An affinity biomimetic chromatography medium with tetrapeptide as functional ligand

A chromatographic medium and ligand technology, applied in the field of protein chromatographic separation technology, can solve the problems of high price of protein A affinity medium, harsh elution conditions, easy detachment of ligands, etc., to avoid antibody aggregation or activity decline, The effect of large adsorption capacity and strong adsorption selectivity

Active Publication Date: 2020-04-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the protein A affinity medium is expensive, and there are disadvantages such as the ligand is easy to fall off, the elution conditions are harsh, and the ligand fall off can easily cause an immune response.

Method used

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  • An affinity biomimetic chromatography medium with tetrapeptide as functional ligand
  • An affinity biomimetic chromatography medium with tetrapeptide as functional ligand
  • An affinity biomimetic chromatography medium with tetrapeptide as functional ligand

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation of Tetrapeptide Affinity Chromatography Medium

[0043] The process of preparing tetrapeptide chromatography medium with agarose gel as matrix mainly includes four steps: matrix activation, bromoalcoholation, spacer coupling and ligand coupling. (1) Matrix activation: first weigh 5g of sodium hydroxide, add 10mL of 20% (v / v) DMSO solution, dissolve, let stand, after the solution is cooled, add 10g of drained agarose gel matrix, and then add 10mL Allyl bromide, activated in a shaker at 180rpm at 30°C for 24 hours, washed with deionized water, and filtered with suction to obtain an activated matrix; (2) bromoalcoholization: mix the activated matrix with 5g NBS, and react in a shaker at 180rpm at 30°C For 3 hours, wash with deionized water and filter with suction to obtain the brominated matrix; (3) space arm coupling: mix the brominated matrix with 3 mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 12), at 30°C at 180rpm React in a sha...

Embodiment 2

[0044] Example 2: Preparation of Tetrapeptide Affinity Chromatography Medium

[0045] The process of preparing tetrapeptide chromatography medium with agarose gel as matrix mainly includes four steps: matrix activation, bromoalcoholation, spacer coupling and ligand coupling. (1) Matrix activation: first weigh 5g of sodium hydroxide, add 10mL of 20% (v / v) DMSO solution, dissolve, let stand, after the solution is cooled, add 10g of drained agarose gel matrix, and then add 10mL Allyl bromide, activated in a shaker at 180rpm at 30°C for 24 hours, washed with deionized water, and filtered with suction to obtain an activated matrix; (2) bromoalcoholization: mix the activated matrix with 5g NBS, and react in a shaker at 180rpm at 30°C For 3 hours, wash with deionized water and filter with suction to obtain the brominated matrix; (3) space arm coupling: mix the brominated matrix with 3 mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 12), at 30°C at 180rpm React in a sha...

Embodiment 3

[0046] Example 3: Preparation of Tetrapeptide Affinity Chromatography Medium

[0047] The process of preparing tetrapeptide chromatography medium with agarose gel as matrix mainly includes four steps: matrix activation, bromoalcoholation, spacer coupling and ligand coupling. (1) Matrix activation: first weigh 5g of sodium hydroxide, add 10mL of 20% (v / v) DMSO solution, dissolve, let stand, after the solution is cooled, add 10g of drained agarose gel matrix, and then add 10mL Allyl bromide, activated in a shaker at 180rpm at 30°C for 24 hours, washed with deionized water, and filtered with suction to obtain an activated matrix; (2) bromoalcoholization: mix the activated matrix with 5g NBS, and react in a shaker at 180rpm at 30°C For 3 hours, wash with deionized water and filter with suction to obtain the brominated matrix; (3) space arm coupling: mix the brominated matrix with 3 mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 12), at 30°C at 180rpm React in a sha...

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Abstract

The invention discloses an affinity biomimetic chromatography medium with a tetrapeptide as a functional ligand, wherein the affinity biomimetic chromatography medium can be used for antibody separation and purification, and the amino acid sequence of the tetrapeptide is phenylalanine-tyrosine-tryptophan-arginine. The preparation method comprises: activating with allyl bromide, carrying out bromination alcoholization, linking a space arm hexanediamine to a polysaccharide gel, and conjugating with an acetylated phenylalanine-tyrosine-tryptophan-arginine tetrapeptide ligand to obtain the tetrapeptide chromatography medium. According to the present invention, the tetrapeptide chromatography medium has good adsorption capacity to antibodies, has high selectivity, can adsorb antibodies under weak alkaline (pH value of 8.0) conditions, can be eluted under weak acidic (pH value of 5.0) conditions, has mild separation conditions, and can be used for isolating immunoglobulins from human serum and isolating monoclonal antibodies in cell culture.

Description

technical field [0001] The invention relates to an affinity biomimetic chromatographic medium with tetrapeptide as a functional ligand, which is expected to be used in the separation and purification of antibodies, and belongs to the protein chromatographic separation technology in the field of biochemical industry. Background technique [0002] Monoclonal antibodies (mAbs) are widely used in the treatment of cancer, autoimmune diseases, etc. In recent years, with the development of antibody engineering, the expression level of cells has been significantly increased, reaching as high as 10g / L. The purity requirements of antibody drugs are high, the separation and purification process is complicated, and the downstream process accounts for a large proportion (50-80%) of the production cost. The development of new cost-effective separation and purification methods has become a research hotspot in the field of antibody preparation. [0003] Currently, the most commonly used an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/30B01D15/08
CPCB01D15/08B01J20/24
Inventor 姚善泾方钰明林东强张其磊关怡新
Owner ZHEJIANG UNIV
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