Affinity chromatography medium employing tetrapeptide as functional ligand and preparation method of affinity chromatography medium

Abstract

The invention discloses an affinity chromatography medium employing tetrapeptide as a functional ligand and a preparation method of the affinity chromatography medium. The method comprises the following steps: adding dry matrix and allyl bromide to a dimethyl sulfoxide solution, activating, and reacting activating matrix with N-bromo succinimide; enabling bromo alcoholized matrix to react with hexamethylendiamine to obtain amino activating matrix; sequentially washing with deionized water, absolute ethyl alcohol and anhydrous N,N-dimethyl formamide, adding an N,N-dimethyl formamide solution containing tetrapeptide, 2-(7-azobenzotriazole)-N,N,N',N'-te-tramethyluronium hexafluorophosphate and N,N-diisopropylethylamine, and coupling a tetrapeptide ligand; and putting a medium coupled to tetrapeptide in a mixed liquid of sodium acetate and acetic anhydride to obtain the affinity chromatography medium employing tetrapeptide as the functional ligand. According to the novel chromatography medium developed by the method, a functional group is tetrapeptide composed of tyrosine, phenylalanine, arginine and histidine, and is designed on the basis of a protein A binding site of an antibody Fc segment; the antibody binding selectivity is greatly improved; and the affinity chromatography medium can be applied to efficient separation of an antibody.

Description

technical field

[0001] The invention relates to an affinity chromatography medium with tetrapeptide as a functional ligand and a preparation method thereof, belonging to protein chromatography separation technology in the field of biochemical industry. Background technique

[0002] Antibody refers to the immunoglobulin produced by the body's immune system from plasma cells proliferated and differentiated from B lymphocytes or memory cells under the stimulation of external antigens, and can specifically bind to the corresponding antigen. Antibodies have a variety of biological activities, including specific binding to antigens, activating complement, and binding to cell Fc receptors. The main uses include disease treatment, in vitro diagnosis and detection, and tumor localization imaging.

[0003] Common antibody separation methods include salting out, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The quality requirements o...

Examples

Embodiment 1

[0022] 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 for 48 hours, and filter with suction. Wash with deionized water to obtain an activated chromatography matrix; then mix the activated matrix and 5g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 180rpm at 30°C for 1 hour, filter with suction, and use deionized Wash with water; then mix the brominated matrix with 3mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 12), react in a shaker at 180rpm at 30°C for 24 hours to obtain the amino-activated matrix; take 1g of the amino-activated matrix, and use Wash with deionized water, absolute ethanol and anhydrous N,N-dimethylformamide, filter with suction, add to 2mL containing 100mg tetrapeptide, 100mg 2-(7-azobenzotriazole)-N,N, N',N'-tetramethyluronium hexafluorophosphate and 62.5 μL of N,N-diisopropylethylamine in ...

Embodiment 2

[0024] Take 10 g of drained agarose gel, add 7.5 mL of 20% (v / v) dimethyl sulfoxide solution, 8 mL of allyl bromide and 4 g of sodium hydroxide, activate in a shaker at 180 rpm at 30 °C for 36 hours, and filter with suction , washed with deionized water to obtain an activated chromatography matrix; then the activated matrix and 4g N-bromosuccinimide were mixed for bromoalcoholization, reacted in a shaker at 180rpm at 30°C for 1 hour, filtered with suction, and used to remove Wash with ionic water; then mix the brominated matrix with 3mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 11), react in a shaker at 180rpm at 30°C for 16 hours to obtain the amino-activated matrix; take 1g of the amino-activated matrix, and use Wash with deionized water, absolute ethanol and anhydrous N,N-dimethylformamide, filter with suction, add to 2mL containing 80mg tetrapeptide, 80mg 2-(7-azobenzotriazole)-N,N ,N',N'-tetramethyluronium hexafluorophosphate and 50 μL N,N-diisopropylethy...

Embodiment 3

[0026]Take 10 g of drained agarose gel, add 5 mL of 20% (v / v) dimethyl sulfoxide solution, 6 mL of allyl bromide and 3 g of sodium hydroxide, activate in a shaker at 180 rpm at 30 ° C for 24 hours, and filter with suction. Wash with deionized water to obtain an activated chromatography matrix; then mix the activated matrix and 3g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 180rpm at 30°C for 1 hour, filter with suction, and use deionized Wash with water; then mix the brominated matrix with 1.8mL hexamethylenediamine and 1M sodium carbonate buffer (pH 11), react in a shaker at 180rpm at 30°C for 12 hours to obtain the amino-activated matrix; take 1g of the amino-activated matrix, and use Wash with deionized water, absolute ethanol and anhydrous N,N-dimethylformamide, filter with suction, add to 2mL containing 60mg tetrapeptide, 60mg 2-(7-azobenzotriazole)-N,N ,N',N'-tetramethyluronium hexafluorophosphate and 37.5 μL N,N-diisopropylethylamine in N,N-dimet...

technical field

[0001] The invention relates to an affinity chromatography medium with tetrapeptide as a functional ligand and a preparation method thereof, belonging to protein chromatography separation technology in the field of biochemical industry. Background technique

[0002] Antibody refers to the immunoglobulin produced by the body's immune system from plasma cells proliferated and differentiated from B lymphocytes or memory cells under the stimulation of external antigens, and can specifically bind to the corresponding antigen. Antibodies have a variety of biological activities, including specific binding to antigens, activating complement, and binding to cell Fc receptors. The main uses include disease treatment, in vitro diagnosis and detection, and tumor localization imaging.

[0003] Common antibody separation methods include salting out, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The quality requirements o...

Examples

Embodiment 1

[0022] 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 for 48 hours, and filter with suction. Wash with deionized water to obtain an activated chromatography matrix; then mix the activated matrix and 5g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 180rpm at 30°C for 1 hour, filter with suction, and use deionized Wash with water; then mix the brominated matrix with 3mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 12), react in a shaker at 180rpm at 30°C for 24 hours to obtain the amino-activated matrix; take 1g of the amino-activated matrix, and use Wash with deionized water, absolute ethanol and anhydrous N,N-dimethylformamide, filter with suction, add to 2mL containing 100mg tetrapeptide, 100mg 2-(7-azobenzotriazole)-N,N, N',N'-tetramethyluronium hexafluorophosphate and 62.5 μL of N,N-diisopropylethylamine in ...

Embodiment 2

[0024] Take 10 g of drained agarose gel, add 7.5 mL of 20% (v / v) dimethyl sulfoxide solution, 8 mL of allyl bromide and 4 g of sodium hydroxide, activate in a shaker at 180 rpm at 30 °C for 36 hours, and filter with suction , washed with deionized water to obtain an activated chromatography matrix; then the activated matrix and 4g N-bromosuccinimide were mixed for bromoalcoholization, reacted in a shaker at 180rpm at 30°C for 1 hour, filtered with suction, and used to remove Wash with ionic water; then mix the brominated matrix with 3mL of hexamethylenediamine and 1M sodium carbonate buffer (pH 11), react in a shaker at 180rpm at 30°C for 16 hours to obtain the amino-activated matrix; take 1g of the amino-activated matrix, and use Wash with deionized water, absolute ethanol and anhydrous N,N-dimethylformamide, filter with suction, add to 2mL containing 80mg tetrapeptide, 80mg 2-(7-azobenzotriazole)-N,N ,N',N'-tetramethyluronium hexafluorophosphate and 50 μL N,N-diisopropylethy...

Embodiment 3

[0026]Take 10 g of drained agarose gel, add 5 mL of 20% (v / v) dimethyl sulfoxide solution, 6 mL of allyl bromide and 3 g of sodium hydroxide, activate in a shaker at 180 rpm at 30 ° C for 24 hours, and filter with suction. Wash with deionized water to obtain an activated chromatography matrix; then mix the activated matrix and 3g of N-bromosuccinimide for bromoalcoholization, react in a shaker at 180rpm at 30°C for 1 hour, filter with suction, and use deionized Wash with water; then mix the brominated matrix with 1.8mL hexamethylenediamine and 1M sodium carbonate buffer (pH 11), react in a shaker at 180rpm at 30°C for 12 hours to obtain the amino-activated matrix; take 1g of the amino-activated matrix, and use Wash with deionized water, absolute ethanol and anhydrous N,N-dimethylformamide, filter with suction, add to 2mL containing 60mg tetrapeptide, 60mg 2-(7-azobenzotriazole)-N,N ,N',N'-tetramethyluronium hexafluorophosphate and 37.5 μL N,N-diisopropylethylamine in N,N-dimet...

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