# US 10,308,706 B2 · Methods of preventing and removing trisulfide bonds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,706 B2 - Document ID: US10308706B2 - Status: Granted - Kind: B2 - Application number: 14/638,215 - Filed: 2015-03-04 - Published: 2015-10-01 - Granted: 2019-06-04 - Earliest claimed: 2009-10-02 - Assignees: Biogen MA Inc. - Applicants: Biogen Ma Inc. - Inventors: David Evans; R. Blake Pepinsky; Dingyi Wen; Rashmi Rohit Kshirsagar; Karin Lucas - Web page: https://patentgrep.com/patent/US10308706B2 ## CPC - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 1/1133: {by redox-reactions involving cystein/cystin side chains} - 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 The present invention pertains to methods of preventing and eliminating trisulfide bonds in proteins such as antibodies. In one embodiment, trisulfide bonds in proteins are converted to disulfide bonds as part of chromatographic purification procedures. In another embodiment, the formation of trisulfide bonds in proteins is inhibited by implementation of methods described herein during the cell culture production of such proteins. In another embodiment, monoclonal antibodies are produced by the methods described herein. ## Claims ### Claim 1 A method for reducing the formation of trisulfide bonds in antibodies or antigen-binding fragments thereof, wherein said method comprises cultivating mammalian cells producing said antibodies or antigen-binding fragments thereof in cell culture media under conditions selected from the group consisting of: (a) maintaining or reducing the concentration of sulfur or sulfur-containing compounds in the cell culture media below a threshold level of 10 millimolar; (b) harvesting the cell culture media when the cell culture is at approximately maximum cell density or at approximately peak production of the antibodies or antigen-binding fragments thereof; and (c) a combination of (a) and (b), wherein the sulfur-containing compound is selected from: (i) hydrogen sulfide; (ii) sodium sulfide; and (iii) sodium hydrogen sulfide. ### Claim 2 (depends on 1) The method of claim 1, wherein said method comprises harvesting the cell culture media when the cell culture is at approximately maximum cell density. ### Claim 3 (depends on 1) The method of claim 1, wherein said method comprises harvesting the cell culture media at approximately peak production of the antibodies or antigen-binding fragments thereof. ### Claim 4 A method for reducing the formation of trisulfide bonds in antibodies or antigen-binding fragments thereof in a mammalian cell culture, wherein said method comprises maintaining the concentration of cysteine and/or cystine in the cell culture at or below a threshold level of 150 micromolar during cell cultivation. ### Claim 5 (depends on 4) The method of claim 4, wherein said threshold level is about 50 micromolar. ### Claim 6 (depends on 1) The method of claim 1, wherein the antibodies or antigen-binding fragments thereof are IgG1, IgG2, IgG3 or IgG4. ### Claim 7 (depends on 1) The method of claim 1, wherein the antibodies or antigen-binding fragments thereof are IgG1. ### Claim 8 (depends on 1) The method of claim 1, wherein the mammalian cells comprise CHO cells. ### Claim 9 (depends on 4) The method of claim 4, wherein the method reduces heat induced fragmentation of the antibodies or antigen-binding fragments thereof compared to the antibodies or antigen-binding fragments thereof produced without maintaining the concentration of cysteine and/or cystine in the cell culture at or below the threshold level of 150 micromolar during cell cultivation, wherein the heat induced fragmentation is measured by denaturing, non-reducing capillary electrophoresis. ### Claim 10 (depends on 4) The method of claim 4, wherein the antibodies or antigen-binding fragments thereof are IgG1, IgG2, IgG3 or IgG4. ### Claim 11 (depends on 10) The method of claim 10, wherein the antibodies or antigen-binding fragments thereof are IgG1. ### Claim 12 (depends on 4) The method of claim 4, wherein the cell culture comprises CHO cells. ### Claim 13 (depends on 2) The method of claim 2, wherein the antibodies or antigen-binding fragments thereof are IgG1, IgG2, IgG3 or IgG4. ### Claim 14 (depends on 3) The method of claim 3, wherein the antibodies or antigen-binding fragments thereof are IgG1, IgG2, IgG3 or IgG4. ### Claim 15 (depends on 13) The method of claim 13, wherein the antibodies or antigen-binding fragments thereof are IgG1. ### Claim 16 (depends on 14) The method of claim 14, wherein the antibodies or antigen-binding fragments thereof are IgG1.