Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof

A technology for immunoglobulin and protein separation, which is applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of expensive ligand prices and elution condition constraints, and achieve low prices, improved purity, and process simple effect

Inactive Publication Date: 2008-05-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although antibody chromatography has preliminarily achieved high-capacity adsorption of target products in a wide range of ionic strengths

Method used

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  • Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof
  • Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof
  • Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026]Weigh 1.0g of Sepharose CL-6B, the agarose gel medium Sepharose CL-6B with an average particle size of 90μm, rinsed and drained by a sand filter funnel, placed in a 50mL Erlenmeyer flask, and then added 0.2mL dimethyl sulfoxide, 0.5mL 4mol / L sodium hydroxide solution and 0.3mL allyl bromide; after mixing uniformly, the gel suspension is placed on a water bath shaker and reacted at 25°C for 24 hours to obtain Sepharose CL-6B agarose gel medium with allyl groups . The allyl chromatography medium is rinsed with deionized water to remove free allyl bromide. The allyl modification density of the allyl chromatography medium is 200 μmol / mL.

[0027] Weigh 1.0g of allyl chromatographic medium and place it in 4mL of a mixed solution of dimethylsulfoxide and water with a volume ratio of 1:3; then, add 0.6g of N-bromosuccinimide, mix well and combine Place it on a water bath shaker and react at 25°C for 1 hour; after the reaction medium is rinsed with deionized water and drained, it i...

Embodiment 2

[0029] Weigh 1.0g of Sepharose CL-6B, the agarose gel medium Sepharose CL-6B with an average particle size of 90μm, rinsed and drained by a sand filter funnel, placed in a 50mL Erlenmeyer flask, and then added 0.2mL dimethyl sulfoxide, 0.5mL 4mol / L sodium hydroxide solution and 0.3 mL allyl bromide; after mixing uniformly, the gel suspension is placed on a water bath shaker and reacted at 25° C. for 24 hours to obtain an allyl-containing Sepharose CL-6B agarose gel medium. The allyl chromatography medium is rinsed with deionized water to remove free allyl bromide. The allyl modification density of the allyl chromatography medium is 200 μmol / mL.

[0030] Weigh 1.0g of allyl chromatographic medium and place it in 4mL of a mixed solution of dimethylsulfoxide and water with a volume ratio of 1:3; then, add 0.5g of N-bromosuccinimide, mix well and combine Place it in a water bath shaker and react at 25°C for 1 hour; after the reaction medium is rinsed with deionized water and drained,...

Embodiment 3

[0032] In Example 1, the amount of N-bromosuccinimide added was 1.2g, and the content of 4-(imidazol-1-yl)aniline in the solution was increased to 1.6mmol; under other conditions unchanged, it can be used for immunoglobulin The coupling density of 4-imidazol-1-yl)aniline in the chromatographic medium for protein separation and purification was 123μmol / mL.

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Abstract

The invention relates to a chromatography which is used for isolating and purifying immunoglobulin protein and a preparation method and belongs to the preparation technology of isolating and purifying a chromatography medium of the immunoglobulin. The chromatography medium refers to the end of a space arm that is coupled with a double-loop compound molecular which has an imidazol group and a benzene ring as a chromatography medium matrix. The preparation method uses an allylic chromatography medium and a bromide alchoholization reaction of N-bromosuccinimide to get an activated chromatography medium which reacts with the amino of the double-loop compound to complete the coupling of the matrix in dimethyl sulfoxide solution. The chromatography medium has higher dynamic adsorption capacity to the antibody within the ionic strength scope from 0.02 mol/L to 2.0 mol/L and is directly applied to the recycling of the antibody in the solid to liquid; at he same time, the medium has more moderate elution condition and can purify the antibody from serum, ascites, cell culture supernatant and other solid to liquid having the antibody. The chromatography medium has the wide application prospect in the process of antibody preparation.

Description

Technical field [0001] The invention relates to a chromatographic medium for separating and purifying immunoglobulin proteins and a preparation method thereof, and belongs to a chromatographic medium technology for separating and purifying immunoglobulins. Background technique [0002] In recent years, antibodies have been widely used in medical diagnosis and disease treatment. In 2006, the global sales of more than 20 antibody drugs approved by the FDA exceeded US$17 billion (Am Biotechnol Lab, 24(8): 8 (2006)). The preparation of antibody drugs is usually completed by precipitation, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography and other methods, and the process cost accounts for about 30% to 40% of the production cost. Taking the currently widely used affinity protein A chromatography as an example, although the specific recognition between protein A and the Fc fragment of the antibody makes the antibody separated by protein A chr...

Claims

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Application Information

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IPC IPC(8): B01J20/281C07K1/16
Inventor 史清洪程征孙彦董晓燕白姝
Owner TIANJIN UNIV
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