Affinity chromatography column and preparation method and application thereof

A chromatographic column and affinity technology, applied in the field of separation and purification of biological macromolecules

Active Publication Date: 2022-08-05
杭州华安生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned affinity chromatography column effectively alleviates the clumping / coagulation of the cyanogen bromide-activated agarose chromatography medium caused by the easy precipitation of the ligand after denaturant treatment in the preparation process of the existing cyanogen bromide-activated agarose affinity chromatography column. block problem

Method used

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  • Affinity chromatography column and preparation method and application thereof
  • Affinity chromatography column and preparation method and application thereof
  • Affinity chromatography column and preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0036] According to one aspect of the present invention, a preparation method of the above-mentioned affinity chromatography column, the preparation method comprises the following steps:

[0037] The denaturant-treated biomacromolecules containing amino ligands, the cyanogen bromide-activated agarose chromatography filler and the surfactant are coupled and incubated in the chromatography column empty column to obtain a reaction column with a stationary phase, and then The reaction column is washed and blocked successively to obtain an affinity chromatography column.

[0038] The present invention provides a method for preparing an affinity chromatography column. The preparation method comprises the steps of placing the amino ligand-containing biological macromolecule treated with a denaturant, a cyanogen bromide-activated agarose chromatography filler and a surfactant in the empty space of the chromatography column. The coupling incubation reaction is carried out in the column...

Embodiment 1

[0046] 1. Prepare the solution:

[0047] 1. Amino-containing ligand solution: The denaturant used in this example is 4M urea, the protein dosage is 2mg, and the concentration is 1mg / ml.

[0048] The specific preparation method is as follows: the protein concentration of the amino-containing ligand to be coupled is 2 mg / ml, the dosage is 2 ml, and the protein buffer solution is 4M urea, pH 7.4.

[0049] 2. 1mM hydrochloric acid solution with pH 3.0: Take 0.1 mL of concentrated hydrochloric acid, add 1.1 mL of UP water, and mix well to prepare 1 M dilute hydrochloric acid. Then take 1 mL of 1M dilute hydrochloric acid, dilute it to 1L, and prepare 1 mM dilute hydrochloric acid, and measure the pH to 3.0.

[0050] 3. 0.1M Tris-HCl blocking solution with pH 8.0: Weigh 6.057 g of Tris and dissolve it in 400 mL of UP water, adjust the pH to 8.0 with HCl and make up to 500 mL.

[0051] 4. Circulating buffer solution A: Weigh 4.1 g of anhydrous CH 3 COONa, 14.61g of NaCl was dissol...

experiment example 1

[0066] In order to show that the affinity chromatography column prepared by the present application can effectively alleviate the easy precipitation of the ligand after the denaturant treatment in the preparation process of the existing cyanogen bromide activated agarose affinity chromatography column, resulting in the cyanogen bromide activated agarose layer. For the problem of agglomeration / clumping of the chromatographic medium, the affinity chromatography columns prepared in Example 1 and Comparative Example 1 are used to chromatographically separate serum and purified IgG antibodies, and the purified serum and IgG antibodies are subjected to chromatographic separation. ELISA test, as follows:

[0067] (1), sample acquisition method: provide amino-containing ligand with a protein concentration of 1 mg / ml, take 0.5 ml of vial and immune adjuvant (complete adjuvant for the first immunization of rabbits, and incomplete adjuvant for subsequent immunization) in a volume ratio of...

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Abstract

The invention provides an affinity chromatography column, a preparation method and application thereof, and relates to the technical field of separation and purification of biological macromolecules. The stationary phase of the above-mentioned affinity chromatography column is mainly prepared from the biological macromolecules containing amino ligands treated with a denaturant, a cyanogen bromide-activated agarose chromatography filler and a surfactant through a coupling incubation reaction. The above-mentioned affinity chromatography column can effectively promote the coupling of the amino ligand-containing biomacromolecule treated with the denaturant and the cyanogen bromide-activated agarose chromatography packing by adding a surfactant to the stationary phase, and effectively alleviate the problem. The problem of agglomeration and agglomeration of the cyanogen bromide activated agarose chromatography medium caused by the easy precipitation of the ligand after the denaturant treatment in the preparation process of the existing cyanogen bromide activated agarose affinity chromatography column is solved.

Description

technical field [0001] The invention relates to the technical field of separation and purification of biological macromolecules, in particular to an affinity chromatography column and a preparation method and application thereof. Background technique [0002] At present, other ligands containing amino groups such as proteins, polypeptides and nucleic acids are coupled to the purification medium fillers on the market, mainly using cyanogen bromide to activate the agarose chromatography medium. The cyanogen bromide activated agarose chromatography medium is an activated intermediate filler, which generates activated cyanate groups through the reaction between cyanogen bromide and the hydroxyl groups on agarose, and the activated cyanate groups interact with proteins, Molecules with amino groups such as polypeptides and amino acids are coupled, and the coupling reaction can be carried out in either an aqueous phase or an organic phase. [0003] At present, the cyanogen bromide...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/20B01D15/38C07K16/06C07K1/22
CPCB01D15/206B01D15/3804C07K16/065C07K1/22
Inventor 徐晓勇田向荣张丰
Owner 杭州华安生物技术有限公司
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