Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells
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.
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 CO 2 concentration of 2-10% and a pH of 6.9-7.4, and controlling dissolved oxygen and/or CO 2 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 CO 2 concentration of 2-10% and a pH of 6.9-7.4, and controlling dissolved oxygen and/or CO 2 concentrations to maintain the pH between 6.9-7.4.
2. The method of claim 1 , wherein in step (b), the packaging cells are inoculated at an inoculation density ranging from about 1×10 6 cells/ml to about 5×10 6 cells/ml.
3. 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.
4. The method of claim 1 , wherein in step (d), the pH is ≤7.0.
5. The method of claim 1 , wherein in step (d), the pH is ≤7.05.
6. The method of claim 1 , wherein in step (c) and/or (h), the culture is performed under a shaking condition.
7. The method of claim 1 , wherein in step (c) and/or (h), the pH is maintained between 7.0 and 7.35.
8. The method of claim 1 , wherein the total time of steps (c), (d) and (e) is 72-216 hours.
9. The method of claim 1 , wherein the method produces 1×10 12 transducing units (TU) lentiviruses.
10. The method of claim 1 , wherein the packaging cells are human embryonic kidney epithelial cells.
11. 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.
12. The method of claim 11 , wherein the ratio of Vq1/Vh1 is 5-10.
13. 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 0.05-2×10 11 cells;
(t2) the cell density is 0.5-5×10 6 cells/mi;
(t3) the cell viability rate is ≥90%; and
(t4) the total culture time is ≥72 hours.
14. 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.
15. The method of claim 14 , wherein in step (i), the pH is ≤7.0.
16. The method of claim 14 , wherein in step (i), the pH is ≤7.05.
17. 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.
18. The method of claim 17 , wherein the ratio of Vq2/Vh2 is 5-10.
19. 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.
20. The method of claim 19 , wherein the one or more plasmids comprise three plasmids or four plasmids.