Lean Perfusion Cell Culture Method

Abstract

Described herein is a low perfusion rate cell culture method for producing recombinant proteins.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority for all purposes to U.S. Provisional Patent Application No. 63 / 403,896, filed September 6, 2022, which is incorporated by reference herein in its entirety.

[0002] The present disclosure relates generally to perfusion culture methods for producing recombinant proteins. [Background technology]

[0003] Biopharmaceutical manufacturing typically begins with the cultivation of mammalian cells engineered to express a desired therapeutic protein. More than 50% of approved biologics are produced using mammalian cells, and this number is increasing annually. While monoclonal antibody biologics currently represent the largest segment of the biopharmaceutical market, new protein modalities with multitarget affinities to treat a wider variety of therapeutic indications are being tested and approved at an ever-increasing rate. Meeting the growing complex needs of patients is a driving force behind the biopharmaceutical industry. With an increasingly complex convergence of product modalities in clinical development, it is imperative to further enhance the adaptability, flexibility, productivity, and cost-efficiency of manufacturing processes and facilities while continuing to produce the highest quality biotherapeutics.

[0004] To meet this need, process intensification through increased volumetric productivity is critical for upstream manufacturing processes. Industrial-scale mammalian cell culture processes typically include batch, fed-batch, and perfusion processes. While these cell culture processes have demonstrated robustness over decades of development and optimization, the ability to intensify is often limited by process scale limitations, such as bioreactor size for batch and fed-batch processes and medium volume for batching and removal in perfusion processes. Therefore, there is a need in the art for new cell culture methods that facilitate process intensification. Summary of the Invention [Means for solving the problem]

[0005] Described herein is a novel cell culture method that overcomes certain limitations associated with current conventional culture methods and allows for process intensification. The method allows for efficient use of the bioreactor working volume during the growth and production phases. Slow perfusion maintains the culture at a high cell density at at least half the normal bioreactor working volume during the growth phase; then, during the production phase, the culture volume is increased to full or near-full bioreactor working volume at a lower cell density. This transition allows for a more efficient culture, reducing feed and waste volumes and increasing titer and productivity.

[0006] The present disclosure provides a method for culturing cells to produce a recombinant protein, the method comprising initiating a culture in a bioreactor with a culture volume that is at least 50% of the final bioreactor working volume; inoculating the culture with cells engineered to express the recombinant protein; perfusing the culture at one or more perfusion rates of 0.5 culture volumes per day (V / d) or less until the culture reaches one or more desired goal criteria; increasing the culture volume to a final culture volume; and once the final culture volume is reached, perfusing the culture at one or more perfusion rates of 0.5 culture volumes per day (V / d) or less until the culture is terminated or harvested.

[0007] In one embodiment, the culture is initiated with a culture volume that is at least 50%-75% of the final bioreactor working volume. In a related embodiment, the culture is initiated with a culture volume that is at least 60%-70% of the final bioreactor working volume.

[0008] In one embodiment, the culture is initiated with a culture volume that is about 50% to about 75% of the final bioreactor working volume. In a related embodiment, the culture is initiated with a culture volume that is about 60% to about 70% of the final bioreactor working volume.

[0009] In another embodiment, the culture contains at least 5 x 10 6 cells / mL ~ approx. 50×10 6 In a related embodiment, the culture is seeded at a cell density of at least 6 x 10 cells / mL. 6 cells / mL ~ approx. 20×10 6 Cells are seeded at a cell density of 1000 cells / mL.

[0010] In another embodiment, the culture is 6 cells / mL ~ approx. 50×10 6 In a related embodiment, the culture is seeded at a cell density of about 6 x 10 cells / mL. 6 cells / mL ~ approx. 20×10 6 Cells are seeded at a cell density of 1000 cells / mL.

[0011] In another embodiment, the bioreactor is operated in batch mode for up to 24 hours after inoculation. In a related embodiment, the bioreactor is operated in batch mode for about 24 hours after inoculation.

[0012] In yet another embodiment, the culture is in a growth phase before increasing the culture volume. In a related embodiment, the duration of the growth phase is 60% or less of the culture duration. In a related embodiment, the culture temperature during the growth phase is 35°C to 37°C.

[0013] In another embodiment, the culture is in a production phase after increasing the culture volume. In a related embodiment, the culture temperature during the production phase is between 28°C and 35°C.

[0014] In yet another embodiment, the perfusion rate is at least 0.05 V / d. In another embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.25 V / d. In related embodiments, the perfusion rate is at least about 0.15 V / d. In related embodiments, the perfusion rate is at least about 0.20 V / d. In related embodiments, the perfusion rate is at least about 0.25 V / d.

[0015] In another embodiment, the perfusion rate is between about 0.10 V / d and about 0.25 V / d. In a related embodiment, the perfusion rate is about 0.15 V / d. In a related embodiment, the perfusion rate is about 0.20 V / d. In a related embodiment, the perfusion rate is about 0.25 V / d.

[0016] In one embodiment, the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d (e.g., 0.1 V / d and 0.5 V / d; 0.15 V / d and 0.5 V / d; 0.2 V / d and 0.5 V / d; 0.25 V / d and 0.5 V / d), where the feed rate and concurrent permeate rate are the same, until the culture reaches one or more desired target criteria. In a related embodiment, the culture is perfused at a perfusion rate between 0.05 V / d and 0.5 V / d, where the feed rate and permeate rate are the same, until the culture reaches one or more desired target criteria.

[0017] In one embodiment, once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d (e.g., 0.1 V / d and 0.5 V / d; 0.15 V / d and 0.5 V / d; 0.2 V / d and 0.5 V / d; 0.25 V / d and 0.5 V / d), with the feed rate and concurrent permeate rate being the same, until the culture is terminated or harvested. In a related embodiment, once the final culture volume is reached, the culture is perfused at a perfusion rate between 0.05 V / d and 0.5 V / d, with the feed rate and permeate rate being the same, until the culture is terminated or harvested.

[0018] In another embodiment, the desired target criterion is selected from culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production, and / or plant schedule. In a related embodiment, the desired target criterion is cell density and / or time post-inoculation. In a related embodiment, the desired target criterion is cell density. In a related embodiment, the desired target criterion is time post-inoculation.

[0019] In a related embodiment, the desired target criteria is at least 100×10 5In another related embodiment, the desired target criteria is a cell density of up to 350 x 10 cells / mL. 5 Cell density is in cells / mL.

[0020] In a related embodiment, the desired target time is at least about 24 hours after seeding. In another related embodiment, the desired target time is at least about 48 hours to about 72 hours after seeding. In another related embodiment, the desired target time is about 48 hours to about 72 hours after seeding.

[0021] In another embodiment, differential perfusion is used to increase the culture volume. In a related embodiment, during differential perfusion, the feed rate is faster than the permeate rate. In another related embodiment, differential perfusion includes one or more feed rates and one or more permeate rates. In one related embodiment, at least one feed rate is 0.50 V / d or less. In yet another related embodiment, at least one feed rate is 0.25 V / d or less. In another related embodiment, at least one feed rate is 0.50 V / d or less and the permeate rate is 0.20 V / d or less. In another embodiment, at least one feed rate is 0.40 V / d or less and the permeate rate is 0.15 V / d or less. In yet another related embodiment, the higher feed rate is maintained for at least 6 hours. In another related embodiment, the feed rate is selected to increase the medium volume in the bioreactor to a volume of 100% or less of the bioreactor working volume in the time required for at least one cell population doubling of the culture. In another embodiment, differential perfusion continues until the culture volume in the bioreactor is less than or equal to about 70%-100% of the final bioreactor working volume.

[0022] In another embodiment, the final working volume of the bioreactor is at least 70%-100% of the bioreactor volume. In a related embodiment, the final working volume of the bioreactor is at least 90% of the bioreactor volume. In a related embodiment, the final working volume of the bioreactor is 100% or less of the bioreactor volume. In a related embodiment, the final working volume of the bioreactor is about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% of the bioreactor volume.

[0023] In one embodiment, the cell density at the start of the production phase is at least twice the seed cell density. In another related embodiment, the cell density at the start of the production phase is at least 200 x 10 5 cells / mL.

[0024] In another embodiment, the production period is at least 10 days, and in yet another related embodiment, the production period is at least 10-20 days.

[0025] In one embodiment, the bioreactor is coupled to a separation system. In a related embodiment, the separation system includes a pumping mechanism and a filter or membrane module. In a related embodiment, the separation system is a tangential flow filtration system. In a related embodiment, the tangential flow filtration system is a recirculating tangential flow filter system or an alternating tangential flow filtration system.

[0026] In one embodiment, the bioreactor volume is between 200 liters and 20,000 liters.

[0027] In one embodiment, the bioreactor is a disposable bioreactor or a stainless steel bioreactor.

[0028] In one embodiment, the cell is a mammalian cell, preferably a CHO cell.

[0029] In one embodiment, the recombinant protein is an antibody, preferably an IgG antibody, hi another embodiment, the recombinant protein is an antibody fragment.

[0030] The present disclosure also provides a method of culturing cells to produce a recombinant protein, the method comprising initiating a culture in a bioreactor having a culture volume that is at least 50% of a final bioreactor working volume, and culturing at least 5×10 cells. 6 The method includes seeding the culture with cells engineered to express a recombinant protein at a cell density of 1000 cells / mL; perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture reaches a desired target standard; increasing the volume of the culture medium by differential perfusion (the feed rate is higher than the permeate rate) to approximately 70%-100% of the final bioreactor working volume; and perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture is terminated or harvested.

[0031] The present disclosure provides a method for producing isolated and purified recombinant proteins, comprising initiating a culture in a bioreactor with a culture volume that is at least 50% of the final bioreactor working volume, and culturing at least 6×10 6 Also provided is a method comprising inoculating a culture with cells expressing a recombinant protein at a cell density of 10 ...

[0032] In one embodiment, at least one of the one or more unit operations is an affinity chromatography unit operation, hi a related embodiment, the affinity chromatography medium is selected from Protein A and immobilized metal affinity chromatography (IMAC) medium.

[0033] In one embodiment, at least one of the one or more unit operations is a polishing chromatography unit operation. In a related embodiment, the polishing chromatography medium is selected from an anion exchange chromatography medium, a cation exchange chromatography medium, a multimodal chromatography medium, a hydrophobic interaction chromatography medium, and a hydroxyapatite chromatography medium. In a related embodiment, the at least one polishing chromatography medium is operated in bind-and-elute mode, flow-through mode, or frontal mode.

[0034] In one embodiment, at least one of the one or more unit operations is selected from viral inactivation, viral filtration, depth filtration, and ultrafiltration / diafiltration (UF / DF).

[0035] In one embodiment, the cell is a mammalian cell, preferably a CHO cell.

[0036] In one embodiment, the recombinant protein is an antibody, preferably an IgG antibody, hi another embodiment, the recombinant protein is an antibody fragment.

[0037] The disclosure also provides a method for producing a recombinant protein, the method comprising initiating a culture in a bioreactor with cells engineered to express the recombinant protein, culturing the cells by perfusing the culture at a rate of 0.5 V / d or less to maintain a culture volume of at least 60% of the final bioreactor working volume, at least 24 hours after inoculation, subjecting the culture to a feed-up phase to increase the culture volume to 100% or less of the final bioreactor working volume, and after the feed-up phase, perfusing the culture at a rate of 0.5 V / d or less to maintain the culture volume at more than 70% of the final bioreactor working volume; and harvesting the recombinant protein.

[0038] In one embodiment, the cell is a mammalian cell, preferably a CHO cell.

[0039] In one embodiment, the recombinant protein is an antibody, preferably an IgG antibody. In some embodiments, the recombinant protein is an antibody fragment.

[0040] Non-limiting examples of the present disclosure also include: E1. A method for culturing cells to produce a recombinant protein, comprising: initiating a culture in a bioreactor with a culture volume that is at least 50% of the final bioreactor working volume; seeding the culture with cells engineered to express the protein; perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture reaches one or more desired goal criteria; Increasing the culture volume to a final culture volume; Once the final culture volume is achieved, perfusing the culture at one or more perfusion rates of less than or equal to 0.5 culture volumes / day (V / d) until the culture is terminated or harvested.

[0041] E2. The method of E1, wherein the culture is initiated with a culture volume that is at least 50% to 75% of the final bioreactor working volume.

[0042] E3. The method of E2, wherein the culture is initiated with a culture volume that is at least 60%-70% of the final bioreactor working volume.

[0043] E4. Culture at least 5 x 10 6 cells / mL ~ approx. 50×10 6 The cells are seeded at a cell density of 1000 cells / mL as described in E1.

[0044] E5. Culture at least 6 x 10 6 cells / mL ~ approx. 20×10 6 The method described in E4 is seeded at a cell density of 1000 cells / mL.

[0045] E6. The method of E1, wherein the bioreactor is operated in batch mode for up to 24 hours after inoculation.

[0046] E7. The method of E1, wherein the culture is in a growth phase before increasing the culture volume.

[0047] E8. The method according to E7, wherein the duration of the proliferation phase is 60% or less of the culture period.

[0048] E9. The method according to E7, wherein the culture temperature during the growth phase is 35°C to 37°C.

[0049] E10. The method according to E1, wherein the culture is in the production phase after increasing the culture volume.

[0050] E11. The method according to E10, wherein the culture temperature during the production phase is 28°C to 35°C.

[0051] E12. The method according to E1, wherein the perfusion rate is at least 0.05 V / d.

[0052] E13. The method of E1, wherein the perfusion rate is at least about 0.10 V / d to at least about 0.25 V / d.

[0053] E14. The method of E1, wherein the desired target criteria is selected from culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production and / or plant schedule.

[0054] E15. The method of E14, wherein the desired target criteria is cell density and / or time since seeding.

[0055] E16. The method of E14, wherein the desired target criterion is a cell density of at least 100E5 cells / mL.

[0056] E17. The method according to E16, wherein the desired target criterion is a cell density of up to 350E5 cells / mL.

[0057] E18. The method of E14, wherein the desired target criterion is at least about 24 hours after seeding.

[0058] E19. The method of E18, wherein the desired target criteria is at least about 48-72 hours after seeding.

[0059] E20. Increase culture volume using differential perfusion, as described in E1.

[0060] E21. The method of E20, wherein during differential perfusion, the feed rate is greater than the permeate rate.

[0061] E22. The method of E21, wherein the differential perfusion comprises one or more feed rates and one or more permeation rates.

[0062] E23. The method of E22, wherein at least one supply rate is 0.50 V / d or less.

[0063] E24. The method according to E22, wherein at least one supply rate is 0.25 V / d or less.

[0064] E25. The method of E22, wherein at least one feed rate is 0.50 V / d or less and the permeation rate is 0.20 V / d or less.

[0065] E26. The method of E22, wherein at least one feed rate is 0.40 V / d or less and the permeation rate is 0.15 V / d or less.

[0066] E27. The method of E21, wherein the higher feed rate is maintained for at least 6 hours.

[0067] E28. The method of E21, wherein the feed rate is selected to increase the medium volume in the bioreactor to a volume of no more than 100% of the bioreactor working volume in a time required for at least one cell population doubling of the culture.

[0068] E29. The method of E1, wherein differential perfusion is continued until the culture volume in the bioreactor is less than or equal to about 70%-100% of the final bioreactor working volume.

[0069] E30. The method of E1, wherein the final working volume of the bioreactor is at least 70%-100% of the bioreactor volume.

[0070] E31. The method according to E10, wherein the cell density at the start of the production phase is at least twice the seeding cell density.

[0071] E32. The cell density at the start of the production phase is at least 200 x 10 5 cells / mL, as described in E10.

[0072] E33a. The method of E10, wherein the production phase is at least 10 days.

[0073] E33b. The method of E32, wherein the production period is at least 10-20 days.

[0074] E34. The method of E1, wherein the bioreactor is connected to a separation system.

[0075] E35. The method of E34, wherein the separation system includes a pumping mechanism and a filter or membrane module.

[0076] E36. The method of E34, wherein the separation system is a tangential flow filtration system.

[0077] E37. The method of E36, wherein the tangential flow filter system is a recirculating tangential flow filter system or an alternating tangential flow filtration system.

[0078] E38. The method of E1, wherein the bioreactor volume is between 200 liters and 20,000 liters.

[0079] E39. The method of E1, wherein the bioreactor is disposable or stainless steel.

[0080] E40. A method for culturing cells to produce a recombinant protein, comprising: Initiating a culture in a bioreactor having a culture volume that is at least 50% of the final bioreactor working volume; Protein at least 6 x 10 6 seeding the culture with cells engineered to express at a cell density of 1000 cells / mL; perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture reaches a desired target standard; Increasing the medium volume to approximately 70% to 100% of the final bioreactor working volume by differential perfusion (feed rate is greater than permeate rate); perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture is terminated or harvested; A method comprising:

[0081] E41. A method for producing an isolated and purified recombinant protein, comprising: initiating a culture in a bioreactor with a culture volume that is at least 50% of the final bioreactor working volume; At least 6 x 10 6 seeding the culture with cells expressing the protein at a cell density of 1000 cells / mL; maintaining the culture in the growth phase by perfusing the culture at a rate of at least 0.05 V / d until the culture reaches the desired target criteria; increasing the culture volume by differential perfusion until the culture volume is at least 70% of the final bioreactor working volume; maintaining the culture in the production phase by perfusing the culture at a rate of at least 0.05 V / d; recovering the recombinant protein; processing the recombinant protein through one or more unit operations; Obtaining an isolated and purified recombinant protein; A method comprising:

[0082] E42. The method of E41, wherein at least one unit operation is an affinity chromatography unit operation.

[0083] E43. The method of E42, wherein the affinity chromatography medium is selected from Protein A or immobilized metal affinity chromatography (IMAC).

[0084] E44. The method of E41, wherein at least one unit operation is a polish chromatography unit operation.

[0085] E45. The method of E44, wherein the polishing chromatography medium is selected from anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

[0086] E46. The method of E45, wherein at least one polished chromatography medium is operated in bind-and-elute mode, flow-through mode, or frontal mode.

[0087] E47. The method of E41, wherein at least one unit operation is selected from viral inactivation, viral filtration, depth filtration, and UF / DF.

[0088] E48. A method for producing a recombinant protein, comprising: initiating a culture in a bioreactor with cells engineered to express the protein; Culturing the cells by perfusing the culture at a rate of 0.5 V / d or less to maintain a culture volume of at least 60% of the final bioreactor working volume; subjecting the culture to a feed-up phase to increase the culture volume to no more than 100% of the final bioreactor working volume for at least 24 hours after inoculation; Following the feed-up phase, perfusing the culture at a rate of 0.5 V / d or less to maintain a culture volume greater than 70% of the final bioreactor working volume; recovering the recombinant protein; and A method comprising:

[0089] Further non-limiting example embodiments / features include: F1. A method of culturing cells to produce a recombinant protein, comprising: initiating a culture in a bioreactor with a culture volume that is at least 50% of the final bioreactor working volume; seeding the culture with cells engineered to express the recombinant protein; perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture reaches one or more desired goal criteria; Increasing the culture volume to a final culture volume; Once the final culture volume is reached, perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until the culture is terminated or harvested; A method comprising:

[0090] F2. The method according to F1, wherein the culture is initiated with a culture volume that is 50%-75% of the final bioreactor working volume.

[0091] F3. The method according to F1 or F2, wherein the culture is initiated with a culture volume that is 60% to 70% of the final bioreactor working volume.

[0092] F4.5×10 6 cells / mL~50×10 6 The method of any one of F1 to F3, wherein the culture is seeded to a cell density of 1000 cells / mL.

[0093] F5.6×10 6 cells / mL~20×10 6 The method of any one of F1 to F4, wherein the culture is seeded to a cell density of 1000 cells / mL.

[0094] F6.6×10 6 cells / mL~12×10 6 The method of any one of F1 to F5, wherein the culture is seeded to a cell density of 1000 cells / mL.

[0095] F7. The method of any one of F1-F6, wherein the bioreactor is operated in batch mode for up to 24 hours after inoculation.

[0096] F8. The method of any one of F1 to F7, wherein the culture is in a growth phase prior to increasing the culture volume to the final culture volume.

[0097] F9. The method according to F8, wherein the duration of the growth phase is 60% or less of the culture period.

[0098] F10. The method according to F8 or F9, wherein the culture temperature during the growth phase is 35°C to 37°C.

[0099] F11. The method according to any one of F1 to F10, wherein the culture is in the production phase after increasing the culture volume to the final culture volume.

[0100] F12. The method according to F11, wherein the culture temperature during the production phase is 28°C to 35°C.

[0101] F13. The method according to F11 or F12, wherein the duration of the production period is 10 to 20 days.

[0102] F14. The method of any one of F11 to F13, wherein the cell density at the start of the production phase is at least twice the seeding cell density.

[0103] F15. The cell density at the start of the production phase is at least 200 x 10 5 The method according to any one of F11 to F14, wherein the concentration is cells / mL.

[0104] F16. Perfusing the culture at one or more perfusion rates between 0.05 V / d and 0.5 V / d, with the feed rate and concurrent permeate rate being the same, until the culture reaches one or more desired goal criteria; and / or The method according to any one of F1 to F15, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d, and the feed rate and the simultaneously used permeate rate are the same, until the culture is terminated or harvested.

[0105] F17. Perfusing the culture at one or more perfusion rates between 0.10 V / d and 0.25 V / d, with the feed rate and concurrent permeate rate being the same, until the culture reaches one or more desired goal criteria; and / or The method of any one of F1 to F16, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.10 V / d and 0.25 V / d, and the feed rate and the simultaneously used permeate rate are the same, until the culture is terminated or harvested.

[0106] F18. The method of any one of F1-F17, wherein the culture is perfused at a constant perfusion rate before increasing the culture volume. F19. The method of any one of F1-F18, wherein once the final culture volume is reached, the culture is perfused at a constant perfusion rate until the culture is terminated or harvested.

[0107] F20. The culture is first perfused at a constant perfusion rate before increasing the culture volume; Once the final culture volume is reached, the culture is perfused at a second constant perfusion rate until the culture is terminated or harvested; The method of any one of F1 to F19, wherein the first constant perfusion rate and the second constant perfusion rate are the same. F21. The method of any one of F1-F20, wherein the one or more desired target criteria are selected from culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production schedule, plant schedule, and any combination of the foregoing.

[0108] F22. One or more desired goal criteria are 100 x 10 5 cells / mL~350×10 5 The method according to any one of F1 to F21, wherein the cell density is cells / mL.

[0109] F23. The method of any one of F1-F22, wherein the one or more desired target criteria is time since seeding, and the time since seeding is between 24 hours and 72 hours.

[0110] F24. The method of any one of F1-F23, wherein the culture volume is increased to the final culture volume using differential perfusion, wherein the differential perfusion comprises one or more feed rates and one or more permeate rates.

[0111] F25. The method according to F24, wherein at least one of the one or more feed rates is 0.50 V / d or less, and at least one of the one or more permeation rates is 0.20 V / d or less, and each of the one or more feed rates is faster than the permeation rate used simultaneously.

[0112] F26. The method of F24 or F25, wherein at least one of the one or more feed rates is 0.40 V / d or less, and at least one of the one or more permeation rates is 0.15 V / d or less, and each of the one or more feed rates is faster than the permeation rates used simultaneously.

[0113] F27. The method of F24, wherein each of the one or more feed rates is 0.50 V / d or less, each of the one or more permeation rates is 0.20 V / d or less, and each of the one or more feed rates is faster than the permeation rates used simultaneously.

[0114] F28. The method of F24 or F25, wherein each of the one or more feed rates is 0.40 V / d or less, each of the one or more permeation rates is 0.15 V / d or less, and each of the one or more feed rates is faster than the permeation rates used simultaneously.

[0115] F29. The method of any one of F24-F28, wherein the one or more feed rates and one or more permeation rates are selected to increase the medium volume in the bioreactor to a volume that is no greater than 100% of the bioreactor working volume in the time required for at least one cell population doubling of the culture.

[0116] F30. The method of any one of F24-F29, wherein the differential perfusion comprises a constant feed rate and a constant permeate rate, the constant feed rate being faster than the constant permeate rate.

[0117] F31. The method according to any one of F24 to F30, wherein differential perfusion is continued until the culture volume in the bioreactor is 70% to 100% of the final bioreactor working volume.

[0118] F32. The method according to any one of F1 to F31, wherein the final working volume of the bioreactor is between 70% and 100% of the bioreactor volume.

[0119] F33. A method of culturing cells to produce a recombinant protein, comprising: Initiating the culture in a bioreactor with a culture volume that is less than 50% to 70% of the final bioreactor working volume; Seeding the culture with cells engineered to express the recombinant protein (5 x 10 6 cells / mL~50×10 6 cells / mL cell density); operating the bioreactor in batch mode for up to 24 hours after inoculation; perfusing the culture for 24 to 72 hours at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less (feed rate and concurrent permeate rate are the same); Increasing the culture volume to 70%-100% of the final bioreactor working volume; Once the final culture volume is reached, perfuse the culture at one or more perfusion rates of 0.5 culture volumes per day (V / d) or less (feed rate and permeate rate used simultaneously are the same) until the culture is terminated or harvested. A method comprising:

[0120] F34.6×10 6 cells / mL~12×10 6 The method described in F33, in which the cultures are seeded at a cell density of 1000 cells / mL.

[0121] F35. The method of F33 or F34, wherein the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d for 24 hours to 60 hours before increasing the culture volume.

[0122] F36. The method of any one of F33 to F35, wherein the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d for 24 to 48 hours before increasing the culture volume.

[0123] F37. The method of any one of F33-F36, wherein the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d for 48 hours before increasing the culture volume.

[0124] F38. The method of F33 or F34, wherein the culture is perfused at one or more perfusion rates between 0.10 V / d and 0.25 V / d for 24 hours to 60 hours before increasing the culture volume.

[0125] F39. The method of any one of F33, F34, or F38, wherein the culture is perfused at one or more perfusion rates of 0.10 V / d to 0.25 V / d for 24 to 48 hours before increasing the culture volume.

[0126] F40. The method of any one of F33, F34, F38, or F39, wherein the culture is perfused at one or more perfusion rates between 0.10 V / d and 0.25 V / d for 48 hours before increasing the culture volume.

[0127] F41. The method of any one of F33 to F40, wherein the culture is perfused at a constant perfusion rate before increasing the culture volume.

[0128] F42. The method of any one of F33 to F41, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d until the culture is terminated or harvested.

[0129] F43. The method of any one of F33 to F42, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.10 V / d and 0.25 V / d until the culture is terminated or harvested.

[0130] F44. The method of any one of F33-F43, wherein once the final culture volume is reached, the culture is perfused at a constant perfusion rate until the culture is terminated or harvested.

[0131] F45. The culture is first perfused at a constant perfusion rate before increasing the culture volume; Once the final culture volume is reached, the culture is perfused at a second constant perfusion rate until the culture is terminated or harvested; The method of any one of F33 to F44, wherein the first constant perfusion rate and the second constant perfusion rate are the same.

[0132] F46. The method of any one of F33-F45, wherein the culture volume is increased to the final culture volume using differential perfusion.

[0133] F47. A method for culturing cells to produce a recombinant protein, comprising: Initiating the culture in the bioreactor at a culture volume that is less than 50% to 70% of the final bioreactor working volume; Seeding the culture with cells engineered to express the recombinant protein (5 x 10 6 cells / mL~50×10 6 cells / mL cell density); operating the bioreactor in batch mode for up to 24 hours after inoculation; Cell density is 100 x 10 5 cells / mL~350×10 5 perfusing the culture at one or more perfusion rates of 0.5 culture volumes / day (V / d) or less until a value in the range of cells / mL is reached (feed rate and simultaneously used permeate rate are the same); Increasing the culture volume to 70%-100% of the final bioreactor working volume; Once the final culture volume is reached, perfusing the culture at one or more perfusion rates of 0.5 culture volumes per day (V / d) or less (feed rate and concurrent permeate rate are the same) until the culture is terminated or harvested; A method comprising:

[0134] F48. Culture is 6 x 10 6 cells / mL~12×10 6The method described in F47 is seeded at a cell density of 1000 cells / mL.

[0135] F49. Cell density is 100 × 10 5 cells / mL~350×10 5 The method of F47 or F48, wherein the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d until a value in the range of cells / mL is reached.

[0136] F50. Cell density is 100 x 10 5 cells / mL~350×10 5 The method of any one of F47-F49, wherein the culture is perfused at one or more perfusion rates between 0.10 V / d and 0.25 V / d until a value in the range of cells / mL is reached.

[0137] F51. Cell density is 100 × 10 5 cells / mL~350×10 5 The method of any one of F47 to F50, wherein the culture is perfused at a constant perfusion rate until a value in the range of cells / mL is reached.

[0138] F52. The method of any one of F47 to F51, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.05 V / d and 0.5 V / d until the culture is terminated or harvested.

[0139] F53. The method of any one of F47 to F52, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates between 0.10 V / d and 0.25 V / d until the culture is terminated or harvested.

[0140] F54. The method of any one of F47-F53, wherein once the final culture volume is reached, the culture is perfused at a constant perfusion rate until the culture is terminated or harvested.

[0141] F55. Before increasing the culture volume, the culture is first perfused at a constant perfusion rate; Once the final culture volume is reached, the culture is perfused at a second constant perfusion rate until the culture is terminated or harvested; the first constant perfusion rate and the second constant perfusion rate are the same; A method according to any one of F47 to F54.

[0142] F56. The method of any one of F47-F55, wherein the culture volume is increased to the final culture volume using differential perfusion.

[0143] F57. The method of any one of F1-F56, wherein the bioreactor is connected to a separation system.

[0144] F58. The method of F57, wherein the separation system is a recirculating tangential flow filter system or an alternating tangential flow filtration system.

[0145] F59. The method according to any one of F1 to F58, wherein the volume of the bioreactor is between 200 liters and 20,000 liters.

[0146] F60. The method of any one of F1-F59, wherein the bioreactor is a disposable bioreactor or a stainless steel bioreactor.

[0147] F61. Recovering recombinant proteins; processing the recombinant protein through one or more unit operations; Obtaining an isolated and purified recombinant protein; The method according to any one of F1 to F60, further comprising:

[0148] F62. The method of F61, wherein at least one of the one or more unit operations is an affinity chromatography unit operation.

[0149] F63. The method of F61 or F62, wherein at least one of the one or more unit operations is a polishing chromatography unit operation.

[0150] F64. The method of any one of F61 to F63, wherein at least one of the one or more unit operations is selected from viral inactivation, viral filtration, depth filtration, and ultrafiltration / diafiltration (UF / DF).

[0151] F65. The method of any one of F1 to F64, wherein the recombinant protein is an antibody.

[0152] F66. The method of any one of F1 to F64, wherein the recombinant protein is an IgG antibody.

[0153] F67. The method of any one of F1 to F64, wherein the recombinant protein is an antibody fragment.

[0154] F68. The method of any one of F1 to F67, wherein the cells are CHO cells. [Brief explanation of the drawings]

[0155] [Figure 1] Figure 1 shows an offset schematic for a non-limiting example lean perfusion cell culture from culture initiation to harvest, highlighting the changes in culture volume and feed and permeate rates. At the start of the culture and during the growth phase, the culture volume is maintained low to at least 50%-75% of the final working volume of the bioreactor. Maintaining the same feed and permeate rates maintains the culture volume stationary. During the feed-up period, the feed rate is increased relative to the permeate rate, resulting in an increase in culture volume to the desired final volume (typically 75%-100% or more of the final working volume), after which the feed and permeate rates are maintained at the same rate to maintain the static culture volume. The top plot shows the change in culture volume from initiation to harvest. The middle and bottom plots show that the feed rate is maintained at the same rate as the permeate rate (rates 1 and 3), except during the feed-up phase. During the feed-up phase, the feed rate (rate 2) is increased to facilitate the increase in culture volume in the bioreactor. [Figure 2]Figure 2 shows a schematic of culture volume change over the course of a culture from initiation to harvest. A typical lean perfusion culture with a single feed-up phase (black line) is compared to a typical fed-batch culture with multiple feeds (gray line). Both cultures are initiated with a low culture volume of at least 50%–75% of the final working volume of the bioreactor. The lean perfusion method utilizes a shortened growth phase at a low culture volume, followed by a transition to a production phase with a feed-up of increasing culture volume, maximizing the bioreactor working volume during both phases. The fed-batch method slowly increases the culture volume by stepwise or intermittent feeding to achieve the final working volume at the desired cell density as the culture begins to decline. [Figure 3] Figure 3 shows the mean viable cell density (VCD) for lean perfusion and fed-batch cultures. Lean perfusion (LP): dark gray solid line for mAb1, black solid line for mAb2, light gray solid line for mAb3. Fed-batch culture (FB): dark gray dashed line for mAb1, black dashed line for mAb2, light gray dashed line for mAb3. [Figure 4-1] Figures 4A, 4B, and 4C show the average specific productivity (pg / cell / day) of lean perfusion and fed-batch cultures. Lean perfusion (LP) black columns, fed-batch (FB) gray columns. mAb1 (Figure 4A), mAb2 (Figure 4B), and mAb3 (Figure 4C). [Figure 4-2] Figures 4A, 4B, and 4C show the average specific productivity (pg / cell / day) of lean perfusion and fed-batch cultures. Lean perfusion (LP) black columns, fed-batch (FB) gray columns. mAb1 (Figure 4A), mAb2 (Figure 4B), and mAb3 (Figure 4C). [Figure 5-1] Figures 5A, 5B, and 5C show the average end-of-day titers for lean perfusion and fed-batch cultures. Lean perfusion (LP) black columns, fed-batch (FB) gray columns. mAb1 (Figure 5A), mAb2 (Figure 5B), and mAb3 (Figure 5C). [Figure 5-2] Figures 5A, 5B, and 5C show the average end-of-day titers for lean perfusion and fed-batch cultures. Lean perfusion (LP) black columns, fed-batch (FB) gray columns. mAb1 (Figure 5A), mAb2 (Figure 5B), and mAb3 (Figure 5C). [Figure 6]Figure 6 shows the average lactate production during lean perfusion and fed-batch cultures. Lean perfusion culture: dark gray solid line for mAb1, black solid line for mAb2, light gray solid line for mAb3. Fed-batch culture: dark gray dashed line for mAb1, black dashed line for mAb2, light gray dashed line for mAb3. [Figure 7] Figure 7 shows a schematic of perfusion rates over the course of a culture from initiation to harvest. A typical lean perfusion culture (black line) maintains a low perfusion rate (<0.5 V / d) throughout the entire culture, while a typical perfusion culture (gray line) can be initiated at a desired final perfusion rate (typically 1.0 V / d or higher) or can optionally be initiated at a lower rate and ramped up to the final rate early in the culture. [Figure 8] Figure 8 shows a schematic diagram of the feed medium used over the culture period from initiation to harvest: lean perfusion culture (black line, low perfusion rate); perfusion culture (gray line, higher perfusion rate). [Figure 9] Figure 9 shows the feed medium volumes used during the culture: lean perfusion culture (LP, black column) and perfusion culture (PC, dark grey column). [Figure 10] Figure 10 shows a schematic of the change in culture volume over time. The increase in culture volume during lean perfusion culture (black line) is compared to static culture volume for perfusion culture (gray line). [Figure 11] Figure 11 shows the mean viable cell density (VCD) of lean perfusion cultures compared to perfusion cultures. Lean perfusion cultures: mAb2 (solid black line), mAb3 (solid gray line). Perfusion cultures: mAb2 (dashed black line), mAb3 (dashed gray line). [Figure 12] Figures 12A and 12B show the mean end-of-day titers of lean perfusion cultures compared to perfusion cultures. Lean perfusion cultures (LP): black columns, perfusion cultures (PR): gray columns. mAb2 (Figure 12A), mAb3 (Figure 12B). [Figure 13] Figures 13A and 13B show the average specific productivity (pg / cell / day) of lean perfusion culture compared to perfusion culture. Lean perfusion culture (LP): black columns, perfusion culture (PR): gray columns. mAb2 (Figure 13A), mAb3 (Figure 13B). [Figure 14] Figures 14A and 14B show the mean packed cell volume (%) of lean perfusion cultures compared to perfusion cultures. Lean perfusion cultures (LP): black columns, perfusion cultures (PR): gray columns. mAb2 (Figure 14A), mAb3 (Figure 14B). [Figure 15] FIG. 15 shows the mean viable cell density (VCD) achieved by mAb4 lean perfusion culture (solid black line) and fed-batch culture (solid gray line) from initiation to harvest. [Figure 16] FIG. 16 shows the mean end-day titers (g / L) for mAb4 lean perfusion cultures (black columns) and fed-batch cultures (gray columns). [Figure 17] FIG. 17 shows a comparison of the average lactate concentrations for mAb4 lean perfusion culture (solid black line) and fed-batch culture (solid gray line) from start to harvest. DETAILED DESCRIPTION OF THE INVENTION

[0156] Described herein is a lean perfusion cell culture method for producing a recombinant protein product of interest. Surprisingly, lean perfusion is a more efficient and productive culture method compared to traditional batch, fed-batch, and perfusion culture methods. This method utilizes perfusion, or medium exchange, to add fresh medium to the culture and remove spent medium from the culture. The perfusion rate throughout the culture, from initiation to harvest, is maintained at 50% or less of the perfusion rate used in typical perfusion culture methods operated at high perfusion rates. Additionally, lean perfusion cultures are initiated at high cell densities in low culture volumes that are at least 50% of the bioreactor's final working volume. This allows for more efficient use of the bioreactor working volume during the growth phase. Once one or more desired target criteria are met, the culture volume is then increased and maintained throughout the production phase until harvest. Due to the increased culture volume and relatively low cell density, lean perfusion cultures efficiently utilize the entire bioreactor working volume during the production phase, which, combined with the low perfusion rate, reduces feed medium usage and waste generation. Compared to high perfusion rate perfusion processes, these features also allow for greater flexibility in bioreactor type and size (e.g., disposable and / or large-scale stainless steel bioreactors with capacities ranging from hundreds of liters up to tens of thousands of liters) and the use of common separation systems. For example, lean perfusion allows for the combination of disposable separation systems, such as alternating tangential flow systems, with large-scale (>5000 L) stainless steel bioreactors.

[0157] As used herein, "bioreactor volume" refers to the actual vessel size of a bioreactor, e.g., a 2000 L disposable bioreactor or a 20,000 L stainless steel bioreactor. Bioreactor volume is divided into working volume space and headspace. As used herein, "working volume" or "bioreactor working volume" refers to the volume within a bioreactor in which a cell culture is operated, typically expressed as a percentage of the bioreactor volume. For example, a 2,000 L stirred-tank disposable bioreactor may have a working volume of 100% of the bioreactor volume. For stainless steel bioreactors, the working volume is typically up to about 90% of the bioreactor volume. Some cell culture operations, including the lean perfusion culture process described herein, utilize different percentages of the available working volume over the course of the culture. For example, fed-batch and lean perfusion cultures are initiated with a culture volume that is 50% or more of the final bioreactor working volume, and as the culture progresses, the culture volume is increased to 100% of the final bioreactor working volume. As used herein, "final working volume" refers to the maximum volume within the bioreactor in which the cell culture is operated, typically during the production phase. As used herein, "culture volume" refers to the volume of all culture components in the bioreactor and associated equipment and flow paths, including culture medium, cells, cell debris, air bubbles, and foam. Culture volume is expressed as a percentage of the final working volume. For perfusion cultures, including lean perfusion cultures, the culture volume includes the working volume and hold-up volume in the perfusion flow path, including the filter. For large-scale disposable (SU) bioreactor cultures, e.g., cultures exceeding 500 L, the hold-up volume is negligible. Both the working volume and the culture volume can be increased over the culture period, depending on the process design.

[0158] Batch cell cultures are carried out in closed bioreactor systems containing a fixed volume of culture medium, which serves as a nutrient source to support the culture for its duration. Because no additional medium is added, a typical batch culture begins with culture medium containing all essential nutrients added at or near 100% of the bioreactor working volume. Large-scale bioreactors operating at volumes up to tens of thousands of liters are preferred. For cultures, cells in the growth phase are seeded at a low cell density to maintain waste levels below thresholds that would affect cell culture viability and productivity for as long as possible. As used herein, "cell density" refers to the number of viable cells in a given volume of culture medium. Harvesting is typically initiated when product quality, cell viability, and / or productivity decline.

[0159] Fed-batch culture is the most commonly used mammalian cell culture method for producing recombinant proteins. Fed-batch culture strategies are flexible, accommodate a wide range of bioreactor types and sizes, and can be scaled up or down to meet supply demands. Fed-batch culture offers an improvement over batch culture methods by supplementing the culture with fresh nutrient medium to replenish nutrients depleted during the course of the culture; however, as with batch culture, spent medium containing waste products is not removed from the bioreactor and accumulates over time. As with batch culture, large-scale bioreactors operating at volumes up to tens of thousands of liters are preferred for fed-batch operations due to low titers and to meet product demands. Harvesting is typically initiated when product quality, cell viability, and / or cell productivity decline.

[0160] A typical fed-batch culture is initiated into a basal culture medium containing essential nutrients in a culture volume that is at least 50% of the final working volume of the bioreactor. Fed-batch cultures are initiated in the growth phase; the culture is maintained at a low cell density (e.g., (2-60) x 10) to maintain waste levels below the threshold that affects cell culture health and viability for as long as possible. 5As the cell concentration increases, supplemental nutrient medium is added to the bioreactor, up to 100% of the bioreactor's final working volume, to extend cell life and improve productivity. These medium supplements can be made at regular intervals or in stages throughout the course of the culture. As with batch cultures, cells, proteins, spent culture medium, product and process-related impurities, waste products, etc. are all retained and accumulated in the bioreactor. Over time, these components accumulate, and the health of the culture begins to decline, which negatively impacts cell mass and titer. Fed-batch cultures are typically maintained for 8-15 days. Harvesting is typically initiated when product quality, cell viability, and / or cell productivity decline.

[0161] In recent years, perfusion, or medium exchange, has gained popularity for use in mammalian cell culture processes. Similar to fed-batch operations, nutrient solutions and / or various medium formulations are fed into the bioreactor over the course of the culture. Unlike batch and fed-batch methods, spent culture medium, including waste products, is removed in the permeate during the culture. Perfusion culture utilizes a separation system, including a pump used to direct the contents of the bioreactor through the separation system. Typically, the separation system includes one or more membrane filters (often operated in tangential flow mode) that selectively retain or return cell culture components to the bioreactor. Spent culture medium, waste products, and impurities, as well as other components, including recombinant proteins, can be selectively removed in the permeate depending on the selection criteria of the separation system. In the case of membrane filters, filter pore sizes or molecular weight cutoffs are used. The permeate can be sent to waste or collected for collection. Generally, perfusion cultures are initiated and maintained at or near 100% of the working volume of the bioreactor, and the culture achieves high cell density and / or compact cell mass due to the continuous exchange of medium.

[0162] As used herein, "packed cell volume" (PCV), also known as "packed cell volume percentage" (PCV%), is the ratio of the volume occupied by cells to the total volume of the cell culture (see Stealer, et al., (2006) Biotechnol. Bioeng. Dec 20:95(6):1228-33). Packed cell volume is a function of cell density and cell diameter, and an increase in packed cell volume can occur by increasing cell density, cell diameter, or both. Packed cell volume is an indicator of the solids content in a cell culture. Generally, cell cultures with a higher solids content require more processing to separate the solid material from the desired product during harvesting and downstream purification steps. Also, the desired product may be trapped in the solids and lost during the harvesting process, resulting in a lower production yield. Because host cells vary in size and cell cultures also contain dead and dying cells and other cell debris, packed cell volume is a more accurate way to represent the solids content in a cell culture than cell density or viable cell density. In addition, under growth arrest, some cells increase in size, and the increase in biomass as a result of cell size expansion may lead to differences in the compacted cell volume before and after growth arrest.

[0163] Similar to batch and fed-batch cultures, in perfusion cultures, cells are typically maintained in a bioreactor throughout the culture period. Unlike batch and fed-batch cultures, perfusion cultures typically have a higher cell mass and therefore generate larger amounts of waste and by-products over the course of the culture compared to batch or fed-batch processes, while also requiring larger amounts of fresh medium replenishment. Cell densities achieved during perfusion cultures are typically (20-120) x 10 6 cells / mL or more. Unlike batch and fed-batch cultures, perfusion cultures are performed in low-volume bioreactors (typically 2000 L or less) due to limitations in separation systems that allow for continuous medium exchange and large media consumption. These separation systems are affected by the high cell densities and high flow rates common in typical perfusion cultures. Furthermore, even in low-volume bioreactors, the high packed cell mass that can be achieved during perfusion culture generally limits harvest options.

[0164] Perfusion culture is based on medium exchange, i.e., the addition of fresh culture medium (feed medium) to the bioreactor and the removal of a similar volume of spent culture medium as a permeate through a separation system. Fresh cell culture medium can be added to the bioreactor in stages, intermittently, and / or continuously. The spent culture medium and selected waste products are removed from the bioreactor using one or more separation systems and then exit in the permeate. Traditional perfusion cultures are operated at high perfusion rates, where fresh medium is added to the bioreactor at 1.0 V / d or more and an equal volume of spent medium is removed. In some cases, perfusion cultures are initiated at perfusion rates of 0.5 V / d or more and rapidly increased in speed. This high perfusion rate consumes a larger amount of feed medium than an equivalent-sized fed-batch culture and also generates an equal volume of spent medium that must be disposed of. Because waste and by-products do not accumulate in the bioreactor to the same extent as they do in batch and fed-batch cultures, these cultures can be maintained for longer periods, e.g., 15 to 90 days or more.

[0165] Perfusion cultures utilize a separation system for medium exchange. The filters and / or pumps of conventional separation systems can be adversely affected by the high flow rates, high cell densities, high protein titers, and / or large amounts of waste and by-products associated with these cultures. Therefore, perfusion cultures are typically performed in smaller bioreactors, e.g., at least 200 L, typically 1000 L to 2000 L, to minimize the impact on the separation system. Additionally, the packed cell volume can be much higher in these cultures, which limits the ability to remove all of the desired protein from the bioreactor, resulting in lower recovery titers.

[0166] The "lean perfusion" or "lean perfusion rate" cell culture method described herein is unique. Like fed-batch cultures, lean perfusion cell cultures are initiated at low culture volumes (50%–75% of the final bioreactor working volume), but unlike fed-batch cultures, lean perfusion cultures are seeded at high cell densities. Lean perfusion methods are similar to typical perfusion cultures in that they utilize medium exchange, but differ in that the perfusion rate is maintained at 0.5 V / d or less throughout the entire process (potentially excluding the feed-up phase). The perfusion rate is maintained at a level sufficient to ensure the removal of waste by-products to avoid culture toxicity while delivering sufficient nutrients via fresh medium to maintain essential cell functions. Unlike both fed-batch and perfusion cultures, lean perfusion cultures maintain lower cell densities at higher culture volumes (up to 100% of the final bioreactor working volume) during the production phase, thereby again reducing the amount of feed required to maintain the culture and the amount of waste in the permeate stream (Figure 1).

[0167] By starting and maintaining a low culture volume at a higher cell density during the growth phase and transitioning to a higher culture volume at a lower cell density before the production phase, lean perfusion efficiently utilizes the bioreactor's working volume during each culture phase. This, combined with a low perfusion rate, optimizes the amount of culture medium used during each phase and reduces the volume of spent medium discarded compared to fed-batch and perfusion cultures. Additionally, maintaining a lower cell density and / or compacted cell mass during the production phase reduces the amount of waste that must be processed during harvest and downstream purification. Lean perfusion allows for flexibility in the type and size of bioreactors that can be used, enabling the use of everything from disposable bioreactors to large-volume stainless steel bioreactors. Additionally, the lower cell density, lower compacted cell mass, and smaller spent medium volume generated during the production phase allow for the use of common separation systems, such as disposable separation systems, for perfusion in combination with larger stainless steel bioreactors (up to 10,000 L to 20,000 L or more), allowing for greater harvest flexibility.

[0168] Lean perfusion also offers advantages over typical fed-batch and perfusion culture methods in that by inoculating a low-volume cell culture at a high cell density, the duration of the growth phase is shortened, desired target criteria, such as cell density, are achieved more quickly, and the amount of feed medium required is reduced. The compressed cell volume during the production phase was reduced by up to 50% compared to typical perfusion culture. The specific productivity of lean perfusion culture increased by 22% to 51% compared to typical fed-batch culture. The final titer increased by more than three-fold compared to typical fed-batch culture, and the final titer and specific productivity were comparable or better than those of typical perfusion culture. Additionally, lean perfusion increased cell mass compared to typical fed-batch culture, while maintaining low production of inhibitory metabolic by-products, such as lactate, through the use of low feed and permeate rates. Lean perfusion maintained comparable or improved performance while overcoming the limitations of fed-batch and perfusion culture methods.

[0169] Lean perfusion culture is initiated in a bioreactor using a cell culture medium suitable for culture. In one embodiment, the cell culture medium is a basal medium containing essential nutrients. In one embodiment, the culture volume in the bioreactor at the start of the culture is at least 50% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 50% to at least about 75% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 50% to at least about 70% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 50% to at least about 65% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 50% to at least about 60% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 50% to at least about 55% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 55% to at least about 75% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 55% to at least about 70% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 55% to at least about 65% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 55% to at least about 60% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 70% to at least about 75% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 60% to at least about 70% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 60% to at least about 65% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 65% to at least about 75% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 70% to at least about 75% of the final bioreactor working volume.

[0170] In one embodiment, the culture volume is at least 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, or 95% or more of the final bioreactor working volume. In one embodiment, the culture volume is greater than 50% of the final bioreactor working volume. In one embodiment, the culture volume is greater than 55% of the final bioreactor working volume. In one embodiment, the culture volume is greater than 60% of the final bioreactor working volume. In one embodiment, the culture volume is greater than 65% of the final bioreactor working volume. In one embodiment, the culture volume is greater than 70% of the final bioreactor working volume. In one embodiment, the culture volume is greater than 75% of the final bioreactor working volume. In one embodiment, the culture volume is at least 66% of the final bioreactor working volume. In one embodiment, the culture volume is at least 67% of the final bioreactor working volume. In one embodiment, the culture volume is at least 68% of the final bioreactor working volume. In one embodiment, the culture volume is at least 69% of the final bioreactor working volume. In one embodiment, the culture volume is at least about 70% of the final bioreactor working volume. In one embodiment, the culture volume is at least 75% of the final bioreactor working volume.

[0171] In one embodiment, the culture volume in the bioreactor at the start of culture is about 50% to about 75% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 50% to about 70% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 50% to about 65% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 50% to about 60% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 50% to about 55% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 55% to about 75% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 55% to about 70% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 55% to about 65% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 55% to about 60% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 70% to about 75% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 60% to about 70% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 60% to about 65% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 65% to about 75% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of culture is about 70% to about 75% of the final bioreactor working volume.

[0172] In one embodiment, the culture volume in the bioreactor at the start of the culture is about 50%, about 55%, about 60%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or more of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 55% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 60% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 65% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 66% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 67% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 68% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 69% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 70% of the final bioreactor working volume. In one embodiment, the culture volume in the bioreactor at the start of the culture is about 75% of the final bioreactor working volume.

[0173] The culture is inoculated with cells engineered to express the desired protein. In one embodiment, the culture volume at inoculation is at least 50% of the final bioreactor working volume, allowing for the use of N-1 seed bioreactors with a wide range of working volumes. In one embodiment, the culture is inoculated at a cell density that allows the culture to meet one or more desired process, production, and / or plant parameters. In one embodiment, the desired parameter is a shortened growth phase. For example, in some embodiments, at least 5.0 x 10 6 The culture is seeded at a cell density of at least 6.0 x 10 cells / mL. In one embodiment, the cell density is at least 6.0 x 10 6 cells / mL~50.0×10 6In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 25.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 20.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 15.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 14.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 13.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 11.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 15.0 x 10 6 In one embodiment, the cell density is at least 6.0 x 10 cells / mL. 6 cells / mL ~ at least 10.0 x 10 6 cells / mL.

[0174] In one embodiment, the seeded cell density is about 6×10 6 cells / mL ~ approx. 50×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 25×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 20×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 15×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6cells / mL ~ approx. 14×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 13×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 12×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 11×10 6 In one embodiment, the seeded cell density is about 6 x 10 cells / mL. 6 cells / mL ~ approx. 10×10 6 cells / mL.

[0175] In one embodiment, the culture contains at least 5 x 10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 10×10 6 , 11×10 6 , 12×10 6 , 13×10 6 , 14×10 6 , 15×10 6 , 20×10 6 , 25×10 6 , 30×10 6 , 35×10 6 , 40×10 6 , 45×10 6 , 50×10 6 In one embodiment, the cell density is at least 7.0 x 10 cells / mL or greater. 6 In one embodiment, the cell density is at least 8.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 9.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 10.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 11.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 12.0 x 10 cells / mL.6 In one embodiment, the cell density is at least 13.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 14.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 15.0 x 10 cells / mL. 6 In one embodiment, the cell density is at least 20.0 x 10 cells / mL. 6 cells / mL.

[0176] In one embodiment, the culture comprises approximately 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , about 9×10 6 , about 10×10 6 , about 11×10 6 , about 12×10 6 , about 13×10 6 , about 14×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 or about 50 x 10 6 Cells are seeded at a cell density of 1000 cells / mL.

[0177] In one embodiment, the seeded cell density is about 7 x 10 6 In one embodiment, the seeded cell density is about 8 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 9 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 10 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 11 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 12 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 13 x 10 cells / mL. 6In one embodiment, the seeded cell density is about 14 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 15 x 10 cells / mL. 6 In one embodiment, the seeded cell density is about 20 x 10 cells / mL. 6 cells / mL.

[0178] In one embodiment, the culture is inoculated with animal cells. Preferably, the cells are mammalian cells, such as, for example, CHO cells.

[0179] In one embodiment, the culture is maintained in batch mode for up to 48 hours after initiation to establish the cells in the culture environment. During batch mode, no medium is added to or removed from the culture. In one embodiment, batch mode is maintained for less than 12 hours. In one embodiment, batch mode is maintained for at least 12 to 24 hours (e.g., about 12.5 to about 24 hours). In one embodiment, batch mode is maintained for 24 to 48 hours. In one embodiment, medium changes are initiated when the culture is initiated.

[0180] Cell cultures typically include two or more distinct phases, including at least one growth phase and at least one production phase, with possible transition phases in between, during which culture conditions are manipulated to support shifts between the growth and production phases.

[0181] As used herein, "growth phase" refers to the period of exponential cell growth (i.e., log phase) during which cells generally divide rapidly. Culture conditions that promote cell growth and survival are established. The length of the growth phase can be based on various factors, including, for example, cell type, cell density, cell growth rate, culture conditions, production and / or plant scheduling or timeline. As used herein, "culture period" refers to the time from the start of the culture to its termination or harvest. The growth phase for a typical fed-batch or perfusion culture can be up to 75% of the culture period. In the lean perfusion method described herein, the growth phase is reduced to up to 60% of the culture period. In one embodiment, the growth phase is about 60% (e.g., 60%) of the culture period. In one embodiment, the growth phase is less than 60% of the culture period. In one embodiment, the growth phase is about 10% to about 60% of the culture period. In one embodiment, the growth phase is about 10% to about 50% of the culture period. In one embodiment, the growth phase is about 10% to about 40% of the culture period. In one embodiment, the growth phase is about 10% to about 30% of the culture period. In one embodiment, the growth phase is about 10% to about 20% of the culture period. In one embodiment, the growth phase is about 20% to about 60% of the culture period. In one embodiment, the growth phase is about 20% to about 50% of the culture period. In one embodiment, the growth phase is about 20% to about 40% of the culture period. In one embodiment, the growth phase is about 20% to about 30% of the culture period. In one embodiment, the growth phase is about 30% to about 60% of the culture period. In one embodiment, the growth phase is about 30% to about 50% of the culture period. In one embodiment, the growth phase is about 30% to about 40% of the culture period. In one embodiment, the growth phase is about 10% of the culture period. In one embodiment, the growth phase is about 20% of the culture period. In one embodiment, the growth phase is about 30% of the culture period. In one embodiment, the growth phase is about 40% of the culture period. In one embodiment, the growth phase is about 50% of the culture period. In one embodiment, the growth phase is about 60% of the culture period. In one embodiment, the growth phase is 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, or about 69% of the culture period. In one embodiment, the growth phase is about 10%, about 20%, about 30%, about 40%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60% of the culture period.

[0182] In one embodiment, the growth phase is at least 10% to at least 50% of the culture period. In one embodiment, the growth phase is at least 10% to at least 40% of the culture period. In one embodiment, the growth phase is at least 10% to at least 30% of the culture period. In one embodiment, the growth phase is at least 10% to at least 20% of the culture period. In one embodiment, the growth phase is at least 20% to at least 60% of the culture period. In one embodiment, the growth phase is at least 20% to at least 50% of the culture period. In one embodiment, the growth phase is at least 20% to at least 40% of the culture period. In one embodiment, the growth phase is at least 20% to at least 30% of the culture period. In one embodiment, the growth phase is at least 30% to at least 60% of the culture period. In one embodiment, the growth phase is at least 30% to at least 50% of the culture period. In one embodiment, the growth phase is at least 30% to at least 40% of the culture period.

[0183] In one embodiment, the growth phase lasts for up to 10% of the culture period. In one embodiment, the growth phase lasts for up to 20% of the culture period. In one embodiment, the growth phase lasts for up to 30% of the culture period. In one embodiment, the growth phase lasts for up to 40% of the culture period. In one embodiment, the growth phase lasts for up to 50% of the culture period.

[0184] In one embodiment, the growth phase is at least 60% of the culture period. In one embodiment, the growth phase is at most 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% of the culture period. In one embodiment, the growth phase is at least 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59% of the culture period.

[0185] Culture volume is maintained by medium exchange, which simultaneously supplies fresh medium to the bioreactor and removes spent medium from the culture. Fresh cell culture medium can be added to the bioreactor in stages, intermittently, and / or continuously. Spent medium can be removed from the bioreactor in stages, intermittently, and / or continuously using one or more separation systems. In one embodiment, the spent medium exits in the permeate stream.

[0186] During the growth and production phases, the static culture volume is maintained by constant and simultaneous exchange of fresh and spent medium. "Medium exchange" and "perfusion" are used interchangeably herein and refer to the simultaneous addition of fresh nutrients and / or medium ("feed") and removal of spent medium ("permeate") from the bioreactor. "Feed rate" and "feed flow rate" are used interchangeably herein and refer to the rate at which fresh nutrients and / or medium are added to the bioreactor. "Permeate rate" and "permeate flow rate" are used interchangeably herein and refer to the rate at which spent medium is removed from the bioreactor through the permeate stream of a separation system. "Spent medium" and "conditioned medium" are used interchangeably herein and refer collectively to medium containing nutrient-depleted culture medium, accumulated waste products, by-products, impurities including product-related impurities and / or process-related impurities, etc. Spent medium may also contain a protein of interest, such as a recombinant protein. "Medium exchange rate" and "perfusion rate" are used interchangeably herein and refer to the rate at which fresh nutrients and / or medium are added ("feed rate") and spent medium is removed from the bioreactor ("permeate rate"). Typically, the feed rate and permeate rate are the same. If there is a difference between the feed rate and permeate rate, the perfusion rate is generally the lower of the two.

[0187] In one embodiment, the perfusion rate consists of the feed flow and permeate flow being maintained at the same rate during the growth phase and production phase. In one embodiment, the culture is perfused at one or more feed and perfusion rates during the growth and / or production phases. In one embodiment, the culture is perfused at the same rate during the growth and production phases. In one embodiment, the culture is perfused at different rates during the growth and production phases.

[0188] Medium exchange rates or perfusion rates are expressed in terms of volume over a period of time. The volume component is expressed as a fraction of the culture volume. The time component is typically expressed relative to 24 hours or a day. One of skill in the art can readily determine a time period appropriate for a manufacturing process. As used herein, medium exchange rates or perfusion rates are expressed in terms of parts of culture volume per day (V / d). In one embodiment, the perfusion rate is 0.5 culture volumes / day (V / d) or less. In one embodiment, the perfusion rate is at least about 0.05 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to less than about 0.5 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.45 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.40 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.35 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.30 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.25 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.20 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.15 V / d. In one embodiment, the perfusion rate is at least about 0.05 V / d to at least about 0.10 V / d. In one embodiment, the perfusion rate is at least about 0.1 V / d to at least about 0.5 V / d.

[0189] In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.45 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.40 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.35 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.30 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.25 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.20 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.15 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to at least about 0.125 V / d.

[0190] In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.5 V / d. In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.45 V / d. In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.40 V / d. In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.35 V / d. In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.30 V / d. In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.25 V / d. In one embodiment, the perfusion rate is at least about 0.15 V / d to at least about 0.20 V / d.

[0191] In one embodiment, the perfusion rate is at least about 0.20 V / d to at least about 0.5 V / d. In one embodiment, the perfusion rate is at least about 0.20 V / d to at least about 0.45 V / d. In one embodiment, the perfusion rate is at least about 0.20 V / d to at least about 0.40 V / d. In one embodiment, the perfusion rate is at least about 0.20 V / d to at least about 0.35 V / d. In one embodiment, the perfusion rate is at least about 0.20 V / d to at least about 0.30 V / d. In one embodiment, the perfusion rate is at least about 0.20 V / d to at least about 0.25 V / d.

[0192] In one embodiment, the perfusion rate is at least about 0.3 V / d to at least about 0.5 V / d. In one embodiment, the perfusion rate is at least about 0.3 V / d to at least about 0.45 V / d. In one embodiment, the perfusion rate is at least about 0.3 V / d to at least about 0.40 V / d. In one embodiment, the perfusion rate is maintained at at least about 0.30 V / d to at least about 0.35 V / d.

[0193] In one embodiment, the perfusion rate is at least about 0.40 V / d to at least about 0.5 V / d. In one embodiment, the perfusion rate is at least about 0.40 V / d to at least about 0.45 V / d.

[0194] In one embodiment, the perfusion rate is about 0.05 V / d to about 0.5 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.45 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.40 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.35 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.30 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.25 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.20 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.15 V / d. In one embodiment, the perfusion rate is about 0.05 V / d to about 0.10 V / d. In one embodiment, the perfusion rate is about 0.1 V / d to about 0.5 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.45 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.40 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.35 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.30 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.25 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.20 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.15 V / d. In one embodiment, the perfusion rate is about 0.10 V / d to about 0.125 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.5 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.45 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.40 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.35 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.30 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.25 V / d. In one embodiment, the perfusion rate is about 0.15 V / d to about 0.20 V / d. In one embodiment, the perfusion rate is about 0.20 V / d to about 0.5 V / d. In one embodiment, the perfusion rate is about 0.20 V / d to about 0.45 V / d. In one embodiment, the perfusion rate is about 0.20 V / d to about 0.40 V / d.In one embodiment, the perfusion rate is about 0.20 V / d to about 0.35 V / d. In one embodiment, the perfusion rate is about 0.20 V / d to about 0.30 V / d. In one embodiment, the perfusion rate is about 0.20 V / d to about 0.25 V / d. In one embodiment, the perfusion rate is about 0.3 V / d to about 0.5 V / d. In one embodiment, the perfusion rate is about 0.3 V / d to about 0.45 V / d. In one embodiment, the perfusion rate is about 0.3 V / d to about 0.40 V / d. In one embodiment, the perfusion rate is about 0.30 V / d to about 0.35 V / d. In one embodiment, the perfusion rate is about 0.40 V / d to about 0.5 V / d. In one embodiment, the perfusion rate is about 0.40 V / d to about 0.45 V / d. In any of these embodiments, the feed rate and permeate rate are the same. In any of these embodiments, the culture is perfused at the same rate during the growth and production phases.

[0195] In one embodiment, the perfusion rate is at least about 0.10 V / d to 0.40 V / d. In one embodiment, the perfusion rate is at least about 0.10 V / d to 0.25 V / d. In one embodiment, the perfusion rate is at least about 0.13 V / d to 0.39 V / d. In one embodiment, the perfusion rate is at least about 0.13 V / d to 0.22 V / d.

[0196] In one embodiment, the perfusion rate is at least about 0.05 V / d, 0.10 V / d, 0.11 V / d, 0.12 V / d, 0.13 V / d, 0.14 V / d, 0.15 V / d, 0.16 V / d, 0.17 V / d, 0.18 V / d, 0.19 V / d, 0.20 V / d, 0.21 V / d, 0.22 V / d, 0.23 V / d, 0.24 V / d, 0.25 V / d, 0.26 V / d, 0.27 V / d, 0.28 V / d, In one embodiment, the perfusion rate is at least about 0.13 V / d. In one embodiment, the perfusion rate is at least about 0.22 V / d.

[0197] In one embodiment, the perfusion rates are about 0.05 V / d, 0.06 V / d, 0.07 V / d, 0.08 V / d, 0.09 V / d, about 0.10 V / d, about 0.11 V / d, about 0.12 V / d, about 0.13 V / d, about 0.14 V / d, about 0.15 V / d, about 0.16 V / d, about 0.17 V / d, about 0.18 V / d, about 0.19 V / d, about 0.20 V / d, about 0.21 V / d, about 0.22 V / d, about 0.23 V / d, about 0.24 V / d, about 0.25 V / d, about 0.26 V / d, about 0.27 V / d, about 0.28 V / d, about 0.29 V / d, about 0.30 V / d, about 0.31 V / d, about 0.32 V / d, about 0.33 V / d, about 0.34 V / d, about 0.35 V / d, about 0.36 V / d, about 0.37 V / d, about 0.38 V / d, about 0.39 V / d, about 0.40 V / d, about 0.41 V / d, about 0.42 V / d, about 0.43 V / d, about 0.44 V / d, about 0.45 V / d, about 0.46 V / d, about 0.47 V / d, about 0.48 V / d, about 0.49 V / d, about 0.50 V / d, about 0.51 V / d, about 0.52 V / d, about 0.53 V / d, about 0.54 V / d, about 0.55 / d, approximately 0.28V / d, approximately 0.29V / d, approximately 0.30V / d, approximately 0.31V / d, approximately 0.32V / d, approximately 0.33V / d, approximately 0.34V / d, approximately 0.35V / d, approximately 0.36V / d, approximately 0.37V / d, approximately 0.38V / d, approximately 0.39 V / d, about 0.40V / d, about 0.41V / d, about 0.42V / d, about 0.43V / d, about 0.44V / d, about 0.45V / d, about 0.46V / d, about 0.47V / d, about 0.48V / d, about 0.49V / d or about 0.50V / d. In one embodiment, the perfusion rate is about 0.13 V / d. In one embodiment, the perfusion rate is about 0.22 V / d. In any of these embodiments, the feed rate and permeate rate are the same. In any of these embodiments, the culture is perfused at the same rate during the growth and production phases.

[0198] The growth phase is carried out at a high cell density in a culture volume that is at least 50% to approximately 75% of the final working volume of the bioreactor, which allows for efficient use of a smaller culture volume and reduces the amount of feed required and waste generated. It also shortens the growth phase to less than 70% of the culture duration. Introducing a feed-up transition between the growth and production phases increases the culture volume and reduces the cell mass in the bioreactor, thereby reducing the amount of feed required and reducing waste in the permeate stream during the production phase.

[0199] When the culture achieves one or more desired target criteria, at least one feed-up phase is initiated to increase the culture volume in the bioreactor to a desired final culture volume that is up to 100% of the final bioreactor working volume. Such target criteria may include one or more time points (e.g., time after inoculation), titer, cell density, packed cell volume, product attributes, process, production, and / or plant schedules.

[0200] Those skilled in the art can determine a suitable method for increasing the culture volume in a bioreactor. In one embodiment, "differential perfusion" is used to increase the culture volume. During differential perfusion, the feed flow rate and the permeate flow rate are not the same. If the feed flow rate is faster than the permeate flow rate, a net increase in culture volume occurs.

[0201] The feed rate and permeate rate can be determined so that the desired culture volume is achieved within a desired time frame. In one embodiment, one or more feed rates can be used during the feed-up phase to achieve the desired culture volume in the bioreactor. In one embodiment, the feed rate is increased relative to the permeate rate. In one embodiment, the permeate rate is held constant and the feed rate is increased during the feed-up phase. In one embodiment, the permeate rate is decreased relative to the feed rate. In one embodiment, the permeate is not harvested until the desired culture volume is achieved.

[0202] During the feed-up phase, the feed rate and permeate rate are each independently maintained at a rate of 0.5 culture volumes per day (V / d) or less, with the feed rate being greater than the permeate rate. In one embodiment, the feed rate or permeate rate is at least about 0.05 V / d to 0.50 V / d. In one embodiment, the feed rate or permeate rate is at least about 0.05 V / d to at least about 0.45 V / d. In one embodiment, the feed rate or permeate rate is at least about 0.05 V / d to at least about 0.40 V / d. In one embodiment, the feed rate or permeate rate is at least about 0.05 V / d to at least about 0.35 V / d. In one embodiment, the feed rate or permeate rate is at least about 0.05 V / d to at least about 0.30 V / d. In one embodiment, the feed rate or permeate rate is at least about 0.05 V / d to at least about 0.25 V / d. In one embodiment, the feed rate or permeation rate is at least about 0.05 V / d to at least about 0.20 V / d. In one embodiment, the feed rate or permeation rate is at least about 0.05 V / d to at least about 0.15 V / d. In one aspect, the feed rate or permeation rate is at least about 0.05 V / d to at least about 0.10 V / d.

[0203] In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.50 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.45 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.40 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.35 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.30 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.25 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.20 V / d. In one embodiment, the supply rate or permeation rate is about 0.05 V / d to about 0.15 V / d. In one embodiment, the feed rate or permeation rate is from about 0.05 V / d to about 0.10 V / d.

[0204] In one embodiment, the supply rate is at least 0.10 V / d to at least 0.50 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.45 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.40 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.35 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.30 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.250 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.20 V / d. In one embodiment, the supply rate is at least 0.10 V / d to at least 0.15 V / d. In one embodiment, the supply rate is at least 0.33 V / d. In one embodiment, the supply rate is at least 0.22 V / d. In one embodiment, the supply rate is at least 0.10V / d, 0.11V / d, 0.12V / d, 0.13V / d, 0.14V / d, 0.15V / d, 0.16V / d, 0.17V / d, 0.18V / d, 0.19V / d, 0.20V / d, 0.21V / d, 0.22V / d, 0.23V / d, 0.24V / d, 0.25V / d, 0.26V / d, 0.27V / d, 0.28V / d, 0.29V / d, 0.30V / d, 0.31V / d, 0.32V / d, 0.33V / d, 0.34V / d, 0.35V / d, 0.36V / d, 0.37V / d, 0.38V / d, 0.39V / d, 0.40V / d, 0.41V / d, 0.42V / d, 0.43V / d, 0.44V / d, 0.45V / d, 0.46V / d, 0.47V / d, 0.48V / d, 0.49V / d, 0.50V / d, 0.51V / d, 0.52V / d, 0.53V / d, 0.54V / d, 0.55V / d, 0.56V / d, 0.57V / d, 0.58V / d, 0.59V / d, 0.60V / d, 0.61V / d, 0.62V / d, 0.63V / d, 0.64V / d, 0.65V V / d, 0.26V / d, 0.27V / d, 0.28V / d, 0.29V / d, 0.30V / d, 0.31V / d, 0.32V / d, 0.33V / d, 0.34 V / d, 0.35V / d, 0.36V / d, 0.37V / d, 0.38V / d, 0.39V / d, 0.40V / d, 0.45V / d or 0.50V / d.

[0205] In one embodiment, the supply rate is about 0.10 V / d to about 0.50 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.45 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.40 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.35 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.30 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.250 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.20 V / d. In one embodiment, the supply rate is about 0.10 V / d to about 0.15 V / d. In one embodiment, the supply rate is about 0.33 V / d. In one embodiment, the supply rate is about 0.22 V / d. In one embodiment, the supply rate is about 0.10 V / d, about 0.11 V / d, about 0.12 V / d, about 0.13 V / d, about 0.14 V / d, about 0.15 V / d, about 0.16 V / d, about 0.17 V / d, about 0.18 V / d, about 0.19 V / d, about 0.20 V / d, about 0.21 V / d, about 0.22 V / d, about 0.23 V / d, about 0.24 V / d, about 0.25 V / d , approx. 0.26V / d, approx. 0.27V / d, approx. 0.28V / d, approx. 0.29V / d, approx. 0.30V / d, approx. 0.31V / d, approx. 0.32V / d, approx. 0.33V / d, approx. 0.34V / d, about 0.35V / d, about 0.36V / d, about 0.37V / d, about 0.38V / d, about 0.39V / d, about 0.40V / d, about 0.45V / d or about 0.50V / d.

[0206] In one embodiment, the permeation rate is from 0 V / d to at least 0.50 V / d. In one embodiment, the permeation rate is at least about 0.45 V / d. In one embodiment, the permeation rate is at least about 0.40 V / d. In one embodiment, the permeation rate is from at least about 0.10 V / d to at least about 0.35 V / d. In one embodiment, the permeation rate is from at least about 0.10 V / d to about 0.30 V / d. In one embodiment, the permeation rate is from at least about 0.10 V / d to about 0.25 V / d. In one embodiment, the permeation rate is from at least about 0.10 V / d to about 0.20 V / d. In one embodiment, the permeation rate is from at least about 0.10 V / d to about 0.15 V / d. In one embodiment, the permeation rate is at least about 0.13 V / d. In one embodiment, the permeation rate is at least 0 V / d, 0.10 V / d, 0.11 V / d, 0.12 V / d, 0.13 V / d, 0.14 V / d, 0.15 V / d, 0.16 V / d, 0.17 V / d, 0.18 V / d, 0.19 V / d, 0.20 V / d, 0.21 V / d, 0.22 V / d, 0.23 V / d, 0.24 V / d, 0.25 V / d, 0.26 V / d, 0.27 V / d, 0.28 V / d, 0.29 V / d, or 0.30 V / d.

[0207] In one embodiment, the permeation rate is 0 V / d to about 0.50 V / d. In one embodiment, the permeation rate is 0 V / d to about 0.40 V / d. In one embodiment, the permeation rate is about 0.10 V / d to about 0.35 V / d. In one embodiment, the permeation rate is about 0.10 V / d to about 0.30 V / d. In one embodiment, the permeation rate is about 0.10 V / d to about 0.25 V / d. In one embodiment, the permeation rate is about 0.10 V / d to about 0.20 V / d. In one embodiment, the permeation rate is about 0.10 V / d to about 0.15 V / d. In one embodiment, the permeation rate is about 0 V / d, about 0.10 V / d, about 0.11 V / d, about 0.12 V / d, about 0.13 V / d, about 0.14 V / d, about 0.15 V / d, about 0.16 V / d, about 0.17 V / d, about 0.18 V / d, about 0.19 V / d, about 0.20 V / d, about 0.21 V / d, about 0.22 V / d, about 0.23 V / d, about 0.24 V / d, about 0.25 V / d, about 0.26 V / d, about 0.27 V / d, about 0.28 V / d, about 0.29 V / d, or about 0.30 V / d. In one embodiment, the permeation rate is about 0.45 V / d. In one embodiment, the permeation rate is about 0.40 V / d. In one embodiment, the permeation rate is about 0.13 V / d.

[0208] In one embodiment, the feed rate is at least 0.30 V / d and the permeation rate is less than 0.20 V / d. In one embodiment, the feed rate is less than 0.40 V / d and the permeation rate is less than 0.20 V / d. In one embodiment, the feed rate is 0.3 V / d to 0.4 V / d and the permeation rate is 0.10 V / d to 0.15 V / d. In one embodiment, the feed rate is about 0.3 V / d to about 0.4 V / d and the permeation rate is about 0.1 V / d to about 0.2 V / d.

[0209] In one embodiment, the feed rate during the feed-up phase is selected to meet or achieve one or more desired target criteria, such as culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production and / or plant schedule.

[0210] In one embodiment, one or more feed rates are selected such that the feed-up phase is maintained for at least 6 hours. In one embodiment, the feed-up phase is maintained for at least 60 hours. In one embodiment, the feed-up period is maintained for at least about 16 hours to at least about 40 hours. In one embodiment, the feed-up phase is maintained for 30 hours ± 24 hours. In one embodiment, the feed-up phase is maintained for 30 hours ± 8 hours. In one embodiment, the feed-up phase is maintained for at least 6, 10, 12, 16, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 48, 50, 54, or 60 hours.

[0211] In one embodiment, the feed-up phase is maintained for about 6 hours to about 60 hours (e.g., about 12 hours to about 48 hours, about 16 hours to about 40 hours, about 6 hours to about 54 hours, etc.). In one embodiment, the feed-up phase is maintained for about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 48, about 50, about 54, or about 60 hours.

[0212] Once the feed-up phase is complete, the feed rate and permeate rate may be returned to the same flow rate of 0.5 V / d or less until harvest. In one embodiment, the feed rate is returned to the same rate as the permeate rate. In one embodiment, the feed rate and permeate rate are returned to the rates prior to the feed-up phase (i.e., the feed rate and permeate rate during the preceding growth phase, which may be the same).

[0213] In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to less than 0.5 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.45 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.40 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.35 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.30 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.25 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.20 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.15 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.05 V / d to at least about 0.10 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.1 V / d to 0.5 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.10 V / d to at least about 0.45 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.10 V / d to at least about 0.40 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.10 V / d to at least about 0.35 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.10 V / d to at least about 0.30 V / d. In one embodiment, the feed rate and permeation rate are at least about 0.10 V / d to at least about 0.25 V / d. In one embodiment, the feed rate and permeate rate are at least about 0.10 V / d to at least about 0.20 V / d. In one embodiment, the feed rate and permeate rate are at least about 0.10 V / d to at least about 0.15 V / d. In any of these embodiments, the feed rate and permeate rate can be the same.

[0214] In one embodiment, the feed rate and permeate rate are increased to greater than 0.20 V / d 24 hours after the end of the feed-up phase. In one embodiment, the feed rate and permeate rate are 0.05 V / d. In one embodiment, the feed rate and permeate rate are 0.10 V / d. In one embodiment, the feed rate and permeate rate are 0.15 V / d. In one embodiment, the feed rate and permeate rate are 0.20 V / d. In one embodiment, the feed rate and permeate rate are 0.25 V / d. In one embodiment, the feed rate and permeate rate are 0.30 V / d. In one embodiment, the feed rate and permeate rate are 0.35 V / d. In one embodiment, the feed rate and permeate rate are 0.40 V / d. In one embodiment, the feed rate and permeate rate are 0.45 V / d. In one embodiment, the feed rate and permeate rate are 0.50 V / d.

[0215] In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.05 V / d to about 0.5 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.05 V / d to about 0.45 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.05 V / d to about 0.40 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.05 V / d to about 0.35 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.05 V / d to about 0.30 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same, and are in the range of about 0.05 V / d to about 0.25 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same, and are in the range of about 0.05 V / d to about 0.20 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same, and are in the range of about 0.05 V / d to about 0.15 V / d. In one embodiment, the feed rate and permeation rate are the same, and are in the range of about 0.05 V / d to about 0.10 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same, and are in the range of about 0.1 V / d to about 0.5 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same, and are in the range of about 0.10 V / d to about 0.45 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.10 V / d to about 0.40 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.10 V / d to about 0.35 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and range from about 0.10 V / d to about 0.30 V / d.In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeate rate are the same and range from about 0.10 V / d to about 0.25 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeate rate are the same and range from about 0.10 V / d to about 0.20 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeate rate are the same and range from about 0.10 V / d to about 0.15 V / d.

[0216] In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and are about 0.05 V / d, about 0.10 V / d, about 0.15 V / d, about 0.20 V / d, about 0.25 V / d, about 0.30 V / d, about 0.35 V / d, about 0.4 V / d, about 0.45 V / d, or about 0.5 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and are about 0.25 V / d, about 0.30 V / d, about 0.35 V / d, about 0.4 V / d, about 0.45 V / d, or about 0.5 V / d. In one embodiment, 24 hours after the end of the feed-up phase, the feed rate and permeation rate are the same and are about 0.05 V / d, about 0.10 V / d, about 0.15 V / d, or about 0.20 V / d.

[0217] In one embodiment, the feed-up phase begins when the culture achieves one or more desired target criteria, including desired culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production and / or plant schedule.

[0218] In one embodiment, the feed-up phase begins when the culture reaches a desired cell density. In one embodiment, the feed-up phase begins when the cell density reaches at least 100 x 10 5 In one embodiment, the cell density is started at a maximum of 350 x 10 cells / mL. 5 In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 ~Approx. 350×10 5In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 ~300×10 5 In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 ~250×10 5 In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 ~200×10 5 In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 ~150×10 5 In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 , 125×10 5 , 150×10 5 , 175×10 5 , 200×10 5 , 225×10 5 , 250×10 5 , 275×10 5 , 300×10 5 , 325×10 5 or 350 x 10 5 In one embodiment, the cell density is at least about 100 x 10 cells / mL. 5 In one embodiment, the cell density is at least about 150 x 10 cells / mL. 5 In one embodiment, the cell density is at least about 200 x 10 cells / mL. 5 In one embodiment, the cell density is at least about 250 x 10 cells / mL. 5 In one embodiment, the cell density is at least about 300 x 10 cells / mL. 5 In one embodiment, the cell density is about 350 x 10 cells / mL. 5 cells / mL.

[0219] In one embodiment, the feed-up phase is about 100×10 5 cells / mL ~ approx. 350×10 5 Cell densities in the range of cells / mL (e.g., approximately 100 x 10 5 , about 125×10 5 , about 150×10 5, about 175×10 5 , about 200×10 5 , about 225×10 5 , about 250×10 5 , about 275×10 5 , about 300×10 5 , about 325×10 5 or approximately 350 x 10 5 It starts when the RT-PCR assay reaches a concentration of 1000kJ / mL (cells / mL).

[0220] In one embodiment, the feed-up phase is initiated at least about 24 hours after inoculation. In one embodiment, the feed-up phase is initiated up to 96 hours after inoculation. In one embodiment, the feed-up phase is initiated about 24 hours to about 96 hours after inoculation. In one embodiment, the feed-up phase is initiated about 24 hours to about 72 hours after inoculation. In one embodiment, the feed-up phase is initiated about 24 hours to about 48 hours after inoculation. In one embodiment, the feed-up phase is initiated about 48 hours to about 96 hours after inoculation. In one embodiment, the feed-up phase is initiated about 48 hours to about 72 hours after inoculation. In one embodiment, the feed-up phase is initiated about 72 hours to about 96 hours after inoculation. In one embodiment, the feed-up phase is initiated about 24, 48, 72, or 96 hours after inoculation. In one embodiment, the feed-up phase is initiated about 24 hours after inoculation. In one embodiment, the feed-up phase is initiated about 48 hours after inoculation. In one embodiment, the feed-up phase is initiated about 72 hours after inoculation. In one embodiment, the feed-up phase is initiated about 96 hours after inoculation.

[0221] The feed-up phase is maintained until one or more desired target criteria are met, including, but not limited to, desired culture volume, final culture volume, desired bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attributes, process, production and / or plant schedule.

[0222] In one embodiment, the feed-up phase is maintained for a desired length of time. In one embodiment, the desired length of time is based on process, production, and / or plant schedules. In one embodiment, the feed-up phase is maintained for a time period that allows for at least one cell population doubling of the culture. In one embodiment, the feed-up phase is maintained for a time period sufficient to avoid shocking the culture due to a rapid increase in medium concentrate.

[0223] In one embodiment, the feed-up phase is maintained until the desired culture volume is achieved. In one embodiment, the desired culture volume is equal to the final working volume of the bioreactor. In one embodiment, the feed-up phase is maintained until the culture volume exceeds 70% of the final working volume of the bioreactor. In one embodiment, the feed-up phase is maintained until the culture volume exceeds 75% of the final working volume of the bioreactor. In one embodiment, feed-up is maintained until the culture volume is greater than 75% to less than or equal to 100% of the final working volume of the bioreactor. In one embodiment, feed-up is maintained until the culture volume is between about 80% of the final working volume of the bioreactor and less than or equal to 100% of the final working volume of the bioreactor. In one embodiment, feed-up is maintained until the culture volume is between about 85% of the final working volume of the bioreactor and less than or equal to 100% of the final working volume of the bioreactor. In one embodiment, feed-up is maintained until the culture volume is between about 90% of the final working volume of the bioreactor and less than or equal to 100% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 90% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 91% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 92% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 93% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 94% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 95% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 96% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 97% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 98% of the final working volume of the bioreactor. In one embodiment, the feed-up is maintained until the culture volume is about 99% of the final working volume of the bioreactor.In one embodiment, the feed-up is maintained until the culture volume is at 100% of the final working volume of the bioreactor.

[0224] The feed-up phase may be maintained until a desired cell density is achieved in the increased culture volume. In one embodiment, the feed-up phase is maintained until a desired cell density is achieved in the desired cell culture volume. In one embodiment, the feed-up phase is maintained until a cell density of at least about 200 x 10 5 In one embodiment, the feed-up phase is continued until the cell density reaches about 600 x 10 cells / mL. 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~About 600×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~At least about 550×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~ At least about 500 × 10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~At least about 450×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~At least about 400×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~At least about 350×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~ At least about 300 × 10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 ~At least about 250×10 5In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 300 x 10 cells / mL. 5 ~At least 600×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches about 400 x 10 cells / mL. 5 ~At least 600×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 500 x 10 cells / mL. 5 ~At least 600×10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 200 x 10 cells / mL. 5 , 225×10 5 , 250×10 5 , 275×10 5 , 300×10 5 , 325×10 5 , 350×10 5 , 375×10 5 , 400×10 5 , 425×10 5 , 450×10 5 , 475×10 5 , 500×10 5 , 525×10 5 , 550×10 5 , 575×10 5 or 600 x 10 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 300 x 10 cells / mL. 5 In one embodiment, the feed-up phase is maintained until the cell density reaches about 400 x 10 cells / mL. 5 In one embodiment, the feed-up phase is maintained until the cell density reaches at least about 500 x 10 cells / mL. 5 cells / mL.

[0225] In one embodiment, the feed-up phase is carried out at a cell density of about 200 x 10 5 cells / mL ~ approx. 600×10 5 cells / mL (e.g., approximately 200 x 10 5, about 225×10 5 , about 250×10 5 , about 275×10 5 , about 300×10 5 , about 325×10 5 , about 350×10 5 , about 375×10 5 , about 400×10 5 , about 425×10 5 , about 450×10 5 , about 475×10 5 , about 500×10 5 , about 525×10 5 , about 550×10 5 , about 575×10 5 or approximately 600 x 10 5 The cell density is maintained at 100% (cells / mL).

[0226] In one embodiment, the growth phase continues during and after the feed-up phase. In one embodiment, the growth phase continues after the feed-up phase until a desired cell density is achieved. In one embodiment, the growth phase continues after the temperature shift.

[0227] The production phase marks the beginning of the stationary phase, where cell growth typically levels off and product titer increases. In one embodiment, the production phase begins when the feed-up phase ends. In one embodiment, the production phase begins when the desired cell culture volume, final cell culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production, and / or plant schedule is achieved.

[0228] In one embodiment, the production phase begins when a desired cell density is achieved. In one embodiment, the desired cell density is at least 200 x 10 5 In one embodiment, the desired cell density is at least 250 x 10 cells / mL. 5 In one embodiment, the desired cell density is at least 300 x 10 cells / mL. 5 In one embodiment, the desired cell density is at least 350 x 10 cells / mL. 5In one embodiment, the desired cell density is at least 400 x 10 cells / mL. 5 In one embodiment, the desired cell density is at least 500 x 10 cells / m. 5 In one embodiment, the desired cell density is at least 550 x 10 cells / mL. 5 In one embodiment, the desired cell density is at least 600 x 10 cells / mL. 5 cells / mL.

[0229] In one embodiment, the production phase begins when a desired cell density is achieved in a desired culture volume or a desired working volume. In one embodiment, the production phase begins when a desired cell density is achieved in a desired culture volume. In one embodiment, the production phase begins when a desired cell density is achieved in a desired final culture volume. In one embodiment, the production phase begins when a desired cell density is achieved in a desired working volume of the bioreactor. In one embodiment, the production phase begins when a desired cell density is achieved in a desired final working volume of the bioreactor. In one embodiment, the production phase begins when a cell density of at least 200 x 10 in the final culture volume. 5 In one embodiment, the production phase begins when the cell density reaches at least 200 x 10 cells / mL in the final working volume of the bioreactor. 5 It begins when the number of cells / mL is reached.

[0230] In one embodiment, the production phase begins when the cell density is at least twice the seeded cell density, hi one embodiment, the production phase begins when the cell density is no more than three times the seeded cell density.

[0231] In one embodiment, the production phase begins when the culture volume is greater than 75% and less than or equal to 100% of the final working volume of the bioreactor.

[0232] To increase production of a desired protein by mammalian cells, cell growth can be limited or stopped. Such methods include, but are not limited to, temperature shift, pH shift, the use of chemical inducers of protein production and cell cycle inhibitors, nutrient limitation, or starvation, either alone or in combination. For example, a lower culture temperature can be used to reduce cell growth and promote protein production and culture longevity. The use of temperature shift from the optimum temperature for the growth phase to the optimum temperature for the production phase is often employed in cell culture strategies. To optimize mammalian cell growth, the culture temperature is typically maintained at a physiological temperature, 35°C to 37°C, during the growth phase. The temperature during the production phase can be about 28°C to about 35°C. Temperature adjustments can be used throughout the culture period to achieve the desired growth and production objectives. A combination of temperature shifts can also be used to transition from a first growth phase to a first production phase, a second growth phase, followed by a second production phase, etc.

[0233] In one embodiment, the temperature is shifted during the culture from an optimum temperature for the growth phase to an optimum temperature for the production phase. In one embodiment, the temperature shift occurs at the end of the growth phase. In one embodiment, the temperature shift occurs at the end of the feed-up phase. In one embodiment, the temperature shift occurs when a desired cell density is reached. In one embodiment, the desired cell density is at least 200 x 10 5 In one embodiment, the temperature shift occurs when the culture volume reaches the desired final bioreactor working volume. In one embodiment, the temperature shift occurs when the culture volume exceeds 75% of the final bioreactor working volume.

[0234] In one embodiment of the present disclosure, the temperature during the growth phase is about 35°C to about 38°C. In one embodiment, the temperature is about 34°C to about 36°C. In one embodiment, the temperature is at least 35°C, 36°C, or 37°C ± 0.5°C. In one embodiment, the temperature is 38°C, 37°C, 36.5°C, 36°C, 35.5°C, 35°C, or 34.5°C.

[0235] In one embodiment of the present disclosure, the temperature during the production phase is about 32°C to about 35°C. In one embodiment, the temperature is about 30°C to about 35°C. In one embodiment, the temperature is about 32°C to about 34°C. In one embodiment, the temperature is at least 30°C, 31°C, 32°C, 33°C, or 34°C ± 0.5°C. In one embodiment, the temperature is 32°C, 32.5°C, 33°C, or 33.5°C.

[0236] In one embodiment, at least one temperature shift after the feed-up phase is beneficial to maintain cell density during the production phase.

[0237] In one embodiment, the production phase begins when the culture temperature is reduced.

[0238] In one embodiment, a pH shift may be used alone or in combination with a temperature shift.

[0239] Another method for maintaining cells in a desired physiological state is to induce cell growth arrest by exposing the cell culture to low L-asparagine conditions and / or asparagine starvation (see, for example, WO 2013 / 006479). Cell growth arrest can be achieved and maintained by maintaining low concentrations of L-asparagine in the cell culture through a culture medium containing a limiting concentration of L-asparagine. Maintaining an L-asparagine concentration of 5 mM or less can be used to induce and maintain cells in a growth-arrested state, thereby improving productivity.

[0240] Additionally, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added before, simultaneously with, and / or after the temperature shift, or instead of the temperature shift. If inducers are added after the temperature shift, they can be added 1 hour to 5 days, optionally 1 to 2 days after the temperature shift. Cell cycle inhibitors, i.e., compounds known or suspected to regulate cell cycle progression and related steps of transcription, DNA repair, differentiation, senescence, and associated apoptosis, are also useful for inducing cell growth arrest. For example, cell cycle inhibitors that interact with the cyclic machinery, such as cyclin-dependent kinases (CDKs), are also useful, as are molecules that interact with proteins from other pathways, such as AKT, mTOR, and other pathways that directly or indirectly affect the cell cycle.

[0241] A shift in pH may be used alone or in combination with temperature and / or chemical inducers.

[0242] The production phase continues until one or more desired target criteria are met, including, for example, culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attributes, process, production and / or plant schedule.

[0243] In one embodiment, the production period is 50 days or more. In one embodiment, the production period is 50 days or less. In one embodiment, the production period is 40 days or less. In one embodiment, the production period is 45 days or less. In one embodiment, the production period is 40 days or less. In one embodiment, the production period is 35 days or less. In one embodiment, the production period is 30 days or less. In one embodiment, the production period is 35 days or less. In one embodiment, the production period is 30 days or less. In one embodiment, the production period is 25 days or less. In one embodiment, the production period is 20 days or less. In one embodiment, the production period is 19 days or less. In one embodiment, the production period is 18 days or less. In one embodiment, the production period is 17 days or less. In one embodiment, the production period is 16 days or less. In one embodiment, the production period is 15 days or less. In one embodiment, the production period is 14 days or less. In one embodiment, the production period is 13 days or less. In one embodiment, the production period is 12 days or less. In one embodiment, the production period is 11 days or less. In one embodiment, the production period is 10 days or less. In one embodiment, the production period is 9 days or less. In one embodiment, the production period is 8 days or less. In one embodiment, the production period is 7 days or less. In one embodiment, the production period is 6 days or less. In one embodiment, the production period is 5 days or less. In one embodiment, the production period is 4 days or less.

[0244] In one embodiment, the production period is about 4 to about 20 days (e.g., about 5 to about 20 days, about 6 to about 20 days, about 7 to about 20 days, about 8 to about 20 days, about 9 to about 20 days, about 10 to about 20 days, about 11 to about 20 days, about 12 to about 20 days, about 13 to about 20 days, about 14 to about 20 days, about 15 to about 20 days, about 16 to about 20 days, about 17 to about 20 days, about 18 to about 20 days, about 19 to about 20 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days).

[0245] In one embodiment, the production period is at least 10-20 days. In one embodiment, the production period is at least 11-20 days. In one embodiment, the production period is at least 12-20 days. In one embodiment, the production period is at least 13-20 days. In one embodiment, the production period is at least 14-20 days. In one embodiment, the production period is at least 15-20 days. In one embodiment, the production period is at least 16-20 days. In one embodiment, the production period is at least 17-20 days. In one embodiment, the production period is at least 18-20 days. In one embodiment, the production period is at least 19-20 days.

[0246] In one embodiment, the culture period from initiation to harvest is at least 10 days. In one embodiment, the culture period is at least 20 days. In one embodiment, the culture period is at least 30 days. In one embodiment, the culture period is at least 40 days. In one embodiment, the culture period is at least 50 days. In one embodiment, the culture period is at least 60 days. In one embodiment, the culture period is 10-20 days. In one embodiment, the culture period is 11-20 days. In one embodiment, the culture period is 12-20 days. In one embodiment, the culture period is 13-20 days. In one embodiment, the culture period is 14-20 days. In one embodiment, the culture period is 15-20 days. In one embodiment, the culture period is 16-20 days. In one embodiment, the culture period is 17-20 days. In one embodiment, the culture period is 18-20 days. In one embodiment, the culture period is 19-20 days.

[0247] Following the production phase, the culture is harvested. Alternatively or additionally, the contents of the bioreactor may be partially harvested one or more times during the production phase.

[0248] Throughout the culture period, the cell culture is sampled as needed to monitor the culture and operating conditions. One or more conditions, attributes, characteristics, etc. of the culture may be monitored, such as cell number, viability, viable cell density, packed cell volume, bioreactor volume, pH, pCO2, dissolved oxygen (DO), glucose, lactate, ammonia, osmolality, titer, amino acids, and product quality.

[0249] The lean perfusion process described herein utilizes one or more separation systems connected to the bioreactor and removing spent medium in the permeate stream. In one embodiment, the recombinant protein is retained in the bioreactor by the separation system and bulk recovered. In one embodiment, some recombinant protein may be removed from the bioreactor by the separation system during the growth and / or production phase prior to bulk recovery. In one embodiment, the recombinant protein may be recovered using the separation system.

[0250] The separation system includes at least one pumping mechanism and at least one filter and / or at least one membrane module. Such separation systems utilize filters, including, for example, membrane filters such as hollow fiber filters. The pumping mechanism draws spent culture fluid from the bioreactor through the filter or membrane module. The filter or membrane module is used to selectively retain or return certain components in the culture fluid (e.g., cells, recombinant proteins) to the bioreactor in the retentate and remove spent medium containing components such as waste products, by-products, impurities, process- and production-related impurities that have accumulated in the permeate.

[0251] The filter or membrane module in the separation system operates by separating components in the spent broth based on the molecular weight of the culture components relative to the pores of the filter or membrane. Tangential flow filtration (TFF), also known as cross-flow filtration, can be used in the separation system. A feed stream of spent broth is passed parallel to the filter or membrane surface by a pumping mechanism. Components of the culture that can pass through the membrane exit the separation system in the permeate stream, while the remainder of the broth is recycled back to the bioreactor in the retentate stream. The tangential flow separation system can be a unidirectional recirculating tangential flow system (RTF) or an alternating tangential flow (ATF) filtration system.

[0252] Hollow fiber filters are commonly used in retention systems. When spent culture medium is introduced into the filter, the hollow fiber material can retain certain culture components (e.g., including cells and desired proteins) on the lumen side (inside) and, based on the pore size or molecular weight cutoff of the hollow fiber material, allow certain components to pass through the filter in the permeate stream. Materials retained on the lumen side of the filter in the retentate stream are returned to the bioreactor. Materials exiting the separation system in the permeate stream are treated as waste. Separation systems can be used to concentrate the desired recombinant protein for recovery. The pore size or molecular weight cutoff (MWCO) can be selected to retain the desired protein in the retentate and return it to the bioreactor.

[0253] In various aspects, hollow fibers have an inner diameter of about 0.5 mm to about 1 mm and can be any suitable length (e.g., about 30 cm to about 110 cm, etc.). Ultrafiltration hollow fibers generally have a pore size range of 0.01 μm to 0.1 μm or a molecular weight cut-off (MWCO) of 300 kDa or less and can be used to retain the desired protein in the retentate and return it to the bioreactor. In one embodiment, the MWCO is at least 5 kDa to at least 300 kDa. In one embodiment, the MWCO is 5 kDa to at least 100 kDa. In one embodiment, the MWCO is 10 kDa to at least 30 kDa. In one embodiment, the MWCO is at least 5 kDa, 10 kDa, 30 kDa, 50 kDa, 100 kDa, or 300 kDa. In one embodiment, the MWCO is 30 kDa. In one embodiment, the MWCO is about 5 kDa to about 300 kDa (e.g., about 10 kDa to about 300 kDa, about 10 kDa to about 30 kDa, about 5 kDa, about 10 kDa, about 30 kDa, about 50 kDa, about 100 kDa, about 300 kDa). Such filters are commercially available, for example, ATF6 30 kDa filter and ATF10 30 kDa filter (Refine Technologies, Hanover, NJ), Xampler (Cytiva, Marlborough, MA), Midikros (Spectrum Laboratories, Inc., Dominguez, CA), XCell ATF (registered trademark), Repligen (Waltham, MA), etc.

[0254] More than one separation system and / or filter may be used at one time. In one embodiment, two or more separation systems or filters are operated in parallel. In one embodiment, two or more separation systems or filters are operated in series.

[0255] The cell culture fluid can be drawn from the bioreactor into the filter module by a pumping system that passes the cell culture through or along the filter (e.g., through the lumen side of the hollow fibers) and returns the retentate to the bioreactor. Examples of cell pumping systems include, but are not limited to, peristaltic pumps, double diaphragm pumps, low shear pumps (Levitronix™ pumps, Zurich, Switzerland), reverse tangential flow, and alternating tangential flow systems (ATF™, Repligen, Waltham, MA). In one embodiment, the permeate can be drawn from the filter using a peristaltic pump.

[0256] "Cell(s)," as used herein, includes any prokaryotic or eukaryotic cell. Cells may be obtained ex vivo, in vitro, or in vivo, either separately or as part of a higher-order structure such as a tissue or organ. Cells are usually derived from lines arising from a primary culture that can be maintained in culture indefinitely. The selection of an appropriate host cell depends on various factors, such as the desired expression level, protein modifications (such as glycosylation or phosphorylation) that are desirable or essential for activity, and the ease of folding into a biologically active molecule.

[0257] Preferably, the cells are eukaryotic cells, such as mammalian cells (e.g., CHO cells). Any mammalian cell suitable for recombinant protein expression is suitable for use in connection with the present disclosure. Suitable mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, mouse myeloma (NS0, Sp2 / 0) cells, baby hamster kidney (BHK) cells, human embryonic kidney (293) cells, fibrosarcoma (HT-1080) cells, human embryonic retina (PER.C6) cells, hybrid kidney and B cell (HKB-11), CEVEC's amniocyte production (CAP) cells, human liver (HuH-7) cells, and any other cells used or suitable for use in clinical and / or commercial manufacturing. CHO cell lines are widely used to produce complex recombinant proteins. Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because efficient DHFR-selectable and amplifiable gene expression systems enable high-level recombinant protein expression in these cells (Kaufman RJ (1990), Meth Enzymol 185:537-566). Also widely used is the glutamine synthetase (GS) knockout CHOK1SV cell line, which utilizes GS-based methionine sulfoximine (MSX) selection. Also included in the present disclosure are CHOK1 cells (ATCC CCL61).

[0258] In a preferred embodiment, the cells are genetically engineered to express a protein of commercial or scientific interest. Methods and materials for genetically engineering cells to express a desired protein are well known to those skilled in the art. As used herein, the term "cell culture medium" (also referred to as "culture medium," "culture medium," "cell culture medium," "tissue culture medium," etc.) refers to any nutrient solution used to grow cells, e.g., mammalian cells. Cell culture media generally provide one or more of the following components: an energy source (e.g., in the form of carbohydrates, e.g., glucose); one or more essential amino acids (e.g., all essential amino acids, the 20 basic amino acids plus cysteine); vitamins and / or other organic compounds that are usually required at low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements that are usually required at very low concentrations, e.g., at concentrations in the micromolar range. As used herein, cell culture media encompasses nutrient solutions typically used and / or known to be used in any cell culture process, including but not limited to batch, extended batch, fed-batch, and / or perfusion, or continuous culture of cells.

[0259] Various media formulations may be used during the cell culture continuum, for example, to initiate the culture, facilitate the transition from one stage (e.g., growth stage or phase) to another stage (e.g., production stage or phase), and / or optimize conditions (e.g., concentrated feed medium) during cell culture. Basal media formulations containing components essential for cell survival and growth are typically used to initiate the cell culture. Growth media formulations are generally used to promote cell growth and minimize protein expression. Production media formulations are typically used to promote production of the desired protein and maintain the cells with minimal cell growth. Feed media, concentrated media formulations to replenish nutrients and amino acids generally consumed during the production phase of the cell culture, may be used to replenish and maintain active cultures, particularly cultures operated in perfusion mode. Such concentrated feed medium may contain some, if not all, of the components of the cell culture medium, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x or about 1000x of their normal amounts or concentrations.

[0260] The disclosed methods can be used as part of a larger-scale production process in which cells are cultured in separate stages. Each stage can be carried out in its own bioreactor vessel or other vessel suitable for cell culture. Alternatively, two or more stages can be carried out in a common vessel. For commercial production of recombinant proteins using mammalian cells, there are typically multiple growth stages, e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10, carried out in different culture vessels before the final N production stage. For example, cells can be cultured in various bioreactors for one or more growth stages prior to an N-1 seed stage, which can then be cultured in one or more N-1 bioreactors. The duration of the N-1 stage can range, for example, from 3 to 14 days, or can be continuous and designed to maintain cells in exponential growth before inoculating the production (N) production bioreactor. Cells from the N-1 bioreactor can be transferred to the (N) production bioreactor and grown under conditions that maximize protein production.

[0261] As used herein, the term "bioreactor" refers to any vessel useful for growing cell cultures (e.g., mammalian cell cultures). Non-limiting examples of bioreactors include stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. Bioreactors can be single-use and / or built-in-place stainless steel vessels. In some embodiments, exemplary bioreactors can perform one or more (e.g., one, two, three, or all) of the following steps: supplying nutrients and / or carbon sources, injecting appropriate gases (e.g., oxygen), inflowing and outflowing cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitating (e.g., stirring), and / or cleaning / sterilizing. Any suitable bioreactor diameter may be used. Unless otherwise indicated by context, in some embodiments, a bioreactor can have a volume between 100 mL and 50,000 L. Unless otherwise indicated, a bioreactor can be any size useful for culturing cells; typically, a bioreactor is sized appropriately for the volume of cell culture to be grown therein. In non-limiting embodiments, and unless otherwise indicated by context, a bioreactor can be at least 1 liter (L), or can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume therebetween. In a preferred embodiment, the bioreactor volume is between 200 liters and 20,000 liters. In one embodiment, the bioreactor volume is between 2,000 liters and 18,000 liters. In one embodiment, the bioreactor volume is between 2,000 liters and 15,000 liters. In one embodiment, the bioreactor volume is between 2,000 liters and 12,000 liters. In one embodiment, the bioreactor volume is between 2,000 liters and 10,000 liters. In one embodiment, the bioreactor volume is between 200 liters and 5,000 liters.In one embodiment, the bioreactor volume is between 2,000 liters and 3,000 liters. In one embodiment, the bioreactor volume is between 10,000 liters and 20,000 liters. In one embodiment, the bioreactor volume is between 10,000 liters and 18,000 liters. In one embodiment, the bioreactor volume is between 10,000 liters and 15,000 liters. In one embodiment, the bioreactor volume is between 10,000 liters and 12,000 liters. In one embodiment, the bioreactor volume is 200, 500, 1,000, 2,000, 3,000, 5,000, 10,000, 12,000, 15,000, 18,000, or 20,000 liters.

[0262] The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during the culture period. Those skilled in the art will be able to recognize and select a suitable bioreactor for use in the methods disclosed herein based on relevant considerations.

[0263] The bioreactor volume is divided into a working volume space and a headspace. The working volume of a bioreactor refers to the volume within the bioreactor in which the cell culture is operated and is typically expressed as a percentage of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 70% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 70%-100% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 75% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 80% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 85% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 90% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 91% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 92% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 93% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 94% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 95% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 96% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 97% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 98% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is at least 99% of the bioreactor volume. In one embodiment, the working volume of the bioreactor is about 100% of the bioreactor volume.

[0264] In some embodiments, the lean perfusion method described herein is implemented using disposable bioreactors instead of traditional stainless steel culture vessels. The use of disposable technology minimizes the infrastructure requirements associated with traditional cell culture, such as steel / glass commercial-scale vessels and related equipment. Disposable bioreactors offer flexibility in the manufacturing process; site assembly, reconstitution, sterilization, and validation of disposable bioreactors are often faster, easier, and less costly than traditional built-in-place stainless steel cell culture plants. Disposable bioreactors contain a disposable plastic sterilization bag supported by a non-disposable support structure. The culture is agitated within the bag by agitation or rocking, and air and oxygen spargers and sensors are also provided to measure and adjust various parameters of the culture, such as pH, temperature, oxygen, and cell density. Disposable bioreactors are commercially available, for example, BioSTR®, Sartorius, Goettingen Germany; MOBIUS®, Millipore, Burlington, MA; XCELLEREX®, Cytiva, Marlborough, MA.

[0265] In some embodiments, the lean perfusion methods disclosed herein are carried out in one or more stainless steel bioreactors, particularly built-in-place large-scale stainless steel bioreactors capable of operating at volumes of 2,000 to 20,000 L or greater.

[0266] A bioreactor system maintains conditions within the bioreactor to support cell culture. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). As used herein, "running" a bioreactor system refers to maintaining conditions in the bioreactor system to support cell culture. A bioreactor "run" typically involves inoculating a prepared bioreactor with a seed culture, subjecting the cells to one or more growth and / or production phases until one or more predetermined parameters (time, viable cell density, packed cell volume) are met, and then harvesting the contents of the bioreactor.

[0267] The recovery operation completely or partially clarifies and / or purifies the target protein from at least one impurity found therewith in the cell culture medium, such as residual cell culture medium, cells, cell debris, undesired cell or medium components, and / or product-related and / or process-related impurities. Methods for recovering recombinant proteins from suspension cell cultures are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation methods such as flocculation, gravity separation, centrifugation, sonication, filtration (including membrane filtration, ultrafilters, microfilters, tangential flow, alternative tangential flow, depth filters, and alluvial filtration filters).

[0268] The harvested cell culture fluid (HCCF) may be stored in a surge tank, holding tank, bag, or other container configured to provide feed to the chromatography column skid and appropriate to infrastructure and / or process requirements.

[0269] The harvested cell culture fluid can be subjected to one or more downstream operations to capture and / or polish the target protein. Downstream operations utilize affinity chromatography, which involves resins and / or membranes containing a substance that binds and / or interacts in some way with at least the desired protein, impurities, or contaminants. Affinity chromatography is typically used in biomanufacturing processes as the first capture step to isolate and typically concentrate the desired protein from the harvested cell culture fluid. Non-limiting examples of such affinity chromatography materials include those utilizing staphylococcal proteins, such as Protein A, Protein G, Protein A / G, and Protein L; substrate-binding capture mechanisms; antibody or antibody fragment-binding capture mechanisms; aptamer-binding capture mechanisms; cofactor-binding capture mechanisms; and the like. Immobilized metal affinity chromatography (IMAC) can be used to capture proteins that have, or have been engineered to have, affinity for metal ions. Protein A affinity chromatography is typically used in the first bulk purification operation. Protein A ligands are highly selective for a wide range of proteins containing antibody Fc regions, resulting in robust removal of process-related impurities and high target protein yields. Protein A materials are commercially available from a number of sources, including, for example, MABSELECT™ SURE Protein A, Protein A Sepharose FAST FLOW™, MABSELECT™ PrismA (Cytiva, Marborough, MA), PROSEP-A™ (Merck Millipore, UK), TOYOPEARL™ HC-650F Protein A (TosoHass Co., Philadelphia, PA), and AP Plus, Purolite, King of Prussia, PA).

[0270] Intermediate and / or polishing unit operations utilize various chromatographic techniques for the continued purification of the desired protein and the elimination of process- and product-related impurities, contaminants, viruses, and the like. These chromatographic unit operations utilize resins and / or membranes containing materials that can be operated in various modes; two commonly used modes are bind-and-elute and flow-through. Frontal chromatography allows for a continuous, high-density feed (containing the protein of interest and at least one impurity) onto a chromatography medium. In frontal chromatography, separation of the protein of interest from impurities and contaminants is driven by the binding affinity of the components in the load feed to the chromatography medium. The amount of protein of interest that can be loaded onto and bound to the chromatography medium in the frontal mode typically depends on the amount of more highly charged impurities / contaminants, such as product-related impurities, in the load feed. Initially, all components in the load feed bind to the chromatography medium. Separation of the product of interest from the impurities / contaminants is driven by affinity for the chromatography medium. When the chromatography medium reaches saturation binding, components in the load feed that have a higher affinity for the chromatography medium (typically product-related impurities such as HMW species) displace proteins with weaker affinities (the product of interest), resulting in the proteins with weaker affinities being separated from the chromatography medium. These proteins exit the column as pure bands in the load flow-through. As loading progresses, bound proteins are successively displaced in order of decreasing affinity for the chromatography medium until the column is saturated or near saturation with proteins with higher affinities than the protein of interest.

[0271] Examples of chromatographic media used in such polishing steps include, but are not limited to, media for ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM); and hydroxyapatite chromatography (HA).

[0272] Multiple purification chromatography unit operations (e.g., one, two, or three chromatography unit operations), each typically performing a different function or operating in a different manner, can be combined depending on the requirements of the manufacturing process. Cation exchange chromatography refers to chromatography performed on a solid phase medium (e.g., a resin or membrane) that is negatively charged and has free cations for exchange with cations in an aqueous solution passing over or through the solid phase. The charge can be imparted by attaching, e.g., covalently, one or more charged ligands to the solid phase. Alternatively, or in addition, the charge can be an inherent property of the solid phase (e.g., as in the case of silica, which has an overall negative charge). CEX chromatography is typically used for the removal of high molecular weight (HMW) contaminants, the removal of process-related impurities, and / or viral clearance. Commercially available cation exchange media include sulfopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™, or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (Cytiva), FRACTOGEL-SO™, FRACTOGEL-SE HICAP™, and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL™ XS, TOYOPEARL™ HS (Tosoh Bioscience, King of Prussia, PA), UNOsphere™ (BioRad, Hercules, CA), S Ceramic Hyper™ DF (Pall, Portland, FL). Washington, NY), POROS™ (ThermoFisher, Waltham, MA), ESHMUNO® CSP and ESHMUNO® CP-FT (Millipore Sigma, Darmstadt, Germany).

[0273] Anion exchange chromatography refers to chromatography performed on a solid phase medium (e.g., a resin or membrane) that is positively charged and has free anions for exchange with anions in an aqueous solution passing over or through the solid phase. AEX chromatography is used, for example, for viral clearance and impurity removal. Commercially available anion exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15 Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Cytiva), FRACTOGEL EDM TMAE™, FRACTOGEL EDM DEAE™ (EMD Merck), TOYOPEARL Super Q® and TOYOPEARL NH2-750F (Tosoh Bioscience), POROS HQ™, POROS XQ™ (ThermoFisher).

[0274] Mixed-mode or multimode chromatography (MMC) refers to chromatography that utilizes two or more types of interactions between a stationary phase and analytes to achieve their separation. MMC differs from single-mode chromatography in that two or more types of interactions, such as electrostatic interactions, hydrogen bonding interactions, and hydrophobic interactions, contribute significantly to solute retention. Commercially available multimodal chromatography media include, but are not limited to, Capto™ Adhere, Capto™ MMC Impress, Capto MMC (Cytiva), PPA Hypercel, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY), Eshmuno HCX (Merk Millipore), and Toyopearl MX-TRP-650M (Tosoh Bioscience).

[0275] Hydrophobic interaction chromatography refers to chromatography performed on a solid phase medium that utilizes interactions between hydrophobic ligands and hydrophobic residues on the surface of the protein of interest. Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sephrose™ (Cytiva), Tosoh Hexyl (Tosoh Bioscience), and Capto™ Phenyl (Cytiva).

[0276] Hydroxyapatite chromatography refers to chromatography performed on a solid phase medium that utilizes positively charged calcium and negatively charged phosphate, which can act as cations or anions depending on the pI of the protein and the pH of the buffer. Commercially available hydroxyapatite media include CA ++ Pure-HA, Tosoh Bioscience, HA ULTROGEL®, Sartorius).

[0277] Unit operations aimed at inactivating, reducing, and / or eliminating viral contaminants may include steps to mitigate viral risks by manipulating the environment and / or using filtration. Viral mitigation measures are important to ensure the safety of protein therapeutics and may be implemented one or more times throughout downstream purification. Viral contaminants can arise from a variety of sources, including the use of animal-derived reagents, adventitious viral contaminants in host cell lines, or system failures in GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped viruses. With enveloped viruses, the envelope allows the virus to identify, bind, invade, and infect target host cells. Therefore, enveloped viruses are more susceptible to inactivation methods. Various methods can be employed for viral inactivation, including, but not limited to, heat inactivation / pasteurization, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, chemical inactivators, the addition of surfactants, and solvent / detergent treatments. Surfactants, such as detergents, can be very effective at specifically inactivating enveloped viruses because they solubilize their membranes. One method for achieving viral inactivation is incubation at low pH (e.g., pH <4). Low pH viral inactivation can be followed by a neutralization operation to readjust the virally inactivated solution to a pH more compatible with the requirements of the next downstream unit operation. Low pH viral inactivation is typically performed after purification of harvested cell culture fluid by affinity chromatography, particularly affinity chromatography utilizing substrate-binding ligands derived from Staphylococcus aureus, such as protein A chromatography, because elution is typically performed at low pH. Non-limiting examples of low pH viral inactivation methods are described in U.S. Patent Application Nos. 63 / 168,608 and 63 / 159,217. Non-limiting examples of detergent inactivation methods are described in WO 2020 / 190985. Low pH viral inactivation can also be followed by filtration, such as depth filtration, for clarification of the neutralized fluid.

[0278] Non-enveloped viruses are more difficult to inactivate without risk to the produced proteins and are removed by filtration. Exemplary processes are described in WO 2020 / 159838. Viral filtration can be performed using microfilters or nanofilters, such as those available from PLAVONA® (Asahi Kasei, Chicago, IL), VIROSART® (Sartorius, Goettingen, Germany), VIRESOLVE® Pro (MilliporeSigma, Burlington, MA), Pegasus™ Prime (Pall Biotech, Port Washington, NY), and CUNO Zeta Plus VR (3M, St. Paul, MN). Viral filtration can occur at one or more downstream steps in the biomanufacturing process. Viral inactivation and filtration can occur at one or more downstream stages. Typically, virus filtration follows an affinity chromatography unit operation, and virus filtration precedes or follows an ultrafiltration / diafiltration (UF / DF) operation, but can also occur after UF / DF.

[0279] Downstream unit operations may also include product concentration and buffer exchange of the desired protein into a desired formulation buffer. UF / DF operations can occur at one or more stages in downstream processing. Typically, UF / DF operations are performed prior to bulk storage of the drug substance. Instead of storage, unit operations related to drug product fill / finish may also immediately follow the UF / DF operation. One or more stability-enhancing excipients can optionally be added directly to the UF / DF retentate feed tank containing the formulated purified protein, resulting in the formulated drug substance, or to the UF / DF eluate pool. An exemplary UF / DF process is described in WO 2020 / 159838. Filters for use in UF / DF operations are well known and common in the art and are commercially available from many sources, such as regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY), etc.

[0280] In all chromatographic steps, multiple filters can be used up to the capacity required to achieve the desired objectives of the purification step, or as the physical setup of the holder, skid, or UF / DF system will allow.

[0281] Upstream and / or downstream unit operations may be performed continuously or in stages. Vessels such as surge receptacles, holding tanks, etc. may be used following one or more unit operations.

[0282] To better inform decisions regarding the performance of each step during production, key attributes and performance parameters of the purified desired protein can be measured. These key attributes and parameters can be monitored in real time, near real time, and / or after unit operations. Key parameters that can be measured during cell culture include, but are not limited to, levels of consumed cell culture medium components (e.g., glucose), accumulating metabolic by-products (e.g., lactate and ammonia), and parameters related to cell maintenance and survival, such as dissolved oxygen content. Key attributes such as specific productivity, viable cell density, packed cell volume, pH, osmolality, appearance, color, aggregation, percent yield, and titer can also be monitored during appropriate steps in the manufacturing process. Monitoring and measurements can be performed using known techniques and commercially available equipment.

[0283] The lean perfusion cell culture methods described herein can be used to produce polypeptides and proteins of interest. The polypeptides and proteins can be of scientific or commercial interest, including protein-based therapeutics. Proteins of interest include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using the cell culture methods described herein and can be referred to as "recombinant proteins." The expressed protein can be produced intracellularly or secreted into the culture medium, from which it can be recovered and / or harvested. The term "isolated protein" or "isolated recombinant protein" refers to a polypeptide or protein of interest that has been purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. Proteins of interest include, but are not limited to, proteins that exert a therapeutic effect by binding to a target, such as those listed below (including targets derived from, related to, and variants thereof).

[0284] Proteins of interest may include, but are not limited to, "antigen-binding proteins." An "antigen-binding protein" refers to a protein or polypeptide that comprises an antigen-binding region or portion that has affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include, but are not limited to, antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including, for example, single-chain variable fragments (scFvs), two-chain (bivalent) scFvs, and IgGscFvs (see, e.g., Orcutt et al., 2010, Protein Eng Des Sel 23:221-228)), hetero-IgGs (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA). Also included are chimeric antigen receptors (CARs, CAR Ts) and T cell receptors (TCRs), such as BiTE® molecules, bispecific T cell engagers, and bispecific T cell engagers with extension.

[0285] As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (approximately 25 kDa each) and two heavy chain polypeptides (approximately 50-70 kDa each). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are connected to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0286] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) connected by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy-light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDRs and FRs of a given antibody can be identified using this system. Other numbering systems for the amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0287] As used in the context of the present invention, an "antigen-binding fragment," which is used interchangeably herein with "binding fragment" or "antibody fragment," is a portion of an antibody that lacks at least some of the amino acids present in a full-length heavy and / or light chain, but is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a heavy-chain-only antibody (e.g., a camelid heavy-chain antibody); VHH fragments; see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and CDR fragments, and may be derived from any mammalian source, such as human, mouse, rat, rabbit, or camel. An antigen-binding fragment can compete with an intact antibody for binding to a target antigen, and can be produced by modification of the intact antibody (e.g., enzymatic cleavage or chemistry) or can be synthesized de novo using recombinant DNA technology or peptide synthesis. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen, such as the heavy chain CDR3 from an antibody that binds to the antigen. In other embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of the antibody that binds the antigen or all three CDRs from the light chain of the antibody that binds the antigen. In yet other embodiments, the antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) from an antibody that binds the antigen.

[0288] Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments (each of which has a single antigen-binding site) and the remaining "Fc" fragment (which contains all but the first domain of the immunoglobulin heavy chain constant region). The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fd fragment" contains the VH domain and CH1 domain from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0289] An "Fc fragment" or "Fc domain" of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. The Fc domain may be derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc domain comprises the CH2 domain and the CH3 domain derived from a human IgG1 or human IgG2 immunoglobulin. The Fc domain may retain effector functions such as Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc domain may be modified to reduce or eliminate effector function.

[0290] A "Fab' fragment" is a Fab fragment that has one or more cysteine ​​residues from the antibody hinge region at the C-terminus of the CH1 domain.

[0291] A "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by inter-heavy chain disulfide bridges at the hinge region.

[0292] An "Fv" fragment is the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy-chain variable region (VH) and one immunoglobulin light-chain variable region (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light- or heavy-chain variable region (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site comprising both the VH and VL.

[0293] A "single-chain variable antibody fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0294] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains naturally occur in certain species of animals, such as nurse sharks, nurse sharks, and Camelidae, including camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region; i.e., these functional antibodies are heavy chain-only homodimers (also called "heavy chain antibodies" or "HCAbs"). Camelized VHHs reportedly contain hinge, CH2, and CH3 domains, but lack the CH1 domain, and are recombined with IgG2 and IgG3 constant regions. Camelized VHH domains have been shown to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold and provide a framework for long transmembrane loop structures. Human heavy chain antibodies can be generated from transgenic animals expressing human immunoglobulin genes, for example, the UniAb™ antibodies produced by UniRat™ transgenic rats.

[0295] In some embodiments, the protein of interest may comprise a colony-stimulating factor, such as, for example, granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are erythropoiesis stimulating agents (ESAs), such as Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methoxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit® ( Also included are epoetin alfa), epoetin alfa Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, and variants or analogs thereof, and biosimilars of any of the foregoing.

[0296] In some embodiments, the protein of interest binds to one or more of the following, alone or in any combination: CD proteins (including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174), HER receptor family proteins (including, for example, HER2, HER3, HER4), and EGF receptor, EGFRvIII, cell adhesion molecules such as LFA-1, Mol , p150, 95, VLA-4, ICAM-1, VCAM and alpha v / beta 3 integrin, growth factors (e.g., including but not limited to, vascular endothelial growth factor ("VEGF")); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitory factor, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factor (e.g., aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), such as, inter alia, TGF-α and TGF-β (including TGF-β1, TGF-β3, TGF-β4, or TGF-β5), insulin-like growth factors-I and -II (IGF-I and IGF-II), including but not limited to, des(1-3)-IGF-I (brain IGF-I) and osteomorphic factor), insulin and insulin-related proteins (insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding proteins; (clotting and coagulation-related proteins, such as, inter alia, Factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin and thrombopoietin receptors, colony-stimulating factors (CSFs) (including, inter alia, M-CSF, GM-CSF, and G-CSF), other blood and serum proteins (albumin,including, but not limited to, IgE and blood group antigens), receptors and receptor-associated proteins (including, for example, flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, and T cell receptor); neurotrophic factors (including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin 3, 4, 5, or -6 (NT-3, NT-4, NT-5, or NT-6)); relaxin A chain, relaxin B chain, and prorelaxin, interferons (including, for example, interferon-alpha, -beta, and -gamma), interleukins (IL), such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor, and / or IL-13 to the receptor (IL-13 to the receptor), IL-13RA2 or IL-17 receptor, IL-1RAP; viral antigens (including but not limited to AIDS envelope viral antigen), lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and β, enkephalinase, BCMA, Ig kappa, ROR-1, ERBB2, mesothelin, RANTES (regulated upon activation, normally expressed and secreted by T cells), mouse gonadotropin-related peptide, DNase, FR-alpha, inhibin and activin, integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay-accelerating factors ( DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1,Programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (Ly), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1), transforming growth factor beta (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRα), sclerostin, and biologically active fragments or variants of any of the foregoing.

[0297] In some embodiments, the protein of interest includes abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bemarituzumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, erenumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab Tiuxetan, infliximab, ipilimumab, lerdelimumab, rumiliximab, ixekizumab (lxdkizumab), mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, ordesukimab, palivizumab, panitumumab, pembrolizumab Mab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargamostim, tocilizumab, tositumomab, tarlatamab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, and zalutumumab, as well as biosimilars of any of the foregoing.

[0298] In some embodiments, the recombinant protein produced by the methods described herein is selected from bemarituzumab, denosumab, erenumab, evolocumab, ordesukimab, panitumumab, romosozumab, and tarlatamab.

[0299] In some embodiments, proteins of interest may also include engineered receptors, such as chimeric antigen receptors (CARs) and T cell receptors (TCRs), as well as other proteins containing antigen-binding molecules that interact with their target antigens. CARs can be engineered to bind to antigens (e.g., cell surface antigens) by incorporating antigen-binding molecules that interact with their target antigens. CARs typically incorporate an antigen-binding domain (such as an scFv) in tandem with one or more costimulatory ("signaling") domains and one or more activation domains.

[0300] Where a range of values ​​is provided herein, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0301] As used herein, the terms "a" and "an" mean "one or more," unless specifically indicated otherwise. Furthermore, "one or more" and "at least one" are used interchangeably herein. Furthermore, unless the context otherwise requires, singular terms include pluralities and plural terms include the singular.

[0302] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprises," and variations such as "comprises" and "comprising," should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" may also be interchanged with the terms "containing" or "including," or, when used herein, with the term "having."

[0303] As used herein, "consisting of" excludes any element, step, or ingredient not specified in an embodiment feature or claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the embodiment feature or claim element.

[0304] In each instance herein, the terms "comprising," "consisting essentially of," and "consisting of," as well as any variations thereof, may be replaced with either of the other two terms or any variations thereof.

[0305] In some embodiments, the term "about," as used herein and when applied to one or more values, refers to a value similar to a stated reference value. For example, in some embodiments, unless otherwise stated or otherwise clear from the context (e.g., unless such number exceeds 100% of possible values), the term "about" refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less (greater or less) of the stated reference value in either direction of the stated reference value.

[0306] All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and journal articles, are hereby expressly incorporated by reference.

[0307] What is described in one embodiment of the present disclosure may be combined with one or more other embodiments of the present disclosure unless the context clearly dictates otherwise. For example, the recitation of "in one embodiment" herein may be replaced with "in some embodiments" or "in any embodiment" unless the context clearly dictates otherwise.

[0308] The disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, but instead as non-limiting exemplifications of particular aspects of the present disclosure. Functionally equivalent methods and components are within the scope of the present disclosure. Indeed, various modifications of the disclosed subject matter in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing and accompanying figures. Such modifications are intended to be within the scope of the disclosed subject matter.

[0309] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for purposes of illustration and are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used herein have been selected to best explain the principles of the embodiments, practical applications, or technical improvements to technology found in the marketplace, and / or to enable others skilled in the art to understand the disclosed subject matter. [Example]

[0310] Example 1 Fed-batch cell cultures were compared to lean perfusion cell cultures

[0311] Fed-batch culture Three CHO cell lines, each expressing a different monoclonal antibody (mAb1, mAb2, and mAb3), were cultured under fed-batch conditions. Each cell line was cultured at approximately 0.8 × 10 cells per cell in a separate 3-liter bioreactor containing approximately 1.5 liters of serum-free defined culture medium. 6 cells / mL~1.2×10 6 Cultures were seeded at 1000 cells / mL (n = 3-16 / mAb). Bioreactors were maintained at 36°C, 5% CO2, 6.90 pH, and 315 rpm. Cultures were fed at 8% post-inoculation volume on days 3 and 6, with supplemental feeds at 4% of the nutrient feed volume on the same days. Glucose solution was added as needed to maintain a glucose concentration >2 g / L throughout the entire culture. mAb2 and mAb3 cultures were harvested on day 11, and mAb1 culture was harvested on day 12.

[0312] Lean Perfusion The same three CHO cell lines expressing mAb1, mAb2, or mAb3 were each cultured in separate 7-liter bioreactors at 6 x 10 cells in an initial culture volume of approximately 3 liters of serum-free synthetic basal culture medium. 6 cells / mL~12×10 6 Cells were seeded at 2–8 cells / mL (n = 2–8 / mAb). The bioreactor was maintained at 36°C, 5% CO2, 6.90 pH, and 315 rpm. Perfusion was initiated on day 1 (or approximately 24 h after seeding) using an alternating tangential flow filter system (Repligen, Waltham, MA) equipped with a 30,000 MWCO filter (Cytiva, Westborough, MA). The culture was perfused with serum-free synthetic feed medium. The feed rate and permeate rate were maintained at 0.13 culture volumes per day (V / d), respectively. On day 3, a feed-up phase was initiated to increase the culture volume in the bioreactor to a final culture volume of approximately 4.4 L, which was approximately 100% of the final working volume of the bioreactor. The feed rate was increased to 0.22 V / d, and the permeate rate was held constant at 0.13 V / d. On the fourth day, the feed-up phase ended, and the feed rate was reduced to 0.13 V / d, the same as the permeation rate. The viable cell density after the feed-up phase was approximately (20-30) × 10 6The cell density was 1.27 cells / mL. The temperature of the culture was decreased to 33.0°C within 48 hours after feed-up. On day 5, the feed rate and permeation rate were increased to 0.22 V / d until harvest. mAb1, mAb2, and mAb3 cultures were all harvested on day 15.

[0313] In-process samples were taken to monitor the culture conditions. Viable cell density (VCD) refers to the number of viable cells in a given volume of culture medium. VCD was determined using a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Indianapolis, IN). Titers were measured by high-performance liquid chromatography (HPLC) via affinity chromatography (Protein A, Waters, Milford, MA).

[0314] Figure 2 shows a schematic diagram of the change in culture volume over the course of a cell culture from initiation to harvest. A typical lean perfusion culture using a single feed-up phase (black line) is compared with a typical fed-batch culture using multiple feeds (gray line). Both cultures are initiated with a culture volume at least 50%–75% of their respective final working volumes. The lean perfusion method utilizes a shortened growth phase at a low culture volume. Once the culture reaches one or more desired target criteria, such as cell density or time since inoculation, the culture volume is increased to the desired final culture volume (typically >75%–100% of the final working volume). Compared to fed-batch processes, where the culture volume is increased slowly in a stepwise or intermittent manner and the maximum working volume of the bioreactor is only available at the end of the culture, once the production phase begins, the lean perfusion method allows the bioreactor's maximum working volume to be efficiently utilized for a longer period during the culture operation. Achieving the desired final culture volume and cell density early in the cell culture process leads to increased production in lean perfusion culture compared to fed-batch culture.

[0315] Figure 3 shows the viable cell densities achieved by lean perfusion and fed-batch cultures for three monoclonal antibody samples. Lean perfusion cultures utilized high cell densities at low culture volumes to achieve and maintain higher viable cell densities during the growth phase compared to fed-batch cultures. Lean perfusion: dark gray solid line for mAb1, black solid line for mAb2, and light gray solid line for mAb3. Fed-batch cultures: dark gray dashed line for mAb1, black dashed line for mAb2, and light gray dashed line for mAb3. The lean perfusion cultures incorporated a single feed-up phase to increase the culture volume and reduce cell mass before the production phase. During the production phase, the lean perfusion cultures maintained a low perfusion rate to provide feed medium and remove waste products, achieving a longer culture period compared to fed-batch cultures. Furthermore, although both cultures were initiated at approximately 50% of the final culture volume, the lean perfusion samples were able to support a 10-fold increase in cell density at inoculation compared to the fed-batch samples.

[0316] Figures 4A-4C show the average specific productivity (pg / cell / day) for each culture type: lean perfusion culture (LP) and fed-batch culture (FB). The specific productivity of lean perfusion culture increased compared to fed-batch culture (27% for mAb1, 22% for mAb2, and 51% for mAb3).

[0317] Figures 5A-5C show the average end-day titer (g / L) for each culture type. Lean perfusion samples showed elevated end-day titers compared to their fed-batch counterparts: mAb1 showed a 3-fold increase, mAb2 showed a 4.4-fold increase, and mAb3 showed a 4.7-fold increase.

[0318] Figure 6 shows a comparison of lactate concentrations for lean perfusion and fed-batch cultures. The lower perfusion rate used in the lean perfusion process reduced the accumulation of cell culture waste by removing spent medium in the permeate stream, as shown by the decrease in lactate concentration over the culture period, compared to the fed-batch process, which simply diluted the lactate in the culture volume.

[0319] Lean perfusion culture efficiently utilized the bioreactor working volume throughout the entire culture period. Inoculating a bioreactor containing a low cell culture medium volume at a high cell density allowed for a shorter growth phase, allowing the majority of the culture time to be spent in production at the desired cell density. Increasing the culture volume before production also reduced the cell mass, thereby reducing the accumulation of impurities such as lactate. Furthermore, a low perfusion rate reduced the amount of feed medium required to maintain the culture during both the growth and production phases. As a result, productivity was improved compared to fed-batch culture. In a fed-batch process, the volume and cell density increased slowly over the course of the culture, achieving maximum capacity only when the culture began to decline and be harvested.

[0320] Example 2 Perfusion Culture (High Perfusion Rate, Constant Culture Volume) Compared to Lean Perfusion Cell Culture perfusion culture Two CHO cell lines (mAb2 and mAb3) were each cultured at approximately 0.5 × 10 cells / mL in separate 3-liter bioreactors in 1.5 liters of serum-free synthetic basal culture medium, approximately 100% of the final working volume of the bioreactor. 6 cells / mL~2×10 6 Cells were seeded at 2000 cells / mL (n = 3–8 / mAb). The bioreactor was maintained at 36°C, 5% CO2, 6.90 pH, and 350 rpm. Perfusion began on day 3 using an alternating tangential flow filter system (Repligen) equipped with a 30,000 MWCO filter (Cytiva). The culture was perfused with serum-free synthetic feed medium. At the start, the feed rate and permeate rate were 0.5 V / d. On day 5, the feed rate and permeate volume were increased to 0.75 V / d. On day 8, the feed rate and permeate rate were increased to 1.0 V / d until harvest on day 15.

[0321] Lean Perfusion Approximately 6 x 10 cells of the same CHO cell line (mAb2 and mAb3) were cultured in separate 7 L bioreactors in an initial culture volume of approximately 3 liters of serum-free synthetic basal culture medium. 6 ~12×10 6Cells were seeded at 2–8 cells / mL (n = 2–8 / mAb). The bioreactor was maintained at 36°C, 5% CO2, 6.90 pH, and 315 rpm. Perfusion began on day 1 (or approximately 24 h after seeding) using an alternating tangential flow filter system (Repligen, Waltham, MA) equipped with a 30,000 NMWC filter (GE Healthcare, Westborough, MA). The culture was perfused with serum-free synthetic feed medium. The feed rate and permeate rate were 0.13 V / d starting 24 h after seeding. On day 3, a feed-up phase was initiated to increase the culture volume in the bioreactor to a final culture volume of approximately 4.4 L, which was approximately 100% of the final working volume of the bioreactor. The feed rate was increased to 0.386 V / d, and the permeate rate was held constant at 0.13 V / d, allowing the full bioreactor volume to be reached in approximately 30 h. On day 4, the feed-up phase was completed and the feed rate was reduced to 0.13 V / d, the same as the permeate rate. On day 5, the temperature was reduced to 33.0°C. The viable cell density at the end of the feed-up phase was approximately 30 x 10 6 On day 5, the feed rate and permeation rate were increased to 0.22 V / d until harvest on day 15.

[0322] Figure 7 shows a schematic diagram of different perfusion rates for a lean perfusion step (black line) compared to a high perfusion rate, high cell density pre-perfusion step (gray line). Like a typical perfusion culture process, lean perfusion culture relies on a perfusion rate where the feed rate and permeate rate are the same, except during the feed-up phase, when the feed rate is increased relative to the permeate rate to result in a net increase in culture volume in the bioreactor. Lean perfusion methods utilize low perfusion rates of 0.5 V / d or less, preferably 0.25 V / d or less, throughout the cell culture process. The perfusion rate can be slower before the feed-up phase (Rate 1) and the same or faster after the feed-up phase (Rate 2). For perfusion culture methods, the rate is typically performed at 1.0 V / d or greater. Some perfusion culture methods utilize one or more rapid incremental rate increases, typically at least 0.5 V / d or greater, in the early stages of the culture, and finish at rates of 1.0 V / d or greater. Utilizing high perfusion rates over the course of a culture run means preparing, storing, and using larger amounts of feed medium and increasing the volume of spent medium waste, increasing disposal and environmental costs.

[0323] Figure 8 shows a schematic of the scale of feed medium used by the lean perfusion process (black line), which maintains low levels of consumption throughout the course of the culture, versus a high perfusion rate process (gray line), which uses feed medium at more than twice the rate of the lean perfusion process.

[0324] Figure 9 shows a schematic of the magnitude of the difference in the amount of feed medium required in the black column for lean perfusion culture (LP) and the dark gray column for perfusion culture operated at a high perfusion rate (PC). Compared to perfusion cultures operated continuously at a higher perfusion rate and a volume equal to the maximum working volume of the bioreactor, the lean perfusion method reduces the volume of feed medium required, sometimes by up to four-fold, due to the lower perfusion rate and smaller initial culture volume.

[0325] Figure 10 shows a schematic of the difference in culture volume over time for lean perfusion culture (black line) compared to high perfusion rate culture (gray line). Compared to high-density perfusion culture, which operates at 100% of the bioreactor's working volume, for lean perfusion, the initial culture volume was set at at least 50%–75% of the bioreactor's working volume throughout the culture period and increased to 100% of the working volume during the feed-up phase. Starting with a smaller culture volume allows for higher inoculation from the N-1 reactor at a smaller working volume.

[0326] Figure 11 shows a comparison of viable cell densities (VCDs) for lean perfusion cultures and perfusion cultures operated at high perfusion rates. Lean perfusion cultures: mAb2 (black solid line), mAb3 (gray solid line). Perfusion cultures: mAb2 (black dashed line), mAb3 (gray dashed line). Lean perfusion cultures started at high cell densities in a culture volume that was less than 60% of the bioreactor working volume, increased the culture volume to approximately 100% of the final working volume by the end of the feed-up phase, and maintained at that volume until harvest. High perfusion rate cultures maintained approximately 0.5 x 10 cells in 100% of the final bioreactor working volume. 6 cells / mL~2×10 6 The lean perfusion cultures were started at 1000 cells / mL. The cell density slowly improved to a greater viable cell density by the end of the culture than the lean perfusion samples. By starting the culture at a lower culture volume and higher cell density, the lean perfusion culture achieved the target cell density more quickly, shortening the time required for the growth phase and maximizing time for production.

[0327] Figures 12A and 12B show the final day titers for lean perfusion cultures (LP, black columns) compared to perfusion cultures operated at higher perfusion rates (PC, gray columns). The lean perfusion cultures had final titers comparable to the perfusion cultures. For mAb2, there was a 1.1-fold increase in titer in the lean perfusion culture compared to the perfusion culture. For mAb3, there was a 0.8-fold decrease in titer in the lean perfusion culture compared to the perfusion culture.

[0328] Figures 13A and 13B show the average specific productivity (pg / cell / day) over the culture period for lean perfusion cultures (LP, black columns) and perfusion cultures operated at high perfusion rates (PC, gray columns). The lean perfusion cultures were run at lower perfusion rates and used less feed medium compared to the high perfusion rate perfusion cultures, yet the cells produced more of the protein of interest. The specific productivity for lean perfusion showed an 18% increase for mAb2 and a 19% increase for mAb3 compared to the high perfusion rate cultures.

[0329] Figures 14A and 14B show the percent packed cell mass of lean perfusion cultures compared to high perfusion rate cultures. Lean perfusion culture (LP): black columns; perfusion culture (PC): gray columns. Lean perfusion packed cell mass showed a 45% decrease for mAb2 (Figure 14A) and a 50% decrease for mAb3 (Figure 14B) when compared to high perfusion rate, high cell density perfusion cultures.

[0330] Again, the lean perfusion culture method efficiently utilized a small culture volume during the growth phase by maintaining a high cell density at a low perfusion rate. The smaller culture volume and low perfusion rate allowed for less feed medium to be used during the growth phase compared to perfusion cultures with a high perfusion rate and a large culture volume. The feed-up phase increased the culture volume and reduced cell density for the production phase, which also reduced the feed required and waste generated compared to perfusion cultures with a high perfusion rate and a large culture volume. The feed-up phase, as a transition between the growth and production phases, reduced cell density and provided a large amount of fresh medium to initiate the production phase. The perfusion rate was sufficient to remove waste while replenishing fresh nutrients to maintain cell productivity throughout the culture run. A temperature shift after the feed-up phase helped maintain the desired cell density for the production phase. Additionally, although the compacted cell volume was reduced by up to 50%, lean perfusion culture produced comparable or higher final titers and specific productivity compared to cultures with a high perfusion rate and higher cell density.

[0331] Example 3: Fed-batch cell culture compared to lean perfusion cell culture Fed-batch culture A CHO cell line expressing a monoclonal antibody (mAb4) was cultured under fed-batch conditions (n=5). Approximately 10 cells of the cell line were cultured in a 3-liter bioreactor containing approximately 1.5 liters of serum-free defined culture medium. 6 The culture was seeded at 1000 cells / mL. The bioreactor was maintained at 36°C, 6.90 pH, and 350 rpm. The culture was fed at 7%, 9%, 9%, and 9% of the post-inoculation volume on days 3, 6, 8, and 11, respectively, and supplemented on the same days at 4% of the nutrient feed volume. Glucose solution was added as needed to maintain a glucose concentration >2 g / L throughout the entire culture. The mAb4 culture was harvested on day 13.

[0332] Lean Perfusion In separate 3-liter bioreactors, approximately 75 x 10 cells were cultured in an initial culture volume of approximately 1.2 liters of serum-free defined basal culture medium. 5 The same CHO cell line expressing mAb4 was seeded at 1000 cells / mL (n=4). The bioreactor was maintained at 36°C, pH 6.90, and 350 rpm. Perfusion began on day 1 (or approximately 24 hours after seeding) using an alternating tangential flow filter system (Repligen, Waltham, MA) equipped with a 30,000 MWCO filter (Cytiva, Westborough, MA). The culture was perfused with serum-free synthetic feed medium. The feed rate and permeate rate were maintained at 0.13 culture volumes per day (V / d), respectively. On day 3, a feed-up phase was initiated to increase the culture volume in the bioreactor to a final culture volume of approximately 1.8 liters, which was approximately 100% of the final working volume of the bioreactor. The feed rate was increased to 0.22 V / d, and the permeate rate was held constant at 0.13 V / d. On day 4, the feed-up phase was terminated and the feed rate was reduced to 0.13 V / d, the same as the permeation rate. After the feed-up phase, the viable cell density was approximately (20-30) × 10 6 On day 5, the feed rate and permeation rate were increased to 0.22 V / d until harvest. The mAb4 culture was harvested on day 15.

[0333] In-process samples were taken to monitor culture conditions. Viable cell density (VCD) refers to the number of viable cells in a given volume of culture medium. VCD was determined using an aCedex HiRes Analyzer (Roche Diagnostics Corporation, Indianapolis, IN). Titers were measured by high-performance liquid chromatography (HPLC) via affinity chromatography (Protein A, Waters, Milford, MA).

[0334] FIG. 15 shows the average viable cell density (mean±SD) achieved by mAb4 lean perfusion culture (solid black line) and fed-batch culture (solid gray line) from initiation to harvest.

[0335] FIG. 16 shows the average end-day titers (g / L) for mAb4 lean perfusion cultures (black columns) and fed-batch cultures (gray columns), with individual run data points shown above each column.

[0336] FIG. 17 shows a comparison of the average lactate concentrations for mAb4 lean perfusion culture (solid black line) and fed-batch culture (solid gray line) from start to harvest.

[0337] mAb4 lean perfusion samples showed an approximately four-fold increase in end-day titer compared to their fed-batch counterparts, reduced accumulation of impurities such as lactate, and increased viable cell density in culture.

Claims

1. A method for culturing cells to produce recombinant proteins, The culture is initiated in the bioreactor with a culture volume that is at least 50% of the bioreactor's final working volume; Cells manipulated to express the recombinant protein are seeded into the culture; The culture is perfused at one or more perfusion rates of 0.5 culture volume / day (V / d) or less until the culture reaches one or more desired target criteria; The culture volume in the bioreactor is increased to the final culture volume; Once the final culture volume is reached, the culture is perfused at one or more perfusion rates of 0.5 culture volume / day (V / d) or less until the culture is finished or harvested. A method that includes this.

2. The method according to claim 1, wherein the culture is started with a culture volume that is 50% to 75% of the final working volume of the bioreactor.

3. The method according to claim 1, wherein the culture is started with a culture volume that is 60% to 70% of the final working volume of the bioreactor.

4. In the culture, 5 × 10 ６ cells / mL~50×10 ６ The method according to claim 1 or claim 2, wherein seeding is performed at a cell density of cells / mL.

5. In the culture, 6 × 10 ６ cells / mL~20×10 ６ The method according to claim 1 or claim 2, wherein seeding is performed at a cell density of cells / mL.

6. The method according to claim 1 or 2, wherein the bioreactor is operated in batch mode for a maximum of 24 hours after sowing.

7. The method according to either claim 1 or claim 2, wherein the culture is in the growth phase before increasing the culture volume to the final culture volume.

8. The duration of the growth phase is 60% or less of the duration of the culture; and / or The culture temperature during the growth phase is 35°C to 37°C. The method according to claim 7.

9. The method according to claim 1 or claim 2, wherein the culture is in the production phase after the culture volume has been increased to the final culture volume.

10. The method according to claim 9, wherein the culture temperature during the production period is 28°C to 35°C.

11. The culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until the culture reaches one or more desired target criteria, provided that the supply rate and the permeation rate used simultaneously are the same; and / or Once the final culture volume is reached, the culture is perfused at one or more perfusion rates of 0.05 V / d to 0.5 V / d until the culture is finished or harvested, and the supply rate and the simultaneously used permeation rate are the same. The antibody according to claim 1 or claim 2.

12. Until the culture reaches one or more desired target criteria, the culture will be 0.10 V / d to 0 - Perfumed at one or more perfusation rates of 25 V / d, wherein the supply rate and the simultaneously used permeation rate are the same; and / or The antibody according to claim 1 or 2, wherein once the final culture volume is reached, the culture is perfused at one or more perfusion rates of 0.10 V / d to 0.25 V / d until the culture is terminated or the antibody is recovered, and the supply rate and the simultaneously used permeation rate are the same.

13. The method according to claim 1 or 2, wherein one or more desired target criteria are selected from culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, compressed cell volume, product attributes, process, production schedule, plant schedule, and any combination thereof.

14. The one or more desired target criteria mentioned above are 100 × 10 ５ cells / mL~350×10 ５ The method according to claim 1 or claim 2, wherein the cell density is cells / mL.

15. The method according to claim 1 or claim 2, wherein the one or more desired target criteria is the time after sowing, and the time after sowing is between 24 hours and 72 hours.

16. The method according to claim 1 or 2, wherein the culture volume is increased to the final culture volume using differential perfusion, and the differential perfusion includes one or more supply rates and one or more permeation rates.

17. The method according to claim 16, wherein at least one of the one or more supply speeds is 0.50 V / d or less, at least one of the one or more transmission speeds is 0.20 V / d or less, and each of the one or more supply speeds is faster than the transmission speed used simultaneously.

18. The method according to claim 16, wherein at least one of the one or more supply speeds is 0.40 V / d or less, at least one of the one or more transmission speeds is 0.15 V / d or less, and each of the one or more supply speeds is faster than the transmission speed used simultaneously.

19. The method according to claim 16, wherein the one or more supply rates and the one or more permeation rates are selected to increase the volume of the culture medium in the bioreactor to a volume of 100% or less of the bioreactor working volume in the time required for doubling of at least one cell population of the culture.

20. The method according to claim 16, wherein the differential perfusion is continued until the culture volume in the bioreactor reaches 70% to 100% of the final bioreactor working volume.

21. The method according to claim 1 or claim 2, wherein the final working volume of the bioreactor is 70% to 100% of the bioreactor volume.

22. The method according to claim 9, wherein the cell density at the start of the production period is at least twice the seeded cell density.

23. The cell density at the start of the aforementioned production period is at least 200 × 10 ５ The method according to claim 9, wherein the amount is cells / mL.

24. The method according to claim 9, wherein the duration of the production period is 10 to 20 days.

25. The method according to claim 1 or 2, wherein the bioreactor is connected to a separation system.

26. The method according to claim 25, wherein the separation system is a recirculating tangential flow filter system or an alternating tangential flow filtration system.

27. The method according to claim 1 or claim 2, wherein the volume of the bioreactor is 200 liters to 20,000 liters.

28. The method according to claim 1 or claim 2, wherein the bioreactor is a disposable bioreactor or a stainless steel bioreactor.

29. To recover the recombinant protein; Processing the recombinant protein through one or more unit operations; To obtain isolated and purified recombinant proteins, The method according to claim 1 or claim 2, further comprising:

30. The method according to claim 29, wherein at least one of the one or more unit operations is an affinity chromatography unit operation.

31. The method according to claim 29, wherein at least one of the one or more unit operations is a polishing chromatography unit operation.

32. The method according to claim 29, wherein at least one of the one or more unit operations is selected from virus inactivation, virus filtration, deep filtration, and ultrafiltration / dialysis filtration (UF / DF).

33. The method according to claim 1 or claim 2, wherein the recombinant protein is an antibody or an antibody fragment.

34. The method according to claim 1 or claim 2, wherein the cells are CHO cells.

35. The method according to claim 34, wherein the CHO cells are glutamine synthetase knockout CHO cells or dihydrofolate reductase-deficient CHO cells.

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