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Building IP: BMY Patent Application re "ONLINE BIOMASS CAPACITANCE MONITORING DURING LARGE SCALE PRODUCTION OF POLYPEPTIDES OF INTEREST"
ONLINE BIOMASS CAPACITANCE MONITORING DURING LARGE SCALE PRODUCTION OF POLYPEPTIDES OF INTEREST The present invention relates to the use of online biomass capacitance monitoring in perfusion cultures as a way to minimize volumes of cell culture media in large-scale manufacturing. In certain embodiments, a biomass capacitance probe is used to measure the viable cell density, and the viable cell density is used in conjunction with a pre-determined constant cell-specific perfusion rate to identify a target perfusion rate.
1. A method of controlling the perfusion rate in a perfusion bioreactor comprising: a) measuring the viable cell density of cells growing in cuture using a biomass capacitance probe; and b) dynamically adjusting the target perfusion rate in view of the viable cell density, wherein the cells are grown to a high cell density during perfusion. 2. A method of minimizing cell culture medium perfusion volume comprising: a) measuring the viable cell density of cells growing in cuture using a biomass capacitance probe; and b) dynamically adjusting the target perfusion rate in view of the viable cell density, wherein the cells are grown to a high cell density during perfusion. 3. The method of claim 1 or 2, wherein the viable cell density is used in combination with a constant cell-specific perfusion rate to determine the target perfusion rate. 4. The method of claim 3, wherein the constant cell-specific perfusion rate is between about 0.01 to 0.15 nL/cell/day. 5. The method of claim 3 or 4, wherein the constant cell-specific perfusion rate is between about 0.02 to 0.10 nL/cell/day. 6. The method of any one of claims 3-5, wherein the constant cell-specific perfusion rate is about 0.03 nl/cell/day 7. The method of any one of claims 3-5, wherein the constant cell-specific perfusion rate is about 0.04-0.05 nl/cell/day. 8. The method of any one of claim 3-5 or 7, wherein the constant cell-specific perfusion rate is about 0.04 nl/cell/day 9. The method of any one of claims 3-5, wherein the constant cell-specific perfusion rate is between about 0.08 to 0.10 nL/cell/day 10. The method of any one of claim 3-5 or 9, wherein the constant cell-specific perfusion rate is about 0.10 nl/cell/day 11. The method of any one of claims 1-10, wherein the calculation of the target perfusion flow rate is performed by a bioreactor controller. 12. The method of any one of claims 1-11, wherein the perfusion flow rate is increased to achieve the target perfusion flow rate following measurement of the viable cell density in the bioreactor. 13. The method of any one of claims 1-11, wherein the perfusion flow rate is decreased to achieve the target perfusion flow rate following measurement of the viable cell density in the bioreactor. 14. The method of any one of claims 1-13, wherein the viable cell density is further used to measure the time point when a target cell density is reached. 15. The method of claim 14, wherein the target cell density is >30.times.10.sup.6 cells/ml, >40 x 10.sup.6 cells/ml, >50.times.10.sup.6 cells/ml, >60.times.10.sup.6 cells/ml, or >70.times.10.sup.6 cells/ml. 16. The method of claim 14 or 15, wherein the target cell density is >40.times.10.sup.6 cell s/ml. 17. The method of any one of claims 1-16, wherein the total perfusate is measured. 18. The method of any one of claims 1-17, wherein the daily perfusion rate is measured. 19. The method of any one of claims 1-18, wherein the capacitance is measured. 20. The method of any one of claims 14-19, wherein the cells are transferred into a production bioreactor upon reaching the target cell density. 21. The method of any one of claims 1-20, wherein the cells produce a polypeptide of interest. 22. The method of claim 21, wherein the polypeptide of interest is an antibody. 23. The method of any one of claims 1-22, wherein the cells are mammalian cells. 24. The method of any one of claims 1-23, wherein the cells are CHO cells. 25. The method of any one of claims 1-24, wherein the perfusion bioreactor is an N-1 perfusion bioreactor. 26. The method of any one of claims 1-25, wherein the perfusion bioreactor is attached to an alternating tangential flow system. 27. The method of any one of claims 1-26, wherein the perfusion bioreactor uses less than about 5, less than about 4.5, less than about 4, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2, less than about 1.5 or less than 1 vessel volumes of perfusion media per day. 28. The method of any one of claims 1-27, wherein the perfusion bioreactor uses only about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the total amount of perfusion media used by a process that does not measure the viable cell density. 29. The method of any one of claims 1-28, wherein the biomass capacitance probe is an INCYTE probe. CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 62/559,142, filed Sep. 15, 2017, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0002] A perfusion culture utilizes a cell separation device or filtration methods to retain cells in a bioreactor, while exchanging out spent media with fresh media. Perfusion rates are typically set between one to five bioreactor volumes per day, and maintained constant on a 24 hour basis. Perfusion rates can be increased based on viable cell density measurements to ensure adequate nutrients for the cells. Due to the fact that this perfusion rate is only adjusted on an infrequent, daily basis, the amount of media exchanged can be either under or over-estimated on a per cell basis. Under estimation of an actual cell-specific perfusion rate could result in depletion of nutrients and thus impact cell growth rates. An over-estimation of cell-specific perfusion rate results in high media perfusion rates without fully utilizing the nutrients provided in the media. [0003] Others have measured viable cell density and the perfusion rate during the production phase. Dowd et al., Cytotechnology 42: 35-45 (2003). However, there is a need in the art for mechanisms to dynamically adjust perfusion rates to meet a target rate, thereby reducing media usage while retaining a constant level of nutrients in a high cell density perfusion system. SUMMARY OF THE INVENTION [0004] In one embodiment, the present invention relates to a method of controlling the perfusion rate in a perfusion bioreactor comprising: a) measuring the viable cell density of cells growing in cuture using a biomass capacitance probe; and b) dynamically adjusting the target perfusion rate in view of the viable cell density, wherein the cells are grown to a high cell density during perfusion. [0005] In one embodiment, the present invention relates to a method of minimizing cell culture medium perfusion volume comprising: a) measuring the viable cell density of cells growing in cuture using a biomass capacitance probe; and b) dynamically adjusting the target perfusion rate in view of the viable cell density, wherein the cells are grown to a high cell density during perfusion. [0006] In some embodiments, the viable cell density is used in combination with a constant cell-specific perfusion rate to determine the target perfusion rate. In certain embodiments, the constant cell-specific perfusion rate is between about 0.01 to 0.15 nL/cell/day. In particular embodiments, the constant cell-specific perfusion rate is between about 0.02 to 0.10 nL/cell/day. In one embodiment, the constant cell-specific perfusion rate is about 0.04 nl/cell/day. In embodiments, the constant cell-specific perfusion rate is about 0.03 nl/cell/day. In some embodiments, the constant cell-specific perfusion rate is about 0.04-0.05 nl/cell/day. In certain embodiments, the constant cell-specific perfusion rate is about 0.04 nl/cell/day. In particular embodiments, the constant cell-specific perfusion rate is between about 0.08 to 0.10 nL/cell/day. In embodiments, the constant cell-specific perfusion rate is about 0.10 nl/cell/day [0007] In some embodiments, the calculation of the target perfusion flow rate is performed by a bioreactor controller. In certain embodiments, the perfusion flow rate is increased to achieve the target perfusion flow rate following measurement of the viable cell density in the bioreactor. In other embodiments, the perfusion flow rate is decreased to achieve the target perfusion flow rate following measurement of the viable cell density in the bioreactor. [0008] In some embodiments, the viable cell density is further used to measure the time point when a target cell density is reached. In certain embodiments, the target cell density is >30.times.106 cells/ml, >40.times.10.sup.6 cells/ml, >50.times.10.sup.6 cells/ml, >60.times.10.sup.6 cells/ml, or >70.times.10.sup.6 cells/ml. In particular embodiments, the target cell density is >40.times.10.sup.6 cells/ml. [0009] In some embodiments, the total perfusate is measured. In embodiments, the daily perfusion rate is measured. In certain embodiments, the capacitance is measured. [0010] In embodiments, the cells are transferred into a production bioreactor upon reaching the target cell density. In certain embodiments, the cells produce a polypeptide of interest. In particular embodiments, the polypeptide of interest is an antibody. [0011] In embodiments, the cells are mammalian cells. In certain embodiments, the cells are CHO cells. [0012] In some embodiments, the perfusion bioreactor is an N-1 perfusion bioreactor. In embodiments, the perfusion bioreactor is attached to an alternating tangential flow system. [0013] In embodiments, the perfusion bioreactor uses less than about 5, less than about 4.5, less than about 4, less than about 3.5, less than about 3.0, less than about 2.5, less than about 2, less than about 1.5 or less than 1 vessel volumes of perfusion media per day. In particular embodiments, the perfusion bioreactor uses only about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the total amount of perfusion media used by a process that does not measure the viable cell density. [0014] In embodiments, the biomass capacitance probe is an INCYTE probe. |
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