# US 9,040,661 B2 · Support for affinity chromatography and method for isolating immunoglobulin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,661 B2 - Document ID: US9040661B2 - Status: Granted - Kind: B2 - Application number: 13/996,978 - Filed: 2011-12-20 - Published: 2014-01-02 - Granted: 2015-05-26 - Earliest claimed: 2010-12-21 - Assignees: Jsr Corporation; The University of Western Ontario - Applicants: Satoshi Nakamura; Tetsuo Fukuta; Yusuke Okano; Tomonori Shiotani; Kouji Tamori; Yu Otani; Shun-Cheng Li; Xing Li; Courtney Voss - Inventors: Satoshi Nakamura; Tetsuo Fukuta; Yusuke Okano; Tomonori Shiotani; Kouji Tamori; Yu Otani; Shun-Cheng Li; Xing Li; Courtney Voss - Web page: https://patentgrep.com/patent/US9040661B2 ## CPC - C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes - B01D 15/3804: {Affinity chromatography} - B01J 20/28004: {Sorbent size or size distribution, e.g. particle size} - B01J 20/28057: {Surface area, e.g. B.E.T specific surface area} - B01J 20/28069: {Pore volume, e.g. total pore volume, mesopore volume, micropore volume} - B01J 20/28078: {Pore diameter} - B01J 20/288: Polar phases - B01J 20/321: {consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds} - B01J 20/3217: {Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond} - B01J 20/3274: {Proteins, nucleic acids, polysaccharides, antibodies or antigens} - B01J 20/3278: {Polymers being grafted on the carrier} - B01J 20/3293: {Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers} - C07K 16/065: {Purification, fragmentation} - C07K 17/08: the carrier being a synthetic polymer - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS ## Abstract Provided are a support for affinity chromatography which has excellent alkali resistance, and a method for isolating immunoglobulin. A support for affinity chromatography, containing an immobilized protein ligand represented by the following formula (1): R—R2 (1) wherein R represents a polypeptide consisting of 4 to 30 amino acid residues that contains an amino acid sequence represented by ATK or ASK; and R2 represents a polypeptide consisting of 50 to 500 amino acid residues containing an immunoglobulin-binding domain consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the partial sequence thereof, or an amino acid sequence having 70% or more identity to these sequences; with the proviso that a terminus at which R2 binds to R is C-terminus or N-terminus of the immunoglobulin-binding domain. ## Claims ### Claim 1 A support immobilizing a protein ligand comprising: an anchor peptide consisting of 4 to 30 amino acid residues containing an amino acid sequence represented by ATK or ASK; and an immunoglobulin binding domain consisting of the amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or at least 90% identical to SEQ ID NO: 2 and that is no longer than 500 amino acid residues; wherein the C- or N-terminus of said immunoglobulin binding domain is bound to said anchor peptide and the anchor peptide is bound to the support, optionally via a spacer; and wherein the anchor peptide comprises the sequence XX-UU-XX (SEQ ID NO:48), where X is K, S or T, and U is an optional amino acid residue which may be any amino acid residue, wherein the anchor peptide comprises the sequence XXX-GA-XX (SEQ ID NO: 49), where X is A, K, S or T, and/or wherein the anchor peptide comprises ASK, ASKATK (SEQ ID NO: 45), ATKASK (SEQ ID NO: 44), or ATKATK (SEQ ID NO: 47); and wherein the support has a dynamic binding capacity for IgG that is higher than an otherwise identical support not comprising said anchor peptide after the support is treated with 0.5 M sodium hydroxide for 15 hrs. ### Claim 2 (depends on 1) The support according to claim 1, wherein the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is the amino acid sequence represented by SEQ ID NO: 3. ### Claim 3 (depends on 1) The support according to claim 1, wherein the anchor peptide contains the amino acid sequence represented by a combination of ATK and ASK. ### Claim 4 (depends on 1) The support according to claim 1, wherein the anchor peptide contains the amino acid sequence represented by ATKASK (SEQ ID NO: 44) or ASKATK (SEQ ID NO: 45). ### Claim 5 (depends on 1) The support according to claim 1, wherein the anchor peptide contains the amino acid sequence represented by ASKASK (SEQ ID NO: 46). ### Claim 6 (depends on 1) The support according to claim 1, wherein the anchor peptide contains the amino acid sequence represented by ATKATK (SEQ ID NO: 47). ### Claim 7 (depends on 1) The support according to claim 1, wherein the protein ligand is immobilized on the support via epoxy group ring-opening reaction between an amino group or a thiol group in the ligand and an epoxy group in the support. ### Claim 8 (depends on 1) The support of claim 1 that is an affinity chromatography support. ### Claim 9 (depends on 8) The support of claim 8 that is in a particulate form. ### Claim 10 (depends on 8) The support of claim 8 that comprises synthetic polymer particles having a particle size ranging from 20 μm to 200 μm. ### Claim 11 (depends on 8) The support of claim 8 that comprises synthetic polymer particles having a particle size ranging from 100 nm to 400 nm. ### Claim 12 (depends on 8) The support of claim 8 that comprises porous particles having a specific surface area of 50 m2/g to 150 m2/g. ### Claim 13 (depends on 8) The support of claim 8 that has a dynamic binding capacity (“DBC”) for human polyclonal IgG ranging from 40 to 45 after contact with 0.5 M sodium hydroxide for 15 hrs. ### Claim 14 (depends on 8) The support of claim 8 that has a retention ratio of the immunoglobulin binding domain ranging from 89.0 to 96.8 after contact with 0.5 M sodium hydroxide for 15 hrs. ### Claim 15 (depends on 8) The support of claim 8 that comprises polysaccharide particles having a particle size ranging from 50 μm to 200 μm. ### Claim 16 (depends on 8) The support of claim 8 that comprises polysaccharide particles having a particle size ranging from 500 nm to 1,400 nm. ### Claim 17 (depends on 8) The support of claim 8, wherein said protein ligand ranges in length from 70 to 1,000 amino acid residues. ### Claim 18 (depends on 8) The support of claim 8, wherein said protein ligand ranges in length from 80 to 600 amino acid residues. ### Claim 19 (depends on 1) The support of claim 1, wherein the immunoglobulin binding domain comprises G29A or an alanine substitution at a residue corresponding to G29. ### Claim 20 (depends on 1) A method for isolating an immunoglobulin comprising: applying a sample containing immunoglobulin through the support for affinity chromatography according to claim 1 to adsorb the immunoglobulin to the support; and eluting the immunoglobulin from the support. ### Claim 21 (depends on 7) A method for isolating an immunoglobulin comprising: applying a sample containing immunoglobulin through the support according to claim 7 to adsorb the immunoglobulin to the support; and eluting the adsorbed immunoglobulin from the support.