Strong cation exchange chromatographic matrix and method for using same

Abstract

A cation exchange chromatographic matrix comprising a base material, and a copolymer with one monomer unit having at least a sulfonic acid group, the copolymer being immobilized on the base material, wherein: the copolymer forms substantially no cross-linked structure, and the copolymer comprises neither acrylamide nor an acrylamide derivative as a monomer unit, or comprises acrylamide or an acrylamide derivative as a monomer unit in a range which has no substantial influence; the ratio of the mass of the copolymer to the mass of the base material is 5% or more and 200% or less; and the density of the sulfonic acid group is higher than 30 mmol/L and 200 mmol/L or lower.

1 . A cation exchange chromatographic matrix comprising
 a base material, and   a copolymer with one monomer unit having at least a sulfonic acid group, the copolymer being immobilized on the base material, wherein:   the copolymer forms substantially no cross-linked structure, and the copolymer comprises neither acrylamide nor an acrylamide derivative as a monomer unit, or comprises acrylamide or an acrylamide derivative as a monomer unit in a range which has no substantial influence;   the ratio of the mass of the copolymer to the mass of the base material is 5% or more and 200% or less; and   the density of the sulfonic acid group is higher than 30 mmol/L and 200 mmol/L or lower.   
     
     
         2 . The cation exchange chromatographic matrix according to  claim 1 , wherein the molar ratio of the monomer unit having the sulfonic acid group in the copolymer is smaller than the molar ratio of a neutral monomer unit having no charge. 
     
     
         3 . The cation exchange chromatographic matrix according to  claim 1 , wherein the mass ratio of the monomer unit having the sulfonic acid group in the copolymer is smaller than the mass ratio of a neutral monomer unit having no charge. 
     
     
         4 . The cation exchange chromatographic matrix according to  claim 1 , wherein the monomer unit having the sulfonic acid group is a glycidyl methacrylate derivative. 
     
     
         5 . The cation exchange chromatographic matrix according to  claim 2 , wherein the neutral monomer unit comprises at least a hydrophilic monomer unit, and the molar ratio of the hydrophilic monomer unit to the neutral monomer unit in the copolymer is 50% or more. 
     
     
         6 . The cation exchange chromatographic matrix according to  claim 2 , wherein the neutral monomer unit comprises at least a hydrophilic monomer unit, and the ratio of the mass of the hydrophilic monomer unit to the total mass of the neutral monomer unit in the copolymer is 50% or more. 
     
     
         7 . The cation exchange chromatographic matrix according to  claim 1 , wherein the cation exchange chromatographic matrix contains no carboxyl group, or has a density of a carboxyl group lower than the density of the sulfonic acid group. 
     
     
         8 . The cation exchange chromatographic matrix according to  claim 1 , wherein the base material is in a membrane form. 
     
     
         9 . A cation exchange chromatographic matrix for biomolecule purification, comprising a cation exchange chromatographic matrix according to  claim 1 . 
     
     
         10 . The cation exchange chromatographic matrix for biomolecule purification according to  claim 9 , wherein the cation exchange chromatographic matrix for biomolecule purification is used for antibody protein purification. 
     
     
         11 . A purification method for purifying a physiologically active substance from a mixed solution containing impurities and the physiologically active substance, comprising
 contacting the mixed solution with a cation exchange chromatographic matrix for biomolecule purification according to  claim 9 .   
     
     
         12 . The purification method according to  claim 11 , wherein the mixed solution is contacted with the matrix in a flow-through mode. 
     
     
         13 . The purification method according to  claim 12 , wherein the pH of the mixed solution is 4.0 or higher and 10.0 or lower. 
     
     
         14 . The purification method according to  claim 11 , wherein the physiologically active substance is a monomer of an antibody protein. 
     
     
         15 . The purification method according to  claim 11 , wherein the impurities include dimeric and higher aggregates of the antibody protein. 
     
     
         16 . The purification method according to  claim 11 , wherein the recovery rate of the physiologically active substance is 80% or more. 
     
     
         17 . The purification method according to  claim 11 , wherein 100 mg or more of the antibody protein including the monomer and the aggregates is purified per mL of the cation exchange chromatographic matrix for biomolecule purification. 
     
     
         18 . The purification method according to  claim 11 , wherein the ratio of the aggregates is reduced by 50% or more when a solution of the antibody protein including the monomer and the aggregates is purified. 
     
     
         19 . The purification method according to  claim 11 , further comprising performing purification using an anion exchange chromatographic matrix before or after the purification step using the cation exchange chromatographic matrix. 
     
     
         20 . The purification method according to  claim 19 , wherein the anion exchange chromatographic matrix is in a membrane form. 
     
     
         21 . The purification method according to  claim 19 , wherein the purification using an anion exchange chromatographic matrix is a flow-through mode. 
     
     
         22 . The purification method according to  claim 19 , further comprising an affinity chromatography step before the purification step using the cation exchange chromatographic matrix and the purification step using the anion exchange chromatographic matrix. 
     
     
         23 . The purification method according to  claim 19 , wherein buffer replacement is not performed in a series of purification steps. 
     
     
         24 . The purification method according to  claim 22 , wherein the affinity chromatography step is carried out in a bind and elute mode, and the physiologically active substance is eluted with a buffer consisting of a monovalent acid in the elution step. 
     
     
         25 . The purification method according to  claim 24 , wherein the electrical conductivity of the buffer consisting of a monovalent acid is 10.0 mS/cm or lower. 
     
     
         26 . The purification method according to  claim 22 , further comprising the step of adjusting the pH of the mixed solution to 4.0 or lower after the affinity chromatography step. 
     
     
         27 . The purification method according to  claim 19 , wherein the electrical conductivity of the mixed solution containing the physiologically active substance is 10 mS/cm or lower in a series of purification steps.