# US 12,595,454 B2 · Methods of continuous cell culture > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,595,454 B2 - Document ID: US12595454B2 - Status: Granted - Kind: B2 - Application number: 17/273,004 - Filed: 2019-08-28 - Published: 2021-07-22 - Granted: 2026-04-07 - Earliest claimed: 2018-09-06 - Assignees: Momenta Pharmaceuticals, Inc. - Applicants: Momenta Pharmaceuticals, Inc. - Inventors: Jin Yin; Ru Zang - Web page: https://patentgrep.com/patent/US12595454B2 ## CPC - C12M 29/20: {Degassing; Venting; Bubble traps (means for collection or storage of gas C12M23/36; gas collection apparatus for laboratory use B01L5/02)} - C07K 16/244: {Interleukins [IL]} - C12M 27/02: {Stirrer or mobile mixing elements} - C12M 29/06: {Nozzles; Sprayers; Spargers; Diffusers (per se B01F23/231, B01J19/26)} - C12M 29/10: {Perfusion} - C12M 35/04: {Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli} - C12M 41/34: {of gas} - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - 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 disclosure provides, among other things, continuous culture methods for producing a cell product, e.g., a recombinant protein, e.g., a glycoprotein, e.g., an antibody agent or a fusion protein. In some instances, methods herein allow large-scale production of a recombinant protein using continuous culture methods. The present disclosure identifies and addresses a problem with current continuous cell culture techniques in that at large-scale culture of certain cells have insufficient viable cell concentrations and impaired cell viability. The present disclosure provides, in part, methods and systems for large-scale continuous culture of shear-sensitive cells. ## Claims ### Claim 1 A method for culturing a population of shear-sensitive cells comprising: culturing a population of cells consisting of shear-sensitive cells in a perfusion bioreactor system with at least 100 L of culture media, wherein the culture media has a level of dissolved carbon dioxide that is at most 80 mmHg, and a gas exit velocity that is 10 m/s or less to achieve a steady state viable cell concentration in the culture media within a range of 20×106 cells/mL to 15×107 cells/mL, wherein the shear-sensitive cells are HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, or SP 2/0 cells. ### Claim 2 (depends on 1) The method of claim 1, wherein the gas exit velocity is controlled at least until the bioreactor system reaches the steady state condition. ### Claim 3 (depends on 2) The method of claim 2, wherein the steady state condition includes having a viable cell concentration that varies at most 20% over a period of 5 days. ### Claim 4 (depends on 1) The method of claim 1, wherein the gas exit velocity is controlled throughout the culturing step. ### Claim 5 (depends on 1) The method of claim 1, wherein the culturing the population of cells consisting of shear-sensitive cells in the perfusion bioreactor system to achieve the steady state viable cell concentration further comprises bleeding or removing excess cells and/or non-viable cells. ### Claim 6 (depends on 1) The method of claim 1, wherein the bioreactor system includes at least 200 L, at least 500 L, at least 1,000 L, or at least 2,000 L of culture media. ### Claim 7 (depends on 1) The method of claim 1, wherein culturing is performed for a duration of 30 to 60 days. ### Claim 8 (depends on 1) The method of claim 1, wherein the method further comprises measuring the viable cell concentration. ### Claim 9 (depends on 8) The method of claim 8, wherein the measured viable cell concentration is within a range of 30×106 cells/mL to 15×107 cells/mL, 40×106 cells/mL to 15×107 cells/mL, or 50×106 cells/mL to 15×107 cells/mL. ### Claim 10 (depends on 1) The method of claim 1, wherein the shear-sensitive cells are human cells or murine cells. ### Claim 11 (depends on 1) The method of claim 1, wherein the bioreactor system includes a cell retention device, wherein the cell retention device is or includes a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin-filter, an ultrasonic and dielectrophoretic separator, and/or a gravity settler. ### Claim 12 (depends on 1) The method of claim 1, wherein the bioreactor system comprises a stirred tank bioreactor tank, wherein the bioreactor tank has a capacity of at least 100 L, at least 200 L, at least 500 L, at least 1,000 L, or at least 2,000 L. ### Claim 13 (depends on 1) The method of claim 1, wherein the bioreactor system includes a sparger. ### Claim 14 (depends on 13) The method of claim 13, wherein the sparger is a drilled hole sparger or an open pipe sparger. ### Claim 15 (depends on 1) The method of claim 1, wherein the culturing the population of cells is performed under conditions to express a cell product that comprises one or more of a nucleic acid, a lipid, a peptide, and a protein. ### Claim 16 (depends on 15) The method of claim 15, wherein the cell product is an antibody agent. ### Claim 17 (depends on 15) The method of claim 15, wherein the method further comprises isolating the cell product from at least a portion of the shear-sensitive cells and/or isolating the cell product from at least a portion of the culture media. ### Claim 18 A continuous culture process for culturing a population of cells consisting of shear-sensitive cells, comprising: controlling gas exit velocity of a bioreactor system so that the gas exit velocity does not exceed a rate of 10 m/s, wherein the bioreactor system comprises at least 100 L of culture media, wherein the culture media has a level of dissolved carbon dioxide that is at most 80 mmHg, and wherein the population of cells achieves a steady state viable cell concentration within a range of 20×106 cells/mL to 15×107 cells/mL, wherein the shear-sensitive cells are HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, Hull-7 cells, NS0 cells, or SP 2/0 cells. ### Claim 19 (depends on 14) The method of claim 14, wherein the drilled hole sparger comprises holes having a diameter of about 0.2 mm to about 0.3 mm.