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Mix-mode antibody affinity separation matrix and purification method using same, and target molecule

A separation matrix and mixed-mode technology, which is applied in the field of affinity separation matrix and new mixed-mode antibody affinity separation matrix, can solve the problems of low separation performance, limitation of aggregate removal process, low separation performance of monomer and aggregate, etc. Achieve the effects of reducing load, improving purity, and improving selective separation characteristics

Pending Publication Date: 2015-05-06
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are difficult to perform strict control, and the separation performance is low, so they cannot be used as general separation methods
[0010] As mentioned above, although the antibody affinity separation matrix shows high specificity for antibodies and can achieve high purification, even if the method of use is carefully set, the separation performance of monomers and aggregates is low, so as aggregates There are restrictions on the removal process

Method used

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  • Mix-mode antibody affinity separation matrix and purification method using same, and target molecule
  • Mix-mode antibody affinity separation matrix and purification method using same, and target molecule
  • Mix-mode antibody affinity separation matrix and purification method using same, and target molecule

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0094] Preparation of protein A immobilization carrier

[0095] Take the Low density Glyoxal 4 Rapid Run (ABT company) that is 4mL amount of wet volume in the reaction vessel as 4% agarose beads, after making the slurry volume reach 5mL with water, add 0.25M sodium citrate (pH3. 5) 1 mL of solution. Then 2 mL of 0.8 M sodium periodate was added, and the mixture was stirred at room temperature for 0.5 hours to obtain a carrier with an aldehyde group introduced therein. The carrier slurry was sufficiently washed with water and Dulbecco's PBS(-) (Nissui) (hereinafter referred to as PBS) as a phosphate buffer solution, and the amount of the slurry was recovered to 5 mL. Then, 5 mL of a mixed solution (pH 6.8) of 0.1 M sodium phosphate, 1 M sodium citrate, and 0.3 M sodium chloride was added, and after mixing, the liquid volume was adjusted to 5.5 mL. 5N aqueous sodium hydroxide solution was added thereto to adjust the pH of the carrier slurry to 11.5-12, then 100 mg of protein A...

preparation example 2

[0109] Preparation of Mixed Mode Antibody Affinity Separation Matrix by Immobilizing Protein A on a Carboxyl-Introduced Carrier

[0110] Take Low density Glyoxal4Rapid Run (ABT Company) with a wet volume of 4 mL in the reaction vessel as 4% agarose beads. After making the slurry volume to 5 mL with water, add 0.25M sodium citrate (pH3.5) Solution 1 mL. Then, 1 mL of a mixed solution (pH 3.5) of 0.1 M citric acid and 0.1 M glutamic acid was added and stirred. Further, 0.5 mL of 0.8 M sodium periodate was added, and the mixture was stirred at room temperature for 1 hour to introduce aldehyde groups. The carrier slurry was washed 5 times with 1 M glutamic acid / PBS (pH 7) diluted 100 times with cold water, and the liquid volume of the slurry was adjusted to 5 mL after recovery. 5 mL of 1M glutamic acid / PBS (pH 7) was added thereto, and after inverting and stirring at room temperature for 2 hours, 0.5 mL of 1M dimethylamine borane aqueous solution was added thereto, and inverting...

preparation example 3

[0125] Preparation of Mixed Mode Antibody Affinity Separation Matrix by Immobilizing Carboxyl Groups on Protein A-Introduced Carriers

[0126]As a protein A immobilization carrier, carboxyl groups were introduced into MabSelect SuRe (GE Healthcare Bio-Sciences, carrier 3) replaced with 0.5M saline. MabSelect SuRe was used as carrier 3, and 4 mL of carrier 3 was taken in the reaction vessel in terms of wet volume, and after the slurry volume reached 5 mL, 1 mL of 0.25 M sodium citrate (pH 3.5) solution was added. Then, 1 mL of 0.1 M citric acid and 0.1 M glutamic acid (pH 3.5) were added and stirred. 0.5 mL of 0.8 M sodium periodate was added thereto, and the mixture was stirred at room temperature for 1 hour to introduce aldehyde groups. The carrier slurry was washed 5 times with 1 M glutamic acid / PBS (pH 7) diluted 100 times with cold water, and the liquid volume of the slurry was adjusted to 5 mL after recovery. 5 mL of 1M glutamic acid / PBS (pH 7) was added thereto, and af...

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Abstract

A mix-mode antibody affinity separation matrix has such a structure that an antibody affinity ligand and a cation exchange group are arranged in a single separation matrix. In a first chromatography step in a process for purifying an antibody or an Fc-containing target molecule, the matrix can improve the purity of the antibody which is the main target substance of the affinity purification, can also improve the selective separation properties of monomers, and can reduce the burden on a subsequent impurity removal step with respect to the removal of aggregates.

Description

technical field [0001] The present invention relates to an affinity separation matrix for specifically purifying target molecules, especially a novel mixed-mode antibody affinity separation matrix in which affinity ligands and other ligands can act simultaneously or continuously on a single matrix . A purification method and a target molecule using the separation matrix. Background technique [0002] The affinity ligand has the function of specifically binding to a specific molecule. The affinity separation matrix formed by immobilizing the ligand on a water-insoluble carrier can be used to culture microorganisms and mammals containing biological components or recombinants. Effective separation and purification of useful substances in cells. Examples of antibody affinity ligands that are actually used industrially include: peptidic peptides composed of protein A, protein G, protein L, etc. derived from microorganisms or functionally modified (analogues) obtained by recombin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C07K1/22C07K16/00
CPCC07K1/165B01J39/26C07K1/18C07K1/22C07K16/00
Inventor 水口和信
Owner KANEKA CORP
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