# US 11,999,791 B2 · Prevention of disulfide bond reduction during recombinant production of polypeptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,999,791 B2 - Document ID: US11999791B2 - Status: Granted - Kind: B2 - Application number: 18/310,448 - Filed: 2023-05-01 - Published: 2023-11-16 - Granted: 2024-06-04 - Earliest claimed: 2007-07-09 - Assignees: Genentech, Inc. - Applicants: Genentech, Inc. - Inventors: Yung-Hsiang Kao; Michael W. Laird; Melody Trexler Schmidt; Rita L. Wong; Daniel P. Hewitt - Web page: https://patentgrep.com/patent/US11999791B2 ## CPC - C07K 16/2887: {against CD20} - A61K 39/39591: {Stabilisation, fragmentation} - A61P 5/00: Drugs for disorders of the endocrine system - A61P 37/02: Immunomodulators - C07K 1/14: Extraction; Separation; Purification - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - 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) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract The invention concerns methods for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures. ## Claims ### Claim 1 A method for the prevention of the reduction of a disulfide bond in a therapeutic IgG1 antibody expressed by a recombinant Chinese Hamster Ovary (CHO) host cell, comprising, following fermentation, lowering the pH of the harvested cell culture fluid (HCCF) of the recombinant CHO host cell, wherein lowering the pH of the HCCF results in prevention of the reduction of the disulfide bond in the antibody, and wherein the pH is lowered to a value of less than pH 6. ### Claim 2 (depends on 1) The method of claim 1, wherein the therapeutic IgG1 antibody has been found to be disulfide bond reduced during processing of the HCCF if the pH is not lowered, wherein disulfide bond reduction of the therapeutic IgG1 antibody was detected by measuring loss of intact antibody. ### Claim 3 (depends on 1) The method of claim 1, wherein the therapeutic IgG1 antibody is produced at a scale of greater than 5,000 L. ### Claim 4 (depends on 1) The method of claim 1, wherein mechanical cell lysis during harvest operations is about 30% to about 70% of total cell lysis. ### Claim 5 (depends on 1) The method of claim 1, wherein the pH is lowered immediately after harvest. ### Claim 6 (depends on 1) The method of claim 1, wherein the recombinant CHO host cell is cultured in fed batch culture procedure. ### Claim 7 (depends on 1) The method of claim 1, wherein the recombinant CHO host cell is cultured in multi-stage culture comprising growth phase culture followed by a production phase culture. ### Claim 8 (depends on 1) The method of claim 1, further comprising supplementing the pre-harvest cell culture fluid or HCCF of the recombinant CHO host cell with a thioredoxin inhibitor, wherein the thioredoxin inhibitor is: (i) a direct inhibitor of thioredoxin; (ii) a specific inhibitor of thioredoxin reductase; (iii) a metal ion (iv) an inhibitor of G6PD (v) an inhibitor of hexokinase activity; (vi) cystine or oxidized glutathione; or (vii) a siRNA, antisense nucleotide, or antibody specifically binding to a thioredoxin reductase. ### Claim 9 (depends on 1) The method of claim 1, further comprising, following fermentation, sparging the pre-harvest cell culture fluid or HCCF of the recombinant CHO host cell with air. ### Claim 10 (depends on 1) The method of claim 1, wherein the therapeutic IgG1 antibody is a full length antibody. ### Claim 11 (depends on 1) The method of claim 1, wherein reduction of inter-chain disulfide bonds is prevented. ### Claim 12 (depends on 1) The method of claim 1, further comprising measuring the amount of intact antibody compared with the amount of heavy and light chain fragments of the antibody to confirm disulfide reduction of the antibody is prevented. ### Claim 13 (depends on 1) The method of claim 1, wherein the therapeutic IgG1 antibody is selected from the group consisting of an anti-HER2 antibody; an anti-CD20 antibody; an anti-IL-8 antibody; an anti-VEGF antibody; an anti-CD40 antibody, an anti-CD11a antibody; an anti-CD18 antibody; an anti-IgE antibody; an anti-Apo-2 receptor antibody; an anti-Tissue Factor (TF) antibody; an anti-human α4β7 integrin antibody; an anti-EGFR antibody; an anti-CD3 antibody; an anti-CD25 antibody; an anti-CD4 antibody; an anti-CD52 antibody; an anti-Fc receptor antibody; an anti-carcinoembryonic antigen (CEA) antibody; an antibody directed against breast epithelial cells; an antibody that binds to colon carcinoma cells; an anti-CD38 antibody; an anti-CD33 antibody; an anti-CD22 antibody; an anti-EpCAM antibody; an anti-GpIIb/IIIa antibody; an anti-RSV antibody; an anti-CMV antibody; an anti-HIV antibody; an anti-hepatitis antibody; an anti-CA 125 antibody; an anti-αvβ3 antibody; an anti-human renal cell carcinoma antibody; an anti-human 17-1A antibody; an anti-human colorectal tumor antibody; an anti-human melanoma antibody R24 directed against GD3 ganglioside; an anti-human squamous-cell carcinoma antibody; an anti-human leukocyte antigen (HLA) antibody; and an anti-HLA DR antibody. ### Claim 14 (depends on 1) The method of claim 1, further comprising the step of recovering the therapeutic IgG1 antibody from the HCCF and/or further comprising the step of purifying the therapeutic IgG1 antibody from the HCCF. ### Claim 15 A method for the prevention of the reduction of a disulfide bond in a therapeutic IgG1 antibody expressed by a recombinant Chinese Hamster Ovary (CHO) host cell, comprising, following fermentation and immediately after harvest, lowering the pH of the harvested cell culture fluid (HCCF) of the recombinant CHO host cell, wherein lowering the pH of the HCCF results in prevention of the reduction of the disulfide bond in the therapeutic IgG1 antibody, and wherein the pH is lowered to a value of less than pH 6. ### Claim 16 (depends on 15) The method of claim 15, wherein the therapeutic IgG1 antibody has been found to be disulfide bond reduced during processing of the HCCF if the pH is not lowered. ### Claim 17 (depends on 16) The method of claim 16, wherein disulfide bond reduction of the therapeutic IgG1 antibody was detected by measuring loss of intact antibody. ### Claim 18 (depends on 15) The method of claim 15, wherein the therapeutic IgG1 antibody expressed in the recombinant CHO host cell is produced at a scale of greater than 5,000 L. ### Claim 19 (depends on 15) The method of claim 15, wherein mechanical cell lysis during harvest operations is about 30% to about 70% of total cell lysis. ### Claim 20 (depends on 15) The method of claim 15, wherein the recombinant CHO host cell is cultured in fed batch culture procedure. ### Claim 21 (depends on 15) The method of claim 15, wherein the recombinant CHO host cell is cultured in multi-stage culture comprising growth phase culture followed by a production phase culture. ### Claim 22 (depends on 15) The method of claim 15, further comprising supplementing the pre-harvest cell culture fluid or HCCF of the recombinant host cell with a thioredoxin inhibitor, wherein the thioredoxin inhibitor is: (i) a direct inhibitor of thioredoxin; (ii) a specific inhibitor of thioredoxin reductase; (iii) a metal ion (iv) an inhibitor of G6PD (v) an inhibitor of hexokinase activity; (vi) cystine or oxidized glutathione; or (vii) a siRNA, antisense nucleotide, or antibody specifically binding to a thioredoxin reductase. ### Claim 23 (depends on 15) The method of claim 15, further comprising, following fermentation, sparging the pre-harvest cell culture fluid or HCCF of the recombinant CHO host cell with air. ### Claim 24 (depends on 15) The method of claim 15, wherein the therapeutic IgG1 antibody is a full length antibody. ### Claim 25 (depends on 15) The method of claim 15, wherein reduction of inter-chain disulfide bonds is prevented. ### Claim 26 (depends on 15) The method of claim 15, further comprising measuring the amount of intact antibody compared with the amount of heavy and light chain fragments of the antibody to confirm disulfide reduction of the antibody is prevented. ### Claim 27 (depends on 15) The method of claim 15, wherein the therapeutic IgG1 antibody is selected from the group consisting of an anti-HER2 antibody; an anti-CD20 antibody; an anti-IL-8 antibody; an anti-VEGF antibody; an anti-CD40 antibody, an anti-CD11a antibody; an anti-CD18 antibody; an anti-IgE antibody; an anti-Apo-2 receptor antibody; an anti-Tissue Factor (TF) antibody; an anti-human α4β7 integrin antibody; an anti-EGFR antibody; an anti-CD3 antibody; an anti-CD25 antibody; an anti-CD4 antibody; an anti-CD52 antibody; an anti-Fc receptor antibody; an anti-carcinoembryonic antigen (CEA) antibody; an antibody directed against breast epithelial cells; an antibody that binds to colon carcinoma cells; an anti-CD38 antibody; an anti-CD33 antibody; an anti-CD22 antibody; an anti-EpCAM antibody; an anti-GpIIb/IIIa antibody; an anti-RSV antibody; an anti-CMV antibody; an anti-HIV antibody; an anti-hepatitis antibody; an anti-CA 125 antibody; an anti-αvβ3 antibody; an anti-human renal cell carcinoma antibody; an anti-human 17-1A antibody; an anti-human colorectal tumor antibody; an anti-human melanoma antibody R24 directed against GD3 ganglioside; an anti-human squamous-cell carcinoma antibody; an anti-human leukocyte antigen (HLA) antibody; and an anti-HLA DR antibody. ### Claim 28 (depends on 15) The method of claim 15, further comprising the step of recovering the therapeutic IgG1 antibody from the HCCF and/or further comprising the step of purifying the therapeutic IgG1 antibody from the HCCF. ### Claim 29 (depends on 1) The method of claim 1, wherein the therapeutic IgG1 antibody comprises an Fc region of a human IgG1 antibody. ### Claim 30 (depends on 15) The method of claim 15, wherein the therapeutic IgG1 antibody comprises an Fc region of a human IgG1 antibody. ### Claim 31 A method for the prevention of the reduction of a disulfide bond in a therapeutic protein comprising an Fc region of an IgG1 antibody expressed by a recombinant Chinese Hamster Ovary (CHO) host cell, comprising, following fermentation, lowering the pH of the harvested cell culture fluid (HCCF) of the recombinant CHO host cell, wherein lowering the pH of the HCCF results in prevention of the reduction of the disulfide bond in the therapeutic protein, wherein the pH is lowered to a value of less than pH 6. ### Claim 32 (depends on 31) The method of claim 31, wherein the therapeutic protein has been found to be disulfide bond reduced during processing of the HCCF if the pH is not lowered, wherein disulfide bond reduction of the therapeutic protein was detected by measuring loss of intact protein. ### Claim 33 (depends on 31) The method of claim 31, wherein the therapeutic protein is produced at a scale of greater than 5,000 L. ### Claim 34 (depends on 31) The method of claim 31, wherein mechanical cell lysis during harvest operations is about 30% to about 70% of total cell lysis. ### Claim 35 (depends on 31) The method of claim 31, wherein the pH is lowered immediately after harvest. ### Claim 36 (depends on 31) The method of claim 31, wherein the recombinant CHO host cell is cultured in fed batch culture procedure. ### Claim 37 (depends on 31) The method of claim 31, wherein the recombinant CHO host cell is cultured in multi-stage culture comprising growth phase culture followed by a production phase culture. ### Claim 38 (depends on 31) The method of claim 31, further comprising supplementing the pre-harvest cell culture fluid or HCCF of the recombinant CHO host cell with a thioredoxin inhibitor, wherein the thioredoxin inhibitor is: (i) a direct inhibitor of thioredoxin; (ii) a specific inhibitor of thioredoxin reductase; (iii) a metal ion (iv) an inhibitor of G6PD (v) an inhibitor of hexokinase activity; (vi) cystine or oxidized glutathione; or (vii) a siRNA, antisense nucleotide, or antibody specifically binding to a thioredoxin reductase. ### Claim 39 (depends on 31) The method of claim 31, further comprising, following fermentation, sparging the pre-harvest cell culture fluid or HCCF of the recombinant CHO host cell with air. ### Claim 40 (depends on 31) The method of claim 31, wherein the therapeutic protein is an immunoadhesin. ### Claim 41 (depends on 31) The method of claim 31, wherein reduction of inter-chain disulfide bonds is prevented. ### Claim 42 (depends on 31) The method of claim 31, further comprising the step of recovering the therapeutic protein from the HCCF and/or further comprising the step of purifying the therapeutic protein from the HCCF. ### Claim 43 (depends on 31) The method of claim 31, wherein the therapeutic protein comprises an Fc region of a human IgG1 antibody.