Combined ligand, combined bionic chromatography medium and preparation method and application thereof

Abstract

The invention relates to a combined ligand, a combined bionic chromatography medium and a preparation method and application thereof. The combined bionic chromatography medium uses hydrophilic porousmicrospheres as chromatography matrix, is activated by allyl bromide, is brominated and alcoholized by N-bromosuccinimide, and then is coupled with the combined ligand. The sequence of the combined ligand is phenylalanine-tyrosine-glutamic acid-5-aminobenzimidazole. The combined bionic chromatography medium has two functional groups including phenylalanine-tyrosine-glutamic acid tripeptide and aminobenzimidazole, introduces a hydrophobicity charge-inducing ligand while retaining the high selectivity characteristic of a polypeptide ligand to an antibody, makes elution conditions milder, and achieves effective antibody isolation.

Description

technical field

[0001] The invention relates to the field of bionic chromatography, in particular to a combination ligand, a combination bionic chromatography medium and a preparation method and application thereof. Background technique

[0002] Antibodies have strong targeting and high biocompatibility, and have great potential to be developed into drugs for the treatment of cancer. With the continuous development of antibody engineering, the scale of upstream antibody expression and preparation continues to increase, and the development of efficient downstream separation and purification technology is the key to the development of the antibody industry.

[0003] At present, protein A affinity chromatography is the most commonly used antibody capture method, which has high selectivity, but the medium is expensive, the elution conditions are harsh, and there is a risk of ligand shedding. Therefore, the development of alternative technologies has become a current hotspot, an...

Examples

Embodiment 1

[0054] Embodiment 1: Preparation of combined ligand

[0055] With the help of computer molecular simulation, analyze and evaluate the key residues of protein A and antibody Fc binding site, screen and design the combinatorial ligand of tripeptide-heterocyclic small molecule, whose sequence is phenylalanine-tyramine Acid-glutamic acid-5-aminobenzimidazole.

[0056] Combined ligand, the structural formula is as follows:

[0057]

[0058] The combinatorial ligand can be synthesized by the chemical synthesis method in the prior art, and the combinatorial ligand in this example is entrusted to China Peptide Biochemical Co., Ltd. to prepare it.

[0059] Carry out high performance liquid chromatography characterization for the combined ligand in embodiment 1, such as figure 1 shown.

Embodiment 2

[0060] Example 2: Preparation of Combined Biomimetic Chromatography Medium

[0061] Take 5.0 g of drained agarose gel, add 5.0 g of 20% (v / v) dimethyl sulfoxide, 2.5 g of allyl bromide and 1.0 g of sodium hydroxide, activate it on a shaker at 150 rpm at 30 ° C for 24 hours, and pump Filter and wash with deionized water to obtain an activated chromatography matrix.

[0062] Mix the activated chromatographic matrix, 10.0g 50% (v / v) acetone and 1.5g N-bromosuccinimide for bromoalcoholization, react on a shaking table at 150rpm at 30°C for 3h, filter with suction, and use deionized Washing with water yields a bromoalcoholated substrate.

[0063] Mix 2.5g dimethyl sulfoxide and 5.0g 1M sodium carbonate buffer, add 0.5g phenylalanine-tyrosine-glutamic acid-5-aminobenzimidazolidine, fully dissolve, then add bromoalcohol The chromatographic matrix was reacted on a shaking table at 150 rpm at 30°C for 12 hours, and was repeatedly washed with deionized water, 0.1M HCl, and 0.1M NaOH t...

Embodiment 3

[0067] Example 3: Preparation of Combined Biomimetic Chromatography Medium

[0068] Take 5.0 g of drained agarose gel, add 2.5 g of 20% (v / v) dimethyl sulfoxide, 0.5 g of allyl bromide and 0.5 g of sodium hydroxide, activate it on a shaker at 150 rpm at 30 ° C for 24 hours, and pump Filter and wash with deionized water to obtain an activated chromatography matrix.

[0069] Mix the activated chromatographic matrix, 5.0g 50% (v / v) acetone and 0.5g N-bromosuccinimide for bromoalcoholization, react on a shaking table at 150rpm at 30°C for 1h, filter with suction, and use deionized Washing with water yields a bromoalcoholated substrate.

[0070] Mix 3.0g dimethyl sulfoxide and 5.0g 1M sodium carbonate buffer, add 0.5g phenylalanine-tyrosine-glutamic acid-5-aminobenzimidazolidine, fully dissolve, then add bromoalcohol The chromatographic matrix was reacted on a shaking table at 150 rpm at 30°C for 8 hours, and was repeatedly washed with deionized water, 0.1M HCl, and 0.1M NaOH to ...

technical field

[0001] The invention relates to the field of bionic chromatography, in particular to a combination ligand, a combination bionic chromatography medium and a preparation method and application thereof. Background technique

[0002] Antibodies have strong targeting and high biocompatibility, and have great potential to be developed into drugs for the treatment of cancer. With the continuous development of antibody engineering, the scale of upstream antibody expression and preparation continues to increase, and the development of efficient downstream separation and purification technology is the key to the development of the antibody industry.

[0003] At present, protein A affinity chromatography is the most commonly used antibody capture method, which has high selectivity, but the medium is expensive, the elution conditions are harsh, and there is a risk of ligand shedding. Therefore, the development of alternative technologies has become a current hotspot, an...

Examples

Embodiment 1

[0054] Embodiment 1: Preparation of combined ligand

[0055] With the help of computer molecular simulation, analyze and evaluate the key residues of protein A and antibody Fc binding site, screen and design the combinatorial ligand of tripeptide-heterocyclic small molecule, whose sequence is phenylalanine-tyramine Acid-glutamic acid-5-aminobenzimidazole.

[0056] Combined ligand, the structural formula is as follows:

[0057]

[0058] The combinatorial ligand can be synthesized by the chemical synthesis method in the prior art, and the combinatorial ligand in this example is entrusted to China Peptide Biochemical Co., Ltd. to prepare it.

[0059] Carry out high performance liquid chromatography characterization for the combined ligand in embodiment 1, such as figure 1 shown.

Embodiment 2

[0060] Example 2: Preparation of Combined Biomimetic Chromatography Medium

[0061] Take 5.0 g of drained agarose gel, add 5.0 g of 20% (v / v) dimethyl sulfoxide, 2.5 g of allyl bromide and 1.0 g of sodium hydroxide, activate it on a shaker at 150 rpm at 30 ° C for 24 hours, and pump Filter and wash with deionized water to obtain an activated chromatography matrix.

[0062] Mix the activated chromatographic matrix, 10.0g 50% (v / v) acetone and 1.5g N-bromosuccinimide for bromoalcoholization, react on a shaking table at 150rpm at 30°C for 3h, filter with suction, and use deionized Washing with water yields a bromoalcoholated substrate.

[0063] Mix 2.5g dimethyl sulfoxide and 5.0g 1M sodium carbonate buffer, add 0.5g phenylalanine-tyrosine-glutamic acid-5-aminobenzimidazolidine, fully dissolve, then add bromoalcohol The chromatographic matrix was reacted on a shaking table at 150 rpm at 30°C for 12 hours, and was repeatedly washed with deionized water, 0.1M HCl, and 0.1M NaOH t...

Embodiment 3

[0067] Example 3: Preparation of Combined Biomimetic Chromatography Medium

[0068] Take 5.0 g of drained agarose gel, add 2.5 g of 20% (v / v) dimethyl sulfoxide, 0.5 g of allyl bromide and 0.5 g of sodium hydroxide, activate it on a shaker at 150 rpm at 30 ° C for 24 hours, and pump Filter and wash with deionized water to obtain an activated chromatography matrix.

[0069] Mix the activated chromatographic matrix, 5.0g 50% (v / v) acetone and 0.5g N-bromosuccinimide for bromoalcoholization, react on a shaking table at 150rpm at 30°C for 1h, filter with suction, and use deionized Washing with water yields a bromoalcoholated substrate.

[0070] Mix 3.0g dimethyl sulfoxide and 5.0g 1M sodium carbonate buffer, add 0.5g phenylalanine-tyrosine-glutamic acid-5-aminobenzimidazolidine, fully dissolve, then add bromoalcohol The chromatographic matrix was reacted on a shaking table at 150 rpm at 30°C for 8 hours, and was repeatedly washed with deionized water, 0.1M HCl, and 0.1M NaOH to ...

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