Functionalized high-density chromatographic substrate, preparation method thereof and application thereof

A high-density layer and functionalization technology, which is applied in the preparation method of peptides, chemical instruments and methods, separation methods, etc., can solve the problem of limiting the adsorption capacity of chromatographic substrates and improving adsorption specificity, reducing the amount of ligand coupling, reaction Uncontrollable problems, to achieve the effect of low cost, low cost, and few steps

Active Publication Date: 2018-08-28
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the functionalization of chromatographic matrix using divinyl sulfone is carried out in alkaline aqueous solution, the reaction efficiency is low, the highest functionalized density can only reach 60 μmol / g resin, and the reaction is uncontrollable, which limits the chromatographic matrix. Enhanced adsorption capacity and adsorption specificity
In addition, under the reaction conditions of alkaline aqueous solution, the vinylsulfone group is easily hydrolyzed, which further reduces the amount of ligand coupling.

Method used

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  • Functionalized high-density chromatographic substrate, preparation method thereof and application thereof
  • Functionalized high-density chromatographic substrate, preparation method thereof and application thereof
  • Functionalized high-density chromatographic substrate, preparation method thereof and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Take 0.3 g of agarose resin (Bastarose 6FF: highly cross-linked 6% agarose, average particle size 90 μm; Bogelong Biotechnology Co., Ltd.), filter it with suction and wash it well with acetonitrile to remove the water in it, add 1 mL containing 4-bis A solution of 10% (v / v) divinyl sulfone in acetonitrile of methylaminopyridine, wherein the molar ratio of 4-dimethylaminopyridine to divinyl sulfone is 1:10, reacted at 25°C for 12h, pumped out after the reaction Filter and wash with acetonitrile to completely remove divinyl sulfone and catalyst residues to obtain VS functionalized agarose resin.

[0034] For the control group, 0.3 g of agarose resin was added to 1 mL of carbonate buffer solution (0.5 M, pH 11) containing 10% (v / v) divinyl sulfone and 10% acetone, and reacted for 12 h at 25°C. After the end, filter with suction and wash with acetonitrile to completely remove divinyl sulfone residues. Take quantitative functionalized agarose resin, add excess cysteine ​​so...

Embodiment 2

[0036]Take 0.3 g of agarose resin (Bastarose 6FF: highly cross-linked 6% agarose, with an average particle size of 90 μm; Borgron Biotechnology Co., Ltd.), filter it with suction and wash it with acetonitrile to remove water, add 1 mL containing triphenyl 10% (v / v) acetonitrile solution of divinyl sulfone of phosphine, wherein the molar ratio of triphenyl phosphine to divinyl sulfone is 1:10, react at 25°C for 1 h, after the reaction is completed, suction filtration and wash with acetonitrile Divinyl sulfone and catalyst residues were thoroughly removed to obtain VS functionalized agarose resin.

[0037] In the control group, 0.3 g of agarose resin was added to 1 mL of carbonate buffer (0.5 M, pH 11) containing 10% (v / v) divinyl sulfone and 10% acetone, and the reaction was carried out at 25 °C for 12 h. After the end, suction filtration and washing with acetonitrile thoroughly remove the residual divinyl sulfone. Take a quantitative functionalized agarose resin, add an exces...

Embodiment 3

[0039] Take 0.3 g of agarose resin (Bastarose 6FF: highly cross-linked 6% agarose, with an average particle size of 90 μm; Borgron Biotechnology Co., Ltd.), filter it with suction and wash it with acetonitrile to remove water, add 1 mL containing triphenyl 10% (v / v) acetonitrile solution of phosphine in divinyl sulfone, wherein the molar ratio of triphenyl phosphine and divinyl sulfone is 1:10, react at 25°C for 2h, after the reaction, suction filtration and wash with acetonitrile Divinyl sulfone and catalyst residues were thoroughly removed to obtain VS functionalized agarose resin.

[0040] In the control group, 0.3 g of agarose resin was added to 1 mL of carbonate buffer (0.5 M, pH 11) containing 10% (v / v) divinyl sulfone and 10% acetone, and the reaction was carried out at 25 °C for 12 h. After the end, suction filtration and washing with acetonitrile thoroughly remove the residual divinyl sulfone. Take a quantitative functionalized agarose resin, add an excess of cystein...

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Abstract

The invention discloses a functionalized high-density chromatographic substrate, a preparation method thereof and application thereof. A chromatographic substrate functionalizing method is characterized in that a hydrophilic microsphere or surface with a polyhydroxyl structure is covered with a hydrophilic microsphere with polyhydroxyl polymer as a chromatographic substrate; under an anhydrous condition, a catalyst is utilized to catalyze reaction between surface hydroxyl of the chromatographic substrate and divinyl sulfone, so that surface hydroxyl of the substrate is functionalized. The chromatographic substrate surface functionalizing method is fewer in steps, is simple in process and is high in functionalizing density; the used reagent and the used solvent are conventional reagents, and can be recycled; byproducts are not generated in a reaction process, so that atom economy is high and cost is low; and density control can be realized by regulating and controlling reaction time andthe catalyst type. The chromatographic substrate prepared by the method has a wide application prospect in affinitive chromatographic stuffing preparation and antibody medicine purification.

Description

technical field [0001] The invention belongs to the technical field of preparation of chromatographic materials in the field of biochemical industry, and in particular relates to a functionalized high-density chromatographic matrix, its preparation method and application. technical background [0002] Antibodies and their related products play an important role in clinical treatment and immunological diagnosis. With the rapid development of upstream cell culture technology in recent years, the requirements for downstream antibody purification processes are constantly increasing. The activation method of chromatography matrix with high reactivity and controllable reaction is of great significance for improving the adsorption capacity of the matrix and optimizing the adsorption specificity. The activating reagent of the chromatographic substrate widely used now is epoxy chloro (bromo) propane and allyl bromide, Harding etc. (J.Chromatogr.A, 1997,775:29) studied epoxy chloro (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/28B01J20/30B01D15/38C07K16/00C07K1/22
CPCB01D15/3804B01J20/24B01J20/28019B01J20/28047C07K1/22C07K16/00B01J20/262B01J20/265B01J20/286B01J20/321B01J20/3212B01J20/3251B01J20/327B01J20/3274C07K16/065C07K16/4291C07K2317/21C07K2317/24Y02P20/582
Inventor 程昉李明洋何炜孙冰冰马晓春曲景平
Owner DALIAN UNIV OF TECH
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