Chromatography medium with aminobenzimidazole as functional ligand and preparation method thereof

An aminobenzimidazole, chromatographic medium technology, applied in chemical instruments and methods, other chemical processes, alkali metal oxides/hydroxides, etc., can solve the problem of low pH, limited electrostatic repulsion, antibody Activity loss and other problems, to achieve the effect of convenient elution, less non-specific adsorption of the medium, and fewer operation steps

Active Publication Date: 2016-06-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These media mainly use sulfhydryl-containing heterocyclic compounds as ligands, which are coupled to the substrate through the sulfhydryl sites of the ligands. The coupling efficiency is low, and the elution can only be carried out by the protonated nitrogen atoms on the heterocycle and between proteins. Electrostatic repulsion is limited, and the pH for complete elution is low, which can easily cause loss of antibody activity

Method used

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  • Chromatography medium with aminobenzimidazole as functional ligand and preparation method thereof
  • Chromatography medium with aminobenzimidazole as functional ligand and preparation method thereof
  • Chromatography medium with aminobenzimidazole as functional ligand and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Take 10 g of drained agarose gel, add 2 g of 20% (v / v) dimethyl sulfoxide, 1 g of allyl bromide and 1 g of sodium hydroxide, activate in a shaker at 150 rpm at 25 °C for 48 hours, filter with suction, and use Wash with deionized water to obtain the activated matrix; then mix the activated matrix and 1g N-bromosuccinimide for bromoalcoholization, react in a 150rpm shaker at 25°C for 5 hours, filter with suction, and wash with deionized water; then bromine The substitute matrix was mixed with 1g of 2-aminobenzimidazole and 1M sodium carbonate buffer (pH12), and reacted in a shaker at 150rpm at 25°C for 24 hours; finally, the medium was suction-filtered, washed with deionized water, added to 10g of ethanolamine, and kept at 25°C React in a shaker at 150 rpm for 8 hours, wash with deionized water, and obtain a chromatographic medium with 2-aminobenzimidazole as a ligand, and the ligand density is 40 μmol / ml.

Embodiment 2

[0028] Take 10 g of drained agarose gel, add 10 g of 20% (v / v) dimethyl sulfoxide, 10 g of allyl bromide, and 5 g of sodium hydroxide, activate it in a shaker at 150 rpm at 25 ° C for 8 hours, filter it with suction, and use it Wash with ionic water to obtain the activated matrix; then mix the activated matrix and 5g N-bromosuccinimide for bromoalcoholization, react in a shaker at 150rpm at 25°C for 1 hour, filter with suction, and wash with deionized water; then bromine The substitute matrix was mixed with 3g of 2-aminobenzimidazole and 0.5M sodium carbonate buffer (pH10), and reacted in a shaker at 150rpm at 25°C for 8 hours; finally, the medium was suction-filtered, washed with deionized water, and added to 50g of ethanolamine, 25 React at 150 rpm shaker at ℃ for 2 hours, wash with deionized water, and obtain a chromatographic medium with 2-aminobenzimidazole as a ligand, and the ligand density is 150 μmol / ml.

Embodiment 3

[0030] Take 10 g of drained agarose gel, add 2 g of 20% (v / v) dimethyl sulfoxide, 1 g of allyl bromide and 1 g of sodium hydroxide, activate in a shaker at 150 rpm at 25 °C for 48 hours, filter with suction, and use Wash with deionized water to obtain the activated matrix; then mix the activated matrix and 1g N-bromosuccinimide for bromoalcoholization, react in a 150rpm shaker at 25°C for 5 hours, filter with suction, and wash with deionized water; then bromine The substitute matrix was mixed with 1g of 4-aminobenzimidazole and 1M sodium carbonate buffer (pH12), and reacted in a shaker at 150rpm at 25°C for 24 hours; finally, the medium was suction-filtered, washed with deionized water, added to 10g of ethanolamine, and kept at 25°C React in a shaker at 150 rpm for 8 hours, wash with deionized water, and obtain a chromatographic medium with 4-aminobenzimidazole as a ligand, and the ligand density is 40 μmol / ml.

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Abstract

The invention discloses a chromatographic medium using amino benzimidazole as a function ligand and a preparation method thereof. Hydrophilic porous microspheres are used as a chromatographic medium, activated by allyl bromide, and coupled with the amino benzimidazole to obtain a medium using the amino benzimidazole as the function ligand; dimethyl sulfoxide and the allyl bromide are sequentially added into a chromatographic matrix for activation; the activated chromatographic matrix is reacted with N-bromo-succinimide for bromo-alcoholization; the bromo-alcoholized chromatographic matrix is mixed with an amino benzimidazole solution for coupling the amino benzimidazole ligand; finally an aqueous ethanol amine solution is used for sealing unreacted bromo-alcoholized ends to obtain a hydrophobic charge induced chromatographic medium using the amino benzimidazole as the function group. The new chromatographic medium is simple in preparation process and high in antibody adsorption capacity, and has the characteristics of non salt dependent adsorption, can realize desorption and recovery by changing the solution pH to weak acid, and can be used for hydrophobic charge induction chromatographic separation of antibodies.

Description

technical field [0001] The invention relates to a chromatographic medium with aminobenzimidazole as a functional ligand and a preparation method thereof, belonging to protein chromatographic separation technology in the field of biochemical industry. Background technique [0002] Antibodies can specifically bind to corresponding antigens to produce various immune effects. With the rapid development of biotechnology and continuous breakthroughs in antibody engineering technology, monoclonal antibodies, polyclonal antibodies, and genetically engineered antibodies have been widely used in the fields of biology and medicine. Antibody uses mainly include disease treatment, in vitro diagnosis and detection, tumor localization imaging, and separation and purification as an affinity ligand. [0003] Antibody products often require high purity and must maintain biological activity, so traditional separation processes are often difficult to meet the requirements. Protein A or protei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/30B01J20/28C08B37/12C08B15/06C07K1/20
Inventor 林东强童红飞姚善泾
Owner ZHEJIANG UNIV
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