# US 2013/0253142 A1 · Separating agent for protein purification and protein purification method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0253142 A1 - Document ID: US20130253142A1 - Status: Published application - Kind: A1 - Application number: 13/896,863 - Filed: 2013-05-17 - Published: 2013-09-26 - Earliest claimed: 2008-06-23 - Assignees: Tosoh Corporation - Applicants: Tosoh Corporation - Inventors: Seiji MASUMOTO; Katsuo KOMIYA; Koji NAKAMURA - Web page: https://patentgrep.com/patent/US20130253142A1 ## CPC - C07K 1/16: by chromatography - B01D 15/361: {Ion-exchange} - B01J 20/3208: {Polymeric carriers, supports or substrates} - B01J 20/321: {consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds} - B01J 20/3219: {involving a particular spacer or linking group, e.g. for attaching an active group} - B01J 20/3253: {comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures} - B01J 20/3255: {comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures} - B01J 20/3285: {Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic} - C07C 211/53: having the nitrogen atom of at least one of the amino groups further bound to a hydrocarbon radical substituted by amino groups - C07C 237/20: the carbon skeleton containing six-membered aromatic rings - C07D 213/56: Amides - C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes - C08F 120/06: Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof - B01D 15/327: {with hydrophobic interaction} - C07B 2200/11: Compounds covalently bound to a solid support - 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 - C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) - C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS - C07B: GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR (preparation of carboxylic acid esters by telomerisation C07C67/47; telomerisation C08F) ## Abstract A novel separating agent for protein purification which not only can adsorb proteins in a sufficient amount for protein purification from a low concentration buffer but also can desorb the adsorbed protein easily just by altering the pH of the buffer and a simple and economical method for its production and a method for protein purification using it. One or two ligands selected from the group consisting of a ligand represented by the following formula (1): (wherein m is an integer of from 2 to 6) and a ligand represented by the following formula (2): (wherein each of R1 and R2 is independently a hydrogen atom or a C1-4 alkyl group, and n is an integer of from 1 to 6) are immobilized on a support via a urethane bond without intervention of a spacer arm. ## Claims ### Claim 1 A separating agent for protein purification which is characterized in that one or two ligands selected from the groups consisting of a ligand represented by the following formula (1): (wherein m is an integer of from 2 to 6) and a ligand represented by the following formula (2): (wherein each of R1 and R2 is independently a hydrogen atom or a C1-4 alkyl group, and n is an integer of from 1 to 6) are bonded to a support via a urethane bond. ### Claim 2 (depends on 1) The separating agent for protein purification according to claim 1, wherein the ligand represented by the formula (2) is represented by the following formula (3): (wherein m is an integer of from 2 to 4). ### Claim 3 (depends on 1) The separating agent for protein purification according to claim 1, wherein the ligand represented by the formula (2) is represented by the following formula (4): (wherein each of R1 and R2 is independently a hydrogen atom or a C1-4 alkyl group, and n is an integer of from 1 to 4). ### Claim 4 (depends on 1) The separating agent for protein purification according to claim 1, wherein the amount of the ligand on the support is at least 100 μmol/ml-support. ### Claim 5 (depends on 1) The separating agent for protein purification according to claim 1, wherein the support is made of a crosslinked polymer. ### Claim 6 (depends on 1) A method for producing the separating agent for protein purification as defined in claim 1, which comprises reacting the support with an activator in an organic solvent to activate hydroxyl groups on the surface of the support, and then reacting the activated support with one or two amines selected from the group consisting of a primary amine represented by the following formula (5): (wherein m is an integer of from 2 to 6) and a primary amine represented by the following formula (6): (wherein each of R1 and R2 is independently a hydrogen atom or a C1-4 alkyl group, and n is an integer of from 1 to 6) in an organic solvent. ### Claim 7 (depends on 6) The method for producing the separating agent for protein purification according to claim 6, wherein the primary amine represented by the formula (5) is represented by the following formula (7): (wherein m is an integer of from 2 to 4). ### Claim 8 (depends on 6) The method for producing the separating agent for protein purification according to claim 6, wherein the primary amine represented by the formula (6) is represented by the following formula (8): (wherein each of R1 and R2 is independently a hydrogen atom or a C1-4 alkyl group, and n is an integer of from 1 to 4). ### Claim 9 (depends on 6) The method for producing the separating agent for protein purification according to claim 6, wherein the activator is carbonyldiimidazole. ### Claim 10 (depends on 1) A method for protein purification, which comprises dissolving a protein to be purified in a first buffer, contacting the resulting protein solution with the separating agent for protein purification as defined in claim 1 to allow the separating agent to adsorb the protein and then passing a second buffer having a different pH from the first buffer through the separating agent to elute the protein adsorbed on the separating agent.