# US 2020/0399670 A1 · Recombinant production method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399670 A1 - Document ID: US20200399670A1 - Status: Published application - Kind: A1 - Application number: 16/924,056 - Filed: 2020-07-08 - Published: 2020-12-24 - Earliest claimed: 2015-12-16 - Assignees: Hoffmann-La Roche Inc. - Applicants: Hoffmann-La Roche Inc. - Inventors: Tobias KILLIAN; Markus NEUBAUER - Web page: https://patentgrep.com/patent/US20200399670A1 ## CPC - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)} - C12N 5/0602: {Vertebrate cells} - C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids} - C12N 2500/30: Organic components - C12N 2501/11: Epidermal growth factor [EGF] - C12N 2501/2301: Interleukin-1 (IL-1) - C12N 2501/25: Tumour necrosing factors [TNF] - C12N 2510/00: Genetically modified cells - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) ## Abstract Herein is reported a method for the recombinant production of a polypeptide in a eukaryotic cell comprising the steps of (i) cultivating a eukaryotic cell comprising a nucleic acid encoding the polypeptide in a cultivation medium comprising a compound selected from the group consisting of trans-2-methyl 2-pentenoic acid, the broad-spectrum HDAC inhibitor Quisinostat, and the subtype-specific HDAC inhibitor Romidepsin, and (ii) recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide in a eukaryotic cell. ## Claims ### Claim 1 A method for the recombinant production of a polypeptide in a eukaryotic cell comprising the steps of: cultivating a eukaryotic cell comprising a nucleic acid encoding the polypeptide in a cultivation medium comprising a compound selected from the group consisting of trans-2-methyl 2-pentenoic acid, the broad-spectrum HDAC inhibitor Quisinostat, and the subtype-specific HDAC inhibitor Romidepsin, and recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide in a eukaryotic cell. ### Claim 2 A method for the recombinant production of a polypeptide in a eukaryotic cell comprising the steps of: cultivating a eukaryotic cell comprising a nucleic acid encoding the polypeptide in a cultivation medium comprising a first and a second compound selected from the group of compounds consisting of trans-2-methyl 2-pentenoic acid, subtype-specific HDAC inhibitors, and canonical and non-canonical NFκB activators, and recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide in a eukaryotic cell. ### Claim 3 (depends on 2) The method according to claim 2, wherein the canonical NFκB activator is selected from the group consisting of TNFalpha, ligands of IL-1R/TLR, lipopolysaccharides and IL-1b. ### Claim 4 (depends on 2) The method according to claim 2, wherein the non-canonical NFκB activator is selected from the group consisting of EGF, PMA, and betulinic acid. ### Claim 5 (depends on 2) The method according to claim 2, wherein the subtype-specific HDAC inhibitor is an HDAC-1 and/or HDAC-2 and/or HDAC-3 inhibitor. ### Claim 6 (depends on 5) The method according to claim 5, wherein the subtype-specific HDAC inhibitor is selected from the group consisting of pimelic diphenylamide 106, Apicidin, and Romidepsin. ### Claim 7 (depends on 2) The method according to claim 2, wherein the first compound is trans-2-methyl 2-pentenoic acid and the second compound is Flagellin. ### Claim 8 (depends on 2) The method according to claim 2, wherein the first compound is trans-2-methyl 2-pentenoic acid and the second compound is Quisinostat. ### Claim 9 (depends on 2) The method according to claim 2, wherein the first compound is Romidepsin and the second compound is trans-2-methyl 2-pentenoic acid or Flagellin. ### Claim 10 (depends on 2) The method according to claim 2, wherein the first compound is added of from one hour to five hours after transfection of the cells. ### Claim 11 (depends on 10) The method according to claim 10, wherein the first compound is added about three hours after the transfection of the cells to the cultivation. ### Claim 12 (depends on 2) The method according to claim 2, wherein the second compound is added of from twenty-one hours to thirty hours after the transfection of the cells to the cultivation. ### Claim 13 (depends on 12) The method according to claim 12, wherein the second compound is added about twenty-four to twenty-seven hours after the transfection of the cells to the cultivation. ### Claim 14 (depends on 2) The method according to claim 2, wherein trans-2-methyl 2-pentenoic acid is added to a final concentration in the cultivation medium of about 6-7 mM. ### Claim 15 (depends on 9) The method according to claim 9, wherein Flagellin is added to a final a concentration of 2 ng/mL and Romidepsin is added to a final concentration of 15 nM. ### Claim 16 (depends on 2) The method according to claim 2, wherein the polypeptide is an antibody. ### Claim 17 (depends on 2) The method according to claim 2, wherein the eukaryotic cell is a CHO cell or a HEK cell. ### Claim 18 (depends on 3) The method according to claim 3, wherein the non-canonical NFκB activator is selected from the group consisting of EGF, PMA, and betulinic acid. ### Claim 19 (depends on 6) The method according to claim 6, wherein the first compound is trans-2-methyl 2-pentenoic acid and the second compound is Flagellin. ### Claim 20 (depends on 1) The method according to claim 1, wherein the compound is added of from one hour to five hours after transfection of the cells. ### Claim 21 (depends on 1) The method according to claim 1, wherein trans-2-methyl 2-pentenoic acid is added to a final concentration in the cultivation medium of about 6-7 mM. ### Claim 22 (depends on 1) The method according to claim 1, wherein the polypeptide is an antibody. ### Claim 23 (depends on 1) The method according to claim 1, wherein the eukaryotic cell is a CHO cell or a HEK cell.