# US 10,501,769 B2 · Method for the production of a glycosylated immunoglobulin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,769 B2 - Document ID: US10501769B2 - Status: Granted - Kind: B2 - Application number: 14/844,570 - Filed: 2015-09-03 - Published: 2016-06-30 - Granted: 2019-12-10 - Earliest claimed: 2009-10-26 - Assignees: Hoffmann-La Roche Inc.; Chugai Seiyaku Kabushiki Kaisha - Applicants: Hoffmann-La Roche Inc.; Chugai Seiyaku Kabushiki Kaisha - Inventors: Reinhard Franze; Chikashi Hirashima; Thomas Link; Yoshinori Takagi; Shinya Takuma; Yuriko Tsuda - Web page: https://patentgrep.com/patent/US10501769B2 ## CPC - C12P 21/005: {Glycopeptides, glycoproteins} - C07K 16/2866: {against receptors for cytokines, lymphokines, interferons} - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/41: Glycosylation, sialylation, or fucosylation - C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding - 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)} - 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 production of an immunoglobulin comprising the following steps: a) providing a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin, b) cultivating the eukaryotic cell in a cultivation medium wherein the amount of glucose available in the cultivation medium per time unit is kept constant and limited to less than 80% of the amount that could maximally be utilized by the cells in the cultivation medium per time unit, and c) recovering the immunoglobulin from the culture. ## Claims ### Claim 1 A method for the production of an immunoglobulin comprising a) cultivating a Chinese Hamster Ovary (CHO) cell comprising a nucleic acid encoding the immunoglobulin in a cultivation medium with restricted glucose feeding wherein the amount of glucose available in the cultivation medium per time unit is kept constant in all time units in which restricted glucose feeding is performed and the degree of glucose limitation (DGL) is limited to a single constant value in a range between 0.5 and 0.1, wherein feeding is started once the amount of glucose present in the cultivation medium has dropped to or below a preset value in the cultivation and wherein said cultivation is a fed-batch cultivating, and b) recovering the immunoglobulin. ### Claim 2 (depends on 1) The method according to claim 1, wherein the restricted glucose feeding is started at day 2 or day 3 of the cultivating. ### Claim 3 (depends on 1) The method according to claim 1, wherein the cultivating is at a pH value from pH 6.5 to 7.5. ### Claim 4 (depends on 3) The method according to claim 3, wherein the cultivating is at a pH value from pH 6.9 to 7.3. ### Claim 5 (depends on 4) The method according to claim 4, wherein the cultivating is at a pH value from pH 6.95 to pH 7.05 or at a pH value from pH 7.15 to pH 7.25. ### Claim 6 (depends on 1) The method according to claim 1, wherein the immunoglobulin is an immunoglobulin of class G or class E. ### Claim 7 (depends on 1) The method according to claim 1, wherein cultivating the CHO cell is performed for six to twenty days. ### Claim 8 (depends on 1) The method according to claim 1, wherein the immunoglobulin is an anti-IL-6R antibody. ### Claim 9 (depends on 1) The method according to claim 1, wherein the DGL is limited to a constant value in a range between 0.45 and 0.2. ### Claim 10 (depends on 1) The method according to claim 1, wherein the DGL is limited to 0.4, or 0.2. ### Claim 11 (depends on 8) The method according to claim 8, wherein the anti-IL-6R antibody comprises tocilizumab. ### Claim 12 (depends on 8) The method according to claim 8, wherein the DGL is limited to a constant value in a range between 0.45 and 0.2. ### Claim 13 (depends on 8) The method according to claim 8, wherein the DGL is limited to 0.4, or 0.2. ### Claim 14 (depends on 11) The method according to claim 11, wherein the DGL is limited to a constant value in a range between 0.45 and 0.2. ### Claim 15 (depends on 11) The method according to claim 11, wherein the DGL is limited to 0.4, or 0.2. ### Claim 16 (depends on 1) The method according to claim 1, wherein the fraction of the immunoglobulin with a mannose-5 glycostructure is 10% or less of the sum comprising the amount of the polypeptide with a mannose-5 glycostructure, the amount of the polypeptide G(0) isoform, the amount of the polypeptide G(1) isoform, and the amount of the polypeptide G(2) isoform after 7 days of cultivation. ### Claim 17 (depends on 1) The method according to claim 1, wherein the fraction of the immunoglobulin with a mannose-5 glycostructure is 6% or less of the sum comprising the amount of the polypeptide with a mannose-5 glycostructure, the amount of the polypeptide G(0) isoform, the amount of the polypeptide G(1) isoform, and the amount of the polypeptide G(2) isoform after 7 days of cultivation. ### Claim 18 A method for the production of an immunoglobulin comprising a) cultivating a Chinese Hamster Ovary (CHO) cell comprising a nucleic acid encoding the immunoglobulin in a cultivation medium with restricted glucose feeding wherein the amount of glucose available in the cultivation medium per time unit is kept constant in all time units in which restricted glucose feeding is performed and the degree of glucose limitation (DGL) is limited to a single constant value in a range between 0.5 and 0.1, and wherein the restricted glucose feeding is started at day 2 or day 3 of the cultivating and wherein said cultivating is a fed-batch cultivating, and b) recovering the immunoglobulin. ### Claim 19 (depends on 1) The method according to claim 1, wherein the time unit is about 1 minute. ### Claim 20 (depends on 1) The method according to claim 1, wherein the time unit is about 1 hour. ### Claim 21 (depends on 1) The method according to claim 1, wherein the time unit is about 6 hours. ### Claim 22 (depends on 1) The method according to claim 1, wherein the time unit is about 12 hours. ### Claim 23 (depends on 1) The method according to claim 1, wherein the time unit is about 24 hours. ### Claim 24 (depends on 1) The method according to claim 1, wherein density of CHO cells at the beginning of the cultivating is about 105 cells per milliliter. ### Claim 25 (depends on 1) The method according to claim 1, wherein density of CHO cells at the beginning of the cultivating is about 8×105 cells per milliliter. ### Claim 26 (depends on 1) The method according to claim 1, wherein density of CHO cells at the beginning of the cultivating is about 10×105 cells per milliliter. ### Claim 27 (depends on 1) The method according to claim 1, wherein density of CHO cells at the beginning of the cultivating is about 12×105 cells per milliliter. ### Claim 28 (depends on 1) The method according to claim 1, wherein density of CHO cells is from about 105 cells per milliliter to about 60×105 cells per milliliter during the cultivation. ### Claim 29 (depends on 18) The method according to claim 18, wherein the time unit is about 1 minute. ### Claim 30 (depends on 18) The method according to claim 18, wherein the time unit is about 1 hour. ### Claim 31 (depends on 18) The method according to claim 18, wherein the time unit is about 6 hours. ### Claim 32 (depends on 18) The method according to claim 18, wherein the time unit is about 12 hours. ### Claim 33 (depends on 18) The method according to claim 18, wherein the time unit is about 24 hours. ### Claim 34 (depends on 18) The method according to claim 18, wherein density of CHO cells at the beginning of the cultivating is about 105 cells per milliliter. ### Claim 35 (depends on 18) The method according to claim 18, wherein density of CHO cells at the beginning of the cultivating is about 8×105 cells per milliliter. ### Claim 36 (depends on 18) The method according to claim 18, wherein density of CHO cells at the beginning of the cultivating is about 10×105 cells per milliliter. ### Claim 37 (depends on 18) The method according to claim 18, wherein density of CHO cells at the beginning of the cultivating is about 12×105 cells per milliliter. ### Claim 38 (depends on 18) The method according to claim 18, wherein density of CHO cells is from about 105 cells per milliliter to about 60×105 cells per milliliter during the cultivation. ### Claim 39 (depends on 1) The method according to claim 1, wherein cultivating the CHO cell is performed for six to fifteen days. ### Claim 40 (depends on 1) The method according to claim 1, wherein cultivating the CHO cell is performed for six to eight days. ### Claim 41 (depends on 1) The method according to claim 1, wherein the fraction of the immunoglobulin with a mannose-5 glycostructure is 8% or less of the sum comprising the amount of the polypeptide with a mannose-5 glycostructure, the amount of the polypeptide G(0) isoform, the amount of the polypeptide G(1) isoform, and the amount of the polypeptide G(2) isoform after 7 days of cultivation.