# US 12,180,513 B2 · Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,180,513 B2 - Document ID: US12180513B2 - Status: Granted - Kind: B2 - Application number: 18/498,879 - Filed: 2023-10-31 - Published: 2024-02-22 - Granted: 2024-12-31 - Earliest claimed: 2018-03-29 - Assignees: Shanghai AbelZeta Ltd. - Applicants: Shanghai Abelzeta Ltd. - Inventors: Yi Hong; Ting Yan; Jiangguo Ying; Haojie Zhang; Luyi Zhang; Li Zhang - Web page: https://patentgrep.com/patent/US12180513B2 ## CPC - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 15/86: Viral vectors - C12N 2740/15043: viral genome or elements thereof as genetic vector - C12N 2740/15051: Methods of production or purification of viral material - C12N 2740/16043: viral genome or elements thereof as genetic vector - C12N 2740/16051: Methods of production or purification of viral material - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Provided is a method for large-scale production of lentivirus by using GMP-level serum-free suspension cells. Said method comprises the following steps: (a) providing a seed solution of packaged cells; (b) inoculating the seed solution in a first culture solution; (c) carrying out subculture of the packaged cells; (d) starting a liquid change operation when a liquid change trigger condition is met; (e) repeating steps (c) and (d) 1, 2 or 3 times; (f) starting a transfection operation when a transfection trigger condition is met; (g) optionally performing liquid change after transfection; (h) cultivating the transfected packaged cells; (i) starting harvesting and liquid change operations when a liquid change trigger condition is met; (j) repeating steps (h) and (i) 1, 2 or 3 times; (k) combining each of the recovered liquids; and (l) performing a purifying treatment. The culture solution used in each step is a serum-free cell culture solution. ## Claims ### Claim 1 A method for producing lentiviruses using cells in serum-free suspension culture, the method comprising: (a) providing packaging cells that grow in suspension; (b) inoculating the packaging cells in a first culture medium, wherein the first culture medium is a serum-free cell culture medium; (c) culturing the inoculated packaging cells, at conditions set at a temperature of 30-38° C., a dissolved oxygen concentration of 35-55%, a CO2 concentration of 2-10% and a pH of 6.9-7.4, and controlling dissolved oxygen and/or CO2 concentrations to maintain the pH between 6.9-7.4; (d) preforming a first liquid change when liquid change trigger conditions are met, wherein the liquid change trigger conditions comprise: (s1) the pH is ≤6.9; and (s2) a culture time is ≥72 hours; (e) repeating steps (c) and (d) n times, wherein n is 0, 1, 2, or 3; (f) performing transfection; (g) optionally carrying out post-transfection liquid change; (h) culturing the transfected packaging cells, at culture conditions set at a temperature of 30-37° C., a dissolved oxygen concentration of 35-55%, a CO2 concentration of 2-10% and a pH of 6.9-7.4, and controlling dissolved oxygen and/or CO2 concentrations to maintain the pH between 6.9-7.4. ### Claim 2 (depends on 1) The method of claim 1, wherein in step (b), the packaging cells are inoculated at an inoculation density ranging from about 1×106 cells/ml to about 5×106 cells/ml. ### Claim 3 (depends on 1) The method of claim 1, wherein in step (b), the first culture medium has a volume ranging from about 3 liters to about 100 liters. ### Claim 4 (depends on 1) The method of claim 1, wherein in step (d), the pH is ≤7.0. ### Claim 5 (depends on 1) The method of claim 1, wherein in step (d), the pH is ≤7.05. ### Claim 6 (depends on 1) The method of claim 1, wherein in step (c) and/or (h), the culture is performed under a shaking condition. ### Claim 7 (depends on 1) The method of claim 1, wherein in step (c) and/or (h), the pH is maintained between 7.0 and 7.35. ### Claim 8 (depends on 1) The method of claim 1, wherein the total time of steps (c), (d) and (e) is 72-216 hours. ### Claim 9 (depends on 1) The method of claim 1, wherein the method produces 1×1012 transducing units (TU) lentiviruses. ### Claim 10 (depends on 1) The method of claim 1, wherein the packaging cells are human embryonic kidney epithelial cells. ### Claim 11 (depends on 1) The method of claim 1, wherein in step (d), the first liquid change comprises: harvesting a supernatant of the culture, wherein the culture before harvesting the supernatant has a volume of Vq1, wherein the culture after harvesting the supernatant has a volume of Vh1, and wherein the ratio of Vq1/Vh1 is 3-15; and adding a second culture medium to the culture, wherein the second culture medium is a serum-free cell culture medium. ### Claim 12 (depends on 11) The method of claim 11, wherein the ratio of Vq1/Vh1 is 5-10. ### Claim 13 (depends on 1) The method of claim 1, wherein in step (f), the transfection is performed when transfection trigger conditions are met, wherein the transfection trigger conditions comprise: (t1) the total number of cells is 0.05-2×1011 cells; (t2) the cell density is 0.5-5×106 cells/mi; (t3) the cell viability rate is ≥90%; and (t4) the total culture time is ≥72 hours. ### Claim 14 (depends on 1) The method of claim 1, further comprising: (i) starting a second liquid change when liquid change trigger conditions are met, wherein the liquid change trigger conditions comprise: (s1) the pH is 56.9; and (s2) a culture time is 272 hours; and (j) repeating steps (h) and (i) m times, wherein m is 0, 1, 2, or 3. ### Claim 15 (depends on 14) The method of claim 14, wherein in step (i), the pH is ≤7.0. ### Claim 16 (depends on 14) The method of claim 14, wherein in step (i), the pH is ≤7.05. ### Claim 17 (depends on 14) The method of claim 14, wherein in step (i), the second liquid change comprises: harvesting a supernatant of the culture, wherein the culture before harvesting the supernatant has a volume of Vq2, wherein the culture after harvesting the supernatant has a volume of Vh2, and wherein the ratio of Vq2/Vh2 is 3-15. ### Claim 18 (depends on 17) The method of claim 17, wherein the ratio of Vq2/Vh2 is 5-10. ### Claim 19 (depends on 1) The method of claim 1, wherein in step (f), the transfection comprises: mixing one or more plasmids with a transfection reagent to form a mixture, and adding the mixture to the culture. ### Claim 20 (depends on 19) The method of claim 19, wherein the one or more plasmids comprise three plasmids or four plasmids.