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Mixed Mode Chromatography Media with Carboxyl and Indolyl Functional Groups

A chromatographic medium and functional group technology, applied in peptide preparation methods, chemical instruments and methods, animal/human proteins, etc., can solve the problems of reduced adsorption efficiency, high cost, and extremely high purity requirements, and achieve high adsorption capacity and selectivity, convenient cleaning and regeneration, and large adsorption capacity

Active Publication Date: 2020-09-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, human serum albumin used in clinical practice is separated from plasma, and there are risks such as pathogen infection, and domestic plasma is limited, resulting in tight supply of human serum albumin products
The use of genetic engineering technology to produce recombinant human albumin has good application prospects, but the purity requirements are extremely high, separation is difficult, and the cost is high
[0003] Patent CN1191545 reports the use of cation exchange medium SP Sepharose FF to capture recombinant human serum albumin from yeast fermentation broth. Due to the poor salt tolerance of the ion exchange medium, it is necessary to dilute the yeast fermentation broth until the conductivity is lower than 5.5mS / cm, resulting in protein concentration Low, large processing capacity, reduced adsorption efficiency

Method used

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  • Mixed Mode Chromatography Media with Carboxyl and Indolyl Functional Groups
  • Mixed Mode Chromatography Media with Carboxyl and Indolyl Functional Groups
  • Mixed Mode Chromatography Media with Carboxyl and Indolyl Functional Groups

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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, and place it in a shaker at 25° C. at 150 rpm for 24 hours. Filter, wash with deionized water, obtain the activated agarose gel; Get the activated agarose gel of 10g, add 5g N-bromosuccinimide and 15g 50% (v / v) acetone, place in 25 ℃, 150rpm shaker reaction for 3 hours, suction filtration, washed with deionized water to obtain bromoalcoholated agarose gel; take 10g of bromoalcoholated agarose gel, add 3g (1H-indole-3 -carbonyl)cysteine ​​and 60g 1M sodium carbonate solution, placed in 25 ℃, 150rpm shaker, reacted for 48 hours, suction filtered, washed with deionized water, to obtain agarose gel as matrix, (1H-indole -3-carbonyl) cysteine ​​is a mixed-mode chromatography medium with ligands, and the density of ligands is 223 μmol / ml.

Embodiment 2

[0030] Take 10 g of drained agarose gel, add 5 g of 20% (v / v) dimethyl sulfoxide, 5 g of allyl bromide and 2.5 g of sodium hydroxide, and place it in a shaker at 25 ° C and 150 rpm for 24 hours to react. Suction filtration, wash with deionized water, obtain the agarose gel of activation; Get the agarose gel of 10g activation, add 2.5g N-bromosuccinimide and 15g 50% (v / v) acetone, place React in a shaker at 25°C and 150rpm for 3 hours, filter with suction, wash with deionized water to obtain bromoalcoholated agarose gel; take 10 g of bromoalcoholated agarose gel, add 1.5 g (1H-ind Indole-3-carbonyl)cysteine ​​and 30g 1M sodium carbonate solution were placed in a shaker at 25°C and 150rpm to react for 48 hours, filtered by suction, and washed with deionized water to obtain agarose gel as matrix, (1H -Indole-3-carbonyl)cysteine ​​is a mixed-mode chromatography medium with ligands, and its ligand density is 115 μmol / ml.

Embodiment 3

[0032] Take 10 g of drained agarose gel, add 1 g of 20% (v / v) dimethyl sulfoxide, 1 g of allyl bromide and 0.5 g of sodium hydroxide, and place it in a shaker at 25° C. at 150 rpm for 24 hours. Suction filtration, wash with deionized water, obtain the agarose gel of activation; Get the agarose gel of 10g activation, add 1g N-bromosuccinimide and 15g 50% (v / v) acetone, place React in a shaker at 25°C and 150 rpm for 3 hours, filter with suction, and wash with deionized water to obtain bromoalcoholated agarose gel; take 10 g of bromoalcoholated agarose gel, add 1 g (1H-indole- 3-carbonyl)cysteine ​​and 20g of 1M sodium carbonate solution were placed in a shaker at 25°C and 150rpm to react for 48 hours, filtered by suction, and washed with deionized water to obtain agarose gel-based, (1H-indole Indole-3-carbonyl) cysteine ​​is a mixed-mode chromatography medium with a ligand, and its ligand density is 50 μmol / ml.

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Abstract

The invention discloses a mixed mode chromatography medium with carboxyl and indolyl as functional groups. The chromatography medium comprises a chromatography matrix and a ligand, wherein the chromatography matrix is a hydrophilic porous microsphere with hydroxyl, and the ligand is (1H-indole-3-carbonyl) cysteine, (2-(1H-indole-3-yl)acetyl) cysteine, (3-(1H-indole-3-yl)propionyl) cysteine, or (4-(1H-indole-3-yl)butyryl) cysteine which are subjected to coupling after allyl bromide activation. The mixed mode chromatography medium has the advantages of large adsorption capacity for human blood albumin, high selectivity, and good salt tolerance, the preparation is simple and convenient, properties are stable, and the chromatography medium can be used for separating human blood albumin.

Description

technical field [0001] The invention relates to a mixed-mode chromatographic medium with carboxyl and indolyl as functional groups, which belongs to protein chromatographic separation technology in the field of biochemical industry. Background technique [0002] Human albumin is the most abundant protein in human plasma. It has the functions of maintaining blood osmotic pressure, transporting endogenous and exogenous substances, regulating fatty acids, hormones, and drugs in blood, and has important uses in clinical treatment. , for the treatment of hypoproteinemia, blood loss, trauma, burns, cerebral edema, liver cirrhosis, etc. At present, human serum albumin used in clinical practice is separated from plasma, and there are risks such as pathogen infection, and domestic plasma is limited, resulting in tight supply of human serum albumin products. The use of genetic engineering technology to produce recombinant human albumin has good application prospects, but the purity r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/285B01D15/08C07K14/765C07K1/16B01J20/30
CPCB01D15/08B01J20/22B01J20/24B01J20/285C07K14/765
Inventor 林东强葛程童褚文宁姚善泾
Owner ZHEJIANG UNIV