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Chromatographic medium using aminobenzamide (aminobenzenesulfonamide) pyridine as functional ligand

An amide pyridine and chromatographic medium technology, applied in the field of protein chromatographic separation technology, can solve the problems of affecting the purity of the separated antibody, poor adsorption selectivity, unfavorable separation application, etc., and achieves strong charge induction effect, high ligand density, and adsorption selection. strong effect

Inactive Publication Date: 2019-10-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Luo et al. (J.Chromatogr.A, 2018, 1533:77) used two-step chromatography filled with ABI-4FF and MMI-4FF media to separate IgM in serum, but a small amount of HSA will also bind to the chromatography medium, affecting the separation Antibody purity
Wang et al. (J.Chromatogr.B,2013,936:33) used mixed-mode media Bestarose Diamond MMA, Bestarose Diamond MMC, MEP HyperCel and PPA HyperCel media to separate IgG from simulated serum, wherein Bestarose Diamond MMA media simultaneously adsorbed IgG and HSA, and adsorbed Poor selectivity; Bestarose Diamond MMC and PPAHyperCel media have difficulties in elution of antibodies, which is not conducive to practical separation applications; MEP HyperCel media mainly adsorbs IgG, but still binds a small amount of HSA, and the selectivity of IgG is average

Method used

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  • Chromatographic medium using aminobenzamide (aminobenzenesulfonamide) pyridine as functional ligand
  • Chromatographic medium using aminobenzamide (aminobenzenesulfonamide) pyridine as functional ligand
  • Chromatographic medium using aminobenzamide (aminobenzenesulfonamide) pyridine as functional ligand

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Take 10g of dry agarose gel, add 2g 20% ​​(v / v) dimethyl sulfoxide, 1g allyl bromide and 1g sodium hydroxide, activate it in a shaker at 150rpm at 25℃ for 48 hours, filter with suction, The activated matrix was washed with deionized water; then the activated matrix and 1g of N-bromosuccinimide were mixed for bromo-alcoholization, reacted in a shaker at 150 rpm at 25°C for 1 hour, filtered with suction, and washed with deionized water; Mix the brominated substrate with 2g 4-amino-N-(2-pyridyl)benzenesulfonamide and 1M sodium carbonate buffer (pH12), and react in a shaker at 150rpm at 25°C for 48 hours; finally, filter the medium with suction. Wash with ionized water, add to 10g ethanolamine, react in a shaker at 150rpm at 25°C for 12 hours, wash with deionized water to obtain a chromatography medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as ligand , The ligand density is 40μmol / ml.

Embodiment 2

[0031] Take 10g of dry agarose gel, add 10g 20%(v / v) dimethyl sulfoxide, 10g allyl bromide and 5g sodium hydroxide, activate it in a shaker at 150rpm at 25℃ for 8 hours, filter with suction, The activated matrix was washed with deionized water; then the activated matrix and 5g of N-bromosuccinimide were mixed for bromo-alcoholization, reacted in a shaker at 150 rpm at 25°C for 5 hours, filtered with suction, and washed with deionized water; Mix the brominated substrate with 6g 4-amino-N-(2-pyridyl)benzenesulfonamide and 0.5M sodium carbonate buffer (pH 10), and react in a shaker at 150rpm at 25°C for 8 hours; finally, filter the medium with suction. Wash with deionized water, add to 50g ethanolamine, react in a shaker at 150rpm at 25°C for 48 hours, wash with deionized water to obtain a layer with 4-amino-N-(2-pyridyl)benzenesulfonamide as ligand Analysis medium, the ligand density is 150μmol / ml. Under different pH and NaCl concentration, the binding capacity of the medium to ...

Embodiment 3

[0033] Take 10g of dry agarose gel, add 10g 20%(v / v) dimethyl sulfoxide, 10g allyl bromide and 4g sodium hydroxide, activate it in a shaker at 150rpm at 25℃ for 10 hours, filter with suction, The activated matrix was washed with deionized water; then the activated matrix and 5g of N-bromosuccinimide were mixed for bromo-alcoholization, reacted in a shaker at 150 rpm at 25°C for 1 hour, filtered with suction, and washed with deionized water; Mix the brominated substrate with 5g 4-amino-N-(2-pyridyl)benzenesulfonamide and 0.5M sodium carbonate buffer (pH 12), and react in a shaker at 150rpm at 25°C for 16 hours; finally, filter the medium with suction. Wash with deionized water, add to 5g ethanolamine, react in a shaker at 150rpm at 25°C for 4 hours, wash with deionized water to obtain a layer with 4-amino-N-(2-pyridyl)benzenesulfonamide as ligand Analyze the medium and the ligand density is 95μmol / ml.

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Abstract

The invention discloses a chromatographic medium using aminobenzamide (aminobenzenesulfonamide) pyridine as a functional ligand. The chromatographic medium is prepared by the following steps: hydrophilic porous microspheres are used as a chromatographic substrate, and dimethyl sulfoxide and allyl bromide are sequentially added into the chromatographic substrate for activation; the activated chromatographic substrate reacts with N-bromosuccinimide, and bromo alcoholization is carried out; the bromo-alcoholized chromatographic substrate is coupled with the aminobenzamide (aminobenzenesulfonamide) pyridine ligand; and finally, the unreacted bromo-alcoholized tail end is closed by adopting an ethanol amine aqueous solution to obtain the mixed-mode chromatographic medium using the aminobenzamide (aminobenzenesulfonamide) pyridine as the functional group. The novel chromatographic medium provided by the invention has the characteristics of a large adsorption capacity of antibodies, high selectivity and non-salt dependent adsorption, desorption and recovery can be realized by changing the pH value of a solution to be weakly acidic, the preparation process is simple and convenient, the price is low, and the novel chromatographic medium can be used for mixed mode chromatography separation of the antibodies.

Description

Technical field [0001] The invention relates to a chromatographic medium using aminobenzene (sulfon)amide pyridine as a functional ligand, and belongs to a protein chromatographic separation technology in the field of biochemical engineering. Background technique [0002] Antibody products often require high purity and must maintain biological activity. Therefore, traditional separation processes are often difficult to meet the requirements. Protein A or protein G affinity chromatography media can specifically bind antibodies, but protein affinity ligands are easily degraded and shed by proteases in the feed solution, the number of repeated uses is low, elution is difficult, the media is expensive, and the use cost is extremely high , Restricting large-scale applications. Although traditional methods such as ion exchange chromatography and hydrophobic interaction chromatography can bind antibodies, they have poor specificity and selectivity, and have many separation steps and li...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01J20/30
CPCB01J20/286
Inventor 林东强褚文宁姚善泾
Owner ZHEJIANG UNIV
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