Provided is an affinity chromatography carrier having an excellent purification purity, including a substrate, a hydrophilic polymer, and an affinity ligand, in which the substrate is constituted of at least one selected from the group consisting of a polysaccharide, an acrylate-based polymer, a methacrylate-based polymer and a styrene-based polymer, the hydrophilic polymer is at least one selected from the group consisting of hydrophilic polysaccharides, the affinity ligand is at least one selected from the group consisting of an antibody-binding protein and an antibody-binding polypeptide, a carboxy group is introduced into the affinity chromatography carrier, and the amount of the carboxy group introduced is 15 mmol/L-gel to 60 mmol/L-gel in terms of ion exchange capacity.

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO 51 or SEQ ID NO 52, wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

An Fc-binding polypeptide of improved alkali stability, comprising a mutant of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO 51 or SEQ ID NO 52, wherein at least the asparagine or serine residue at the position corresponding to position 11 in SEQ ID NO: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

The subject invention pertains to proteins are purified by a mixed-mode chromatography system formed by attaching a ligand with cation exchange and hydrophobic 1,3-dioxoisoindolin-2-yl group functionalities to a large-pore support matrix, the only linkage between the ligand and the support matrix being a chain having a backbone of one, two, three, four, or five atoms between the hydrophobic group and the support matrix.

The subject invention pertains to proteins are purified by a mixed-mode chromatography system formed by attaching a ligand with cation exchange and hydrophobic 1,3-dioxoisoindolin-2-yl group functionalities to a large-pore support matrix, the only linkage between the ligand and the support matrix being a chain having a backbone of one, two, three, four, or five atoms between the hydrophobic group and the support matrix.

The present invention relates to composite materials useful for purifying proteins obtained from biological feedstocks. The composite materials of the invention comprise a porous support having an average pore size of 5 to 500 nm, said porous support being filled with a polymer which is cross-linked, wherein the polymer is selected from polyvinylamines or polyallylamines having a weight average molecular weight (Mw) of 2,000 to 500,000 Da and a hydrolysis degree of the formamide groups of at least 66%, with the proviso that a polyvinylamine having a weight average molecular weight (Mw) of 27,200 Da and a hydrolysis degree of 70% and a polyvinylamine having a weight average molecular weight (Mw) of 50,000 Da and a hydrolysis degree of 95% are excluded.

The present invention relates to composite materials useful for purifying proteins obtained from biological feedstocks. The composite materials of the invention comprise a porous support having an average pore size of 5 to 500 nm, said porous support being filled with a polymer which is cross-linked, wherein the polymer is selected from polyvinylamines or polyallylamines having a weight average molecular weight (Mw) of 2,000 to 500,000 Da and a hydrolysis degree of the formamide groups of at least 66%, with the proviso that a polyvinylamine having a weight average molecular weight (Mw) of 27,200 Da and a hydrolysis degree of 70% and a polyvinylamine having a weight average molecular weight (Mw) of 50,000 Da and a hydrolysis degree of 95% are excluded.

A chromatography column for the use of separation of acidic sugar chains, wherein the column comprises a first column and a second column, the second column connected by a flow path downstream of an outlet of the first column, and selected from the following (1) or (2): (1) the carrier of the first column is hydrophobically modified silica having a group containing a primary amine, a secondary amine or/and a tertiary amine, and the carrier of the second column is a resin having a group containing a primary amine, a secondary amine or/and a tertiary amine; (2) the carrier of the first column is a resin having a group containing a primary amine, a secondary amine or/and a tertiary amine, and the carrier of the second column is hydrophobically modified silica having a group containing a primary amine, a secondary amine, or/and a tertiary amine.

A chromatography column for the use of separation of acidic sugar chains, wherein the column comprises a first column and a second column, the second column connected by a flow path downstream of an outlet of the first column, and selected from the following (1) or (2): (1) the carrier of the first column is hydrophobically modified silica having a group containing a primary amine, a secondary amine or/and a tertiary amine, and the carrier of the second column is a resin having a group containing a primary amine, a secondary amine or/and a tertiary amine; (2) the carrier of the first column is a resin having a group containing a primary amine, a secondary amine or/and a tertiary amine, and the carrier of the second column is hydrophobically modified silica having a group containing a primary amine, a secondary amine, or/and a tertiary amine.

A method of separating biomolecules in an aqueous mixture is disclosed comprising a obtaining a separation vessel containing separation media, wherein the separation media comprises a porous support with a hydrophobic monomer grafted thereon, the hydrophobic monomer having the structure:

CH.sub.2CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O)XR.sup.3

wherein n is an integer of 0 or 1; R.sup.1 is independently selected from at least one of: a hydrogen atom, alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total of 10 carbon atoms or less; R.sup.3 is a hydrophobic group selected from at least one of alkyls, aryls, alkylaryls and ethers, wherein the alkyls, aryls, alkylaryls and ethers have a total number of carbon atoms ranging from 4 to 30; R.sup.4 is H or CH.sub.3; and X is O or NH; wherein the hydrophobic monomer is derived from an amine or an alcohol (HXR.sup.3) that has a hydrophilicity index of 25 or less; and
(b) passing the aqueous mixture through the separation vessel thereby separating the biomolecules. Such methods can be used to separate proteins, antibodies, fusion proteins, vaccines, peptides, enzymes, DNA, and/or RNA.