Antibody separating and purifying medium and preparation method thereof

A technology of separation, purification and medium, applied in the field of biological separation materials, to achieve the effect of improving selectivity

Active Publication Date: 2014-01-15
汪志友
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to propose a medium for separating and purifying antibodies and a preparation method thereof in view of many problems of affinity separation media currently used for separation and purification of antibody drugs

Method used

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  • Antibody separating and purifying medium and preparation method thereof
  • Antibody separating and purifying medium and preparation method thereof
  • Antibody separating and purifying medium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020]

[0021] As shown in the above chemical reaction simple formula, first use s-triazine to activate the agarose gel microspheres. Dissolve 10 g of s-triazine (sym-triazine, colorless crystals, melting point 86°C, boiling point 114°C, CAS4719-04-4) in 100ml of dichloromethane, filter, use a rotary evaporator to evaporate the solvent, and remove the residual solid Redissolve in 100ml of acetone and cool to -5°C in an ice-salt bath. Take 10 g of agarose gel microspheres (cross-linking degree 6%, average particle diameter 90 μm) and add to 50 ml of NaOH solution, stir at room temperature for 15 minutes, and filter to remove NaOH solution. The gel was resuspended in cold NaOH solution at 4°C and cooled to 0°C in an ice-salt bath. To this solution was added 20 ml of a cold s-triazine / acetone solution, and the stirring was continued for 70 minutes on an ice bath and 30 minutes at room temperature. Filter, wash with 50ml of acetic acid aqueous solution (1:1 volume ratio) and...

Embodiment 2

[0023] Mix 8.5 volumes of 0.2M K2HPO4 solution with 1.5 volumes of 0.2M KH2PO4 solution to make the coupling buffer. Take 10 g of the s-triazine-activated sepharose microspheres in Example 1 and suspend them in 10 ml of coupling buffer. Then, 4-(2-aminoethyl)-pyridine was added at a ratio of 10 mg / ml as the small molecule affinity ligand of the antibody. Stirring (or shaking, shaking) was continued at room temperature for 24 hours. Wash with coupling buffer until five absorption peaks at 293nm wavelength. The ligand density of the obtained medium was 130 μmol / ml. It was stored in sodium acetate solution (0.2M, pH 5.4) containing 0.02% sodium azide (w / v) until use.

Embodiment 3

[0025] Mix 8.5 volumes of 0.2M K2HPO4 solution with 1.5 volumes of 0.2M KH2PO4 solution to make the coupling buffer. Take 10 g of the s-triazine-activated sepharose microspheres in Example 1 and suspend them in 10 ml of coupling buffer. Then, 4-(2-hydroxyethyl)-pyridine was added at a ratio of 20 mg / ml as the small-molecule affinity ligand of the antibody. Stirring (or shaking, shaking) was continued at room temperature for 24 hours. Wash with coupling buffer until five absorption peaks at 293nm wavelength. The ligand density of the obtained medium was 150 μmol / ml. It was stored in sodium acetate solution (0.2M, pH 5.4) containing 0.02% sodium azide (w / v) until use.

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Abstract

The invention discloses a novel bioseparation material and a preparation method thereof. The preparation method comprises the following steps: processing hydrophilic gel microspheres in a sym-triazine activator as a matrix to prepare an activated matrix, and bonding the activated matrix with two micro-molecular ligands comprising 4-(2-hydroxyethyl)-pyridine and the like through a coupling process to obtain the medium having a high specificity and a good affinity selectivity. The separation medium is used for the industrialized large-scale antibody production through cooperating with affinity chromatography chromatogram or expanded bed adsorption, is highly cost-competitive, allows highly pure antibody products to be directly extracted from biological raw materials comprising blood, serum, blood plasma, blood plasma fractions, ascites, cell culturing solutions, cell homogenates, milk liquids, colostrum and the like only through one step, and realizes time saving and convenience.

Description

technical field [0001] The invention relates to a medium for separating and purifying antibodies and a preparation method thereof, belonging to the field of biological separation materials. Specifically, it relates to a method for extracting blood, serum, plasma, plasma components, ascites, cell culture fluid, cell homogenization fluid, milk, colostrum and other biological materials by affinity chromatography or extended bed adsorption method. A novel bioseparation material for separating and purifying antibodies and a preparation method thereof. Background of the invention [0002] Bioseparation media are a class of materials specifically used for the purification and refinement of biologically related products. Separation media are widely used in the separation of pharmaceutical industry, fermentation industry, biological products, blood products, vaccines and genetic engineering products. When purifying products, the separation medium has good separation effect, high pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/22B01J20/26B01J20/28B01J20/30C07K1/22
Inventor 汪志友
Owner 汪志友
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