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Tetrapeptide chromatography medium taking phenylalanine-tyrosine-histidine-glutamic acid as functional ligand, and application of tetrapeptide chromatography medium

A phenylalanine and chromatographic medium technology, applied in protein chromatographic separation technology, tetrapeptide chromatographic medium and antibody separation application fields, can solve the problems of weak salt-tolerant binding ability, low dynamic antibody loading, etc., to avoid The effect of antibody aggregation, high affinity, and large adsorption capacity

Active Publication Date: 2018-11-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Huang and Zhao et al. (CN 103014880A; Journalof Chromatography A.2014,1359:100-111) used molecular simulation design and screening to obtain octapeptide ligands (phenylalanine-tyrosine-tryptophan-histidine-semi cystine-leucine-aspartic acid-glutamic acid, FYWHCLDE), this ligand has high adsorption selectivity for antibodies, and can be applied to isolate antibodies from cell culture supernatants and human serum, but The dynamic load of the antibody is not high, and the binding to the antibody is mainly dominated by electrostatic interaction, so the salt-resistant binding ability is weak, and the feed solution may need to be diluted during separation

Method used

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  • Tetrapeptide chromatography medium taking phenylalanine-tyrosine-histidine-glutamic acid as functional ligand, and application of tetrapeptide chromatography medium
  • Tetrapeptide chromatography medium taking phenylalanine-tyrosine-histidine-glutamic acid as functional ligand, and application of tetrapeptide chromatography medium
  • Tetrapeptide chromatography medium taking phenylalanine-tyrosine-histidine-glutamic acid as functional ligand, and application of tetrapeptide chromatography medium

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: Take 10 g of drained agarose gel, add 10 mL of 20% (v / v) dimethyl sulfoxide solution, 10 mL of allyl bromide and 5 g of sodium hydroxide, activate in a shaker at 180 rpm at 30 °C After 36 hours, filter with suction and wash with deionized water to obtain the activated matrix; (2) bromoalcoholization: the activated matrix and 5g N-bromosuccinimide are mixed for bromoalcoholation, and placed in a 180rpm shaker at 30°C React for 1 hour, filter with suction, and wash with deionized water 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), and React in a shaker at 180rpm for 24 hours t...

Embodiment 2

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

[0045] The process of preparing tetrapeptide chromatography medium with cellulose microspheres as matrix mainly includes four steps: matrix activation, bromoalcoholation, spacer coupling and ligand coupling. (1) Matrix activation: take 10 g of cellulose microspheres, add 10 mL of 20% (v / v) dimethyl sulfoxide solution, 10 mL of allyl bromide and 5 g of sodium hydroxide, and activate in a shaker at 180 rpm at 35 °C For 48 hours, filter with suction and wash with deionized water to obtain the activated matrix; (2) bromoalcoholization: the activated matrix and 5g N-bromosuccinimide are mixed for bromoalcoholization, and placed in a 180rpm shaker at 30°C React for 1 hour, filter with suction, and wash with deionized water 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), and React in a shaker at 18...

Embodiment 3

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

[0047] The process of preparing tetrapeptide chromatography medium with polymethacrylate microspheres as matrix mainly includes four steps: matrix activation, bromoalcoholation, spacer coupling and ligand coupling. (1) Matrix activation: Take 10 g of drained agarose gel, add 10 mL of 20% (v / v) dimethyl sulfoxide solution, 10 mL of allyl bromide and 5 g of sodium hydroxide, and activate it in a shaker at 180 rpm at 30 °C for 30 Hours, suction filtration, wash with deionized water, obtain activated matrix; (2) bromoalcoholization: activate matrix, 5g N-bromosuccinimide are mixed and carry out bromoalcoholation, react in 180rpm shaker under 30 ℃ For 1 hour, filter with suction and wash with deionized water to obtain the brominated matrix; (3) space arm coupling: mix the brominated matrix with 3 mL of hexamethylenediamine and 1M sodium carbonate buffer (pH12), shake at 180 rpm at 30°C React in bed for ...

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Abstract

The invention discloses a tetrapeptide chromatography medium taking phenylalanine-tyrosine-histidine-glutamic acid as a functional ligand, and application of the tetrapeptide chromatography medium; the tetrapeptide chromatography medium can be used for antibody separation. The tetrapeptide chromatography medium is obtained by taking hydrophilic porous microspheres as chromatography matrix, adopting allyl bromide activation and bromo-alcoholization, linking hexamethylenediamine to form a space arm, and then coupling phenylalanine-tyrosine-histidine-glutamic acid as the tetrapeptide ligand. Thetetrapeptide chromatography medium provided by the invention has good binding ability and absorption selection property to an antibody, can absorb the antibody under the neutral condition and efficiently dissociate the antibody under the weak acidic condition, is mild in separation conditions, and can be applied to separation of monoclonal antibodies from cell culture supernatant and separation ofimmunoglobulins from human serum.

Description

technical field [0001] The invention relates to a tetrapeptide chromatographic medium with phenylalanine-tyrosine-histidine-glutamic acid as functional ligands and antibody separation application, which belongs to protein chromatographic separation technology in the field of biochemical industry. Background technique [0002] Antibodies have the advantages of high specificity, strong targeting and good biocompatibility, and are widely used in therapeutic drugs and diagnostic reagents. With the continuous development of genetic engineering, the cell expression volume and culture scale of the antibody production process have been gradually expanded, which has continuously increased the upstream production capacity and brought great pressure to the downstream separation and purification. At present, protein A affinity chromatography is widely used in the process of antibody separation and purification, which has very high specificity and can efficiently capture antibodies, but ...

Claims

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Application Information

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IPC IPC(8): B01J20/24B01J20/26B01J20/28B01J20/30C07K1/22
CPCB01J20/22B01J20/24B01J20/261B01J20/28021C07K1/22
Inventor 姚善泾陈圣刚林东强
Owner ZHEJIANG UNIV
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