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Chromatographic medium with aminobenzene (sulf)amide pyridine as functional ligand and application of chromatographic medium

An amide pyridine and chromatography medium technology, applied in the field of chromatography medium and protein chromatography separation, can solve the problems of affecting the purity of the isolated antibody, difficult antibody elution, unfavorable separation and application, etc., and achieves strong charge induction effect and good separation effect. , the effect of strong adsorption selectivity

Active Publication Date: 2020-09-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 with aminobenzene (sulf)amide pyridine as functional ligand and application of chromatographic medium
  • Chromatographic medium with aminobenzene (sulf)amide pyridine as functional ligand and application of chromatographic medium
  • Chromatographic medium with aminobenzene (sulf)amide pyridine as functional ligand and application of chromatographic medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 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 an activated matrix; then mix the activated matrix and 1 g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 150 rpm at 25°C for 1 hour, filter with suction, and wash with deionized water; Mix the brominated matrix with 2g of 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; Wash with deionized water, add in 10g ethanolamine, react in a 150rpm shaker at 25°C for 12 hours, wash with deionized water, and obtain a chromatographic medium with 4-amino-N-(2-pyridyl)benzenesulfonamide as a ligand , The ligand density is 40μmol / ml.

Embodiment 2

[0034] 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 in a shaker at 150 rpm at 25 ° C for 8 hours, filter with suction, and use Wash with deionized water to obtain an activated matrix; then mix the activated matrix and 5 g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 150 rpm at 25°C for 5 hours, filter with suction, and wash with deionized water; then The brominated matrix was mixed with 6g of 4-amino-N-(2-pyridyl)benzenesulfonamide and 0.5M sodium carbonate buffer (pH 10), and reacted in a shaker at 150rpm at 25°C for 8 hours; finally, the medium was suction-filtered, Wash with deionized water, add to 50g ethanolamine, react in a 150rpm shaker at 25°C for 48 hours, wash with deionized water, and obtain a layer with 4-amino-N-(2-pyridyl)benzenesulfonamide as the ligand Analysis medium with a ligand density of 150 μmol / ml. At different pH and NaCl concentrations, th...

Embodiment 3

[0036] Take 10 g of drained agarose gel, add 10 g of 20% (v / v) dimethyl sulfoxide, 10 g of allyl bromide and 4 g of sodium hydroxide, activate in a shaker at 150 rpm at 25° C. for 10 hours, filter with suction, and use Wash with deionized water to obtain the activated matrix; then mix the activated matrix and 5g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 150rpm at 25°C for 1 hour, filter with suction, and wash with deionized water; The brominated matrix was mixed with 5g of 4-amino-N-(2-pyridyl)benzenesulfonamide and 0.5M sodium carbonate buffer (pH 12), and reacted in a shaker at 150rpm at 25°C for 16 hours; finally, the medium was suction-filtered, Wash with deionized water, add to 5g ethanolamine, react in a 150rpm shaker at 25°C for 4 hours, wash with deionized water, and obtain a layer with 4-amino-N-(2-pyridyl)benzenesulfonamide as the ligand Analysis medium with a ligand density of 95 μmol / ml.

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Abstract

The invention discloses a chromatographic medium with aminobenzene (sulf)amide pyridine as a functional ligand and application of the chromatography medium. A preparation method for the chromatographic medium comprises the following steps: with hydrophilic porous microspheres as a chromatographic matrix, sequentially adding dimethyl sulfoxide and allyl bromide into the chromatographic matrix for activation; enabling the activated chromatographic matrix to react with N-bromo-succinimide, and carrying out bromo-alcoholization; coupling the chromatographic matrix having undergone bromo-alcoholization with the aminobenzene (sulf)amide pyridine ligand; and finally, closing an unreacted bromo-alcoholized tail end by adopting an aqueous ethanol amine solution to obtain the mixed-mode chromatographic medium with aminobenzene (sulf)amide pyridine as a functional group. The novel chromatographic medium disclosed by the invention can be combined with IgG type broad-spectrum antibody molecules andhas the characteristics of high antibody adsorption capacity, high selectivity and non-salt dependent adsorption; desorption and recovery can be realized by changing the pH value of a solution to beslightly smaller than 7; preparation process is simple and convenient, and price is low; and the chromatographic medium can be used for chromatographic separation of broad-spectrum IgG type antibodies, including polyclonal antibodies in blood and monoclonal antibody drugs expressed by cells.

Description

technical field [0001] The invention relates to a chromatographic medium using aminobenzene (sulfon)amide pyridine as a functional ligand and an application thereof, which belongs to protein chromatographic separation technology in the field of biochemical industry. Background technique [0002] 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 protein G affinity chromatography media can specifically bind to the conserved region of antibodies and have a broad-spectrum antibody separation ability, but protein affinity ligands are easily degraded by proteases in the feed solution and fall off, the number of repeated uses is low, and the elution It is more difficult, the medium is expensive, and the use cost is extremely high, which limits the large-scale application. Although traditional methods such as ion exchange chromatography and hydrophobic int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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