and the reaction of the amino on the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon to obtain filler of the present invention.Method one:
Method two:
Method three:
3 kinds of preparation method's concrete steps of high-effective affinity chromatography filler of the present invention are as follows: method one:
1. liquaemin is dissolved among the 0.1M HCl, concentration is 1%-20% (w/V), places boiling water bath to heat 10-60min.
2. above-mentioned solution is joined in the ethanol, have a large amount of white precipitates to generate, the volume of ethanol is 1-10 a times of above-mentioned solution.
3. filter, under 30-80 ℃ of temperature, be dried to filter residue dried.
4. above-mentioned dried product exhibited is dissolved in the Na of 0.3-1.0mol/L
2CO
3In the solution, solution concentration is 0.5%-10% (w/V), adds the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon again, the weight ratio of heparin and poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon is 30: 1-3: 1, preferable is 20: 1-5: 1, stir, at 40-80 ℃ of reaction 24-96h.
5. filter, under 30-80 ℃ of temperature, be dried to filter residue dried.Method two:
1. prepare amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon
The poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon is added in the ammoniacal liquor, and the w/v of the two is 1: 6-1: 2, stir, and behind 30-60 ℃ of insulation 5-10h, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon.
2.0.05-0.20mol/L add heparin in the HCl solution, concentration is 1%-40% (w/V), add amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide again, the weight ratio of amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide is 10: 1-1: 1, preferable is 8: 1-3: 1, stir, regulate pH=2-8, at room temperature react 24-96h.
3. filter, filter residue is washed to neutrality with massive laundering drained.
4. add 0.1-0.5mol/Na
2CO
3Solution, stir into uniform suspension after, add acetic anhydride, the volume of acetic anhydride be suspension 0.5-5 doubly, at room temperature react 10-60min.
5. filter, the filter residue water fully washs after the pH value is neutrality and drains.Method three:
1, same method two (1)
2. heparin is dissolved in 0.05-0.50mol/LK
2HPO
4In, the concentration of heparin is 1%-10% (w/V), adds amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon and sodium cyanoborohydride again, the weight ratio of the two is 100: 1-10: 1, after stirring into uniform suspension, in 30-100 ℃ of heating 5-15h, temperature of reaction is preferably 50-95 ℃.
3. filter, the filter residue water fully washs to neutral final vacuum and is evacuated to dried.
4. add 0.1-0.5mol/Na
2CO
3Solution, stir into uniform suspension after, add acetic anhydride, the volume of acetic anhydride be suspension 0.5-5 doubly, at room temperature react 10-60min.
5. filter, water fully washs to neutral final vacuum suction strainer extremely dried.
Of the present invention is the high-effective affinity chromatography filler of aglucon with the heparin, at first by diffuse-aggregate method, the even-grained poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of preparation micron order, again with its activation, after active function groups introduced the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, by several different methods heparin is bonded in the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microsphere surface, prepares high-effective affinity chromatography filler.
The preparation of poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon; be by with glytidyl methacrylate; azoisobutyronitrile; polyvinylpyrrolidone; ethanol and toluene mix; glytidyl methacrylate; azoisobutyronitrile; polyvinylpyrrolidone is respectively the 1wt%-10wt% of above-mentioned overall material quantity; 0.1wt%-1.0wt%; 0.5wt%-5.0wt%; the ratio of ethanol and toluene is 2/8-9/1 (V/V); stir; under nitrogen protection, react 5-48h down in 20-70 ℃; after reaction finishes; remove with sedimentation and to desolvate; and fully wash with ethanol, obtain the monodispersed poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of particle diameter.
By scanning electron microscope as can be known, high-effective affinity chromatography filler particle size of the present invention is 7 μ m, and particle size distribution is extremely even, is single dispersed.When filling in this filler in the stainless steel column of φ 4.0 * 7.0cm with 20Mpa pressure, and when being connected in highly effective liquid phase chromatographic system, pressure is 1.5-4.0MPa (flow velocity is 1mL/min) behind its post, and when being sample with the Antithrombin III, its affine capacity is the affine filler of 1.2-4.0mg/g.When with the human serum albumins be sample determination its during to the non-specific adsorption of protein, protein recovery is greater than 90%.Experimental example 1:
With the heparin-poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon resin of following embodiment 1-4 preparation is filler, and with homogenate method dress post, column jecket is under 20MPa pressure: the stainless steel column of φ 4.0 * 7.0cm or optional.Carrying out high performance liquid chromatography under following condition characterizes and efficient affine separation: equilibrium liquid: 0.004M Tris-HCl, 0.10M NaCl, the pH=4.0 eluent: 0.02M Tris-HCl, 2.0M NaCl, pH=7.35 flow velocity: 0.1mL/min～10.0mL/min detects wavelength: 280nm
Of the present invention is that the high-effective affinity chromatography filler of aglucon has good physical strength with the heparin, can be in the following steady operation of 30MPa, thus can be used for high flow rate drip washing; Chemical property is stable, can tolerate the environment of pH1.0-12.0; Antithrombin III is had high affine capacity and low non-specific adsorption, and mass recovery reaches 94%, and is as shown in table 1.
Table 1
Embodiment 1:
1g glytidyl methacrylate, 0.01g azoisobutyronitrile, 0.1g polyvinylpyrrolidone are mixed with 9.4mL ethanol and 9.4mL toluene; stir; under nitrogen protection, react 12h down in 40 ℃; after reaction finishes; remove with sedimentation and to desolvate; and fully wash with ethanol, obtain the monodispersed poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of particle diameter.The 150mg liquaemin is dissolved among the 1.0mL0.1MHCl, place boiling water bath heating 60min after, reactant liquor is joined in the ethanol, have a large amount of white precipitates to generate, the volume of ethanol is 2 times of above-mentioned solution.Filter, under 40 ℃ of temperature, be dried to filter residue dried.Above-mentioned dried product exhibited 50mg is dissolved in the Na of the 0.5mol/L of 2mL
2CO
3In the solution, add 1.0g poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon again, stir, behind 40 ℃ of reaction 48h, filter, after filter residue is washed with water, under 30-80 ℃ of temperature, be dried to dried.Resulting is that the concentration of heparin is the 1.6mg/g microballoon in the high-effective affinity chromatography filler of aglucon with the heparin, is the 3.6mg/g filler to the affine capacity of Antithrombin III, and protein recovery is 94%.
Embodiment 2:
In the ammoniacal liquor of 2mL, add the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of 1.0g embodiment 1 preparation, stir, behind 30 ℃ of insulation 5h, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon.The heparin of 100mg is dissolved among the 1mL 0.1M HCl, adds l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide of above-mentioned amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of 1.0g and 250mg, stir, transfer pH=7, at room temperature react 24h.Filter, filter residue is washed to neutrality with massive laundering drained.The 0.5mol/Na that adds 1mL
2CO
3Solution, stir into uniform suspension after, add the 1mL acetic anhydride, at room temperature react 60min after, filter, the filter residue water fully washs after pH value is neutrality and drains.Resulting is that the concentration of heparin is the 10.2mg/g microballoon in the high-effective affinity chromatography filler of aglucon with the heparin, is the 1.8mg/g filler to the affine capacity of Antithrombin III, and protein recovery is 93.6%.
Embodiment 3:
In the ammoniacal liquor of 2mL, add the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of 1.0g EXAMPLE l preparation, stir, behind 30 ℃ of insulation 5h, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon.The heparin of 130mg is dissolved among the 2mL 0.1M HCl, adds 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide of above-mentioned amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of 1.0g and 150mg, stir, regulate pH=4-5, at room temperature react 48h.Filter, filter residue is washed to neutrality with massive laundering drained.The 0.5mol/L Na that adds 1mL
2CO
3Solution, stir into uniform suspension after, add the 2mL acetic anhydride, at room temperature react 60min after, filter, the filter residue water fully washs after pH value is neutrality and drains.Resulting is that the concentration of heparin is the 1.4mg/g microballoon in the high-effective affinity chromatography filler of aglucon with the heparin, is the 2.4mg/g filler to the affine capacity of Antithrombin III, and protein recovery is 94.8%.
Embodiment 4:
In the ammoniacal liquor of 2mL, add the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of 1.0g EXAMPLE l preparation, stir, behind 30 ℃ of insulation 5h, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon.The 30mg heparin is dissolved in 1.0mL 0.1mol/L K
2HPO
4In, add amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon of 1.0g and 30mg sodium cyanoborohydride again, stir into uniform suspension after, in 50 ℃ the heating 8h.Filter, the filter residue water fully washs to neutral final vacuum and is evacuated to dried.The 0.5mol/L Na that adds 1mL
2CO
3Solution, stir into uniform suspension after, add the 2mL acetic anhydride, at room temperature react 60min after, filter, the filter residue water fully washs after pH value is neutrality and drains.Resulting is that the concentration of heparin is the 1.0mg/g microballoon in the high-effective affinity chromatography filler of aglucon with the heparin, is the 1.1mg/g filler to the affine capacity of Antithrombin III, and protein recovery is 94%.
Claims (4)
1. high-effective affinity chromatography filler as matrix, is characterized in that containing the polymerized unit of following formula with the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon in described filler:
Wherein Hep is a heparin, and the structure of P is:
2. the preparation method of the filler of claim 1, it is characterized in that with hydrochloric acid heparin being handled earlier, and then heparin is bonded directly on the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, obtain the filler of claim 1, wherein the heparin treatment step comprises heparin is dissolved in the 0.1M hydrochloric acid, heating joins in the ethanol then.
3. the preparation method of the filler of claim 1, it is characterized in that earlier the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon being carried out amination, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, and then described amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon and heparin reacted in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide, obtain the filler of claim 1.
4. the preparation method of the filler of claim 1, it is characterized in that earlier the poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon being carried out amination, obtain amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon, and then described amidized poly (glycidylmethacrylate--co-ethylene dimethacrylate) microballoon and heparin carried out reduction amination in the presence of sodium cyanoborohydride, obtain the filler of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB001326473A CN1176940C (en) | 2000-11-20 | 2000-11-20 | High-effective affinity chromatography filler and its preparation method |
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|---|---|---|---|
| CNB001326473A CN1176940C (en) | 2000-11-20 | 2000-11-20 | High-effective affinity chromatography filler and its preparation method |
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| CN1354365A true CN1354365A (en) | 2002-06-19 |
| CN1176940C CN1176940C (en) | 2004-11-24 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308067C (en) * | 2003-05-21 | 2007-04-04 | 中国科学院化学研究所 | Affinity chromatographic stuffing with sulfadimidine as ligand |
| CN101306353B (en) * | 2008-02-02 | 2010-06-09 | 中国人民解放军南京军区南京总医院 | Heparin affinity column and preparation method and use thereof |
| CN104927078A (en) * | 2015-05-05 | 2015-09-23 | 中北大学 | Preparation method and application of heparin functionalized microspheres |
| CN106422415A (en) * | 2016-09-12 | 2017-02-22 | 福州大学 | Mucopolysaccharide functionalized hydrophilic solid-phase microextraction monolithic column |
| CN109364900A (en) * | 2018-12-21 | 2019-02-22 | 中国科学院兰州化学物理研究所 | A kind of preparation of the compound silica gel of polyacrylamide package and application as chromatograph packing material |
| CN109865496A (en) * | 2019-02-26 | 2019-06-11 | 北京大学深圳研究生院 | Magnetic silica filler, magnetic affine filler and preparation method and purposes |
| CN115646466A (en) * | 2022-11-11 | 2023-01-31 | 南通裕弘分析仪器有限公司 | Preparation method of organic-inorganic hybrid particles with core-shell structure |
-
2000
- 2000-11-20 CN CNB001326473A patent/CN1176940C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308067C (en) * | 2003-05-21 | 2007-04-04 | 中国科学院化学研究所 | Affinity chromatographic stuffing with sulfadimidine as ligand |
| CN101306353B (en) * | 2008-02-02 | 2010-06-09 | 中国人民解放军南京军区南京总医院 | Heparin affinity column and preparation method and use thereof |
| CN104927078A (en) * | 2015-05-05 | 2015-09-23 | 中北大学 | Preparation method and application of heparin functionalized microspheres |
| CN104927078B (en) * | 2015-05-05 | 2018-05-01 | 中北大学 | A kind of preparation method and applications of test tube of hepari functional microsphere |
| CN106422415A (en) * | 2016-09-12 | 2017-02-22 | 福州大学 | Mucopolysaccharide functionalized hydrophilic solid-phase microextraction monolithic column |
| CN109364900A (en) * | 2018-12-21 | 2019-02-22 | 中国科学院兰州化学物理研究所 | A kind of preparation of the compound silica gel of polyacrylamide package and application as chromatograph packing material |
| CN109865496A (en) * | 2019-02-26 | 2019-06-11 | 北京大学深圳研究生院 | Magnetic silica filler, magnetic affine filler and preparation method and purposes |
| CN115646466A (en) * | 2022-11-11 | 2023-01-31 | 南通裕弘分析仪器有限公司 | Preparation method of organic-inorganic hybrid particles with core-shell structure |
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| CN1176940C (en) | 2004-11-24 |
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