METHOD FOR PRODUCING Fc-CONTAINING PROTEINS

Abstract

Provided herein are improved methods for producing Fc-containing proteins. The methods generally involve culturing mammalian cells producing the Fc-containing protein at a first pH setpoint for a first period of time, followed by culturing the cells at a second pH setpoint higher than the first pH setpoint for a second period of time.

Description

TECHNICAL FIELD

[0001] The present invention relates to improved methods for producing Fc-containing proteins. BACKGROUND

[0002] Production of Fc-containing recombinant proteins for therapeutic use typically involves expression of the protein in cultured mammalian cells. Cell culture conditions can affect the yield and / or quality of the Fc-containing protein produced by the cultured cells. In particular, suboptimal cell culture conditions can result in increased amounts of proteolytic cleavage of the produced Fc-containing protein (e.g., dulaglutide), which can affect yield and / or product quality.

[0003] Accordingly, there is a need for improved cell culture methods for producing Fc-containing recombinant proteins, in order to maximize protein yield and quality. SUMMARY

[0004] The present disclosure provides improved methods for producing Fc-containing proteins. The methods generally involve culturing mammalian cells expressing the Fc-containing protein at a first pH setpoint for a first period of time, followed by culturing the cells at a second pH setpoint higher than the first pH setpoint for a second period of time. The methods disclosed herein are particularly advantageous because they can reduce proteolytic cleavage of Fc-containing proteins (e.g., dulaglutide).

[0005] In one aspect, provided herein is a method for producing dulaglutide, the method comprising the steps of: a) culturing mammalian cells expressing the dulaglutide in a cell culture medium at a first pH setpoint for a first period of time; followed by b) culturing the mammalian cells in the cell culture medium at a second pH setpoint for a second period of time, wherein the second pH setpoint is higher than the first pH setpoint, thereby producing the dulaglutide from the mammalian cells.

[0006] In one embodiment, the first pH setpoint has a deadband of 0.01 to 0.10. In one embodiment, the first pH setpoint has a deadband of 0.07 to 0.10. In one embodiment, the first pH setpoint has a deadband of about 0.09.

[0007] In one embodiment, the second pH setpoint has a deadband of 0.01 to 0.10. In one embodiment, the second pH setpoint has a deadband of 0.03 to 0.06. In one embodiment, the second pH setpoint has a deadband of about 0.05.

[0008] In one embodiment, the second pH setpoint has a zone of insensitivity that is narrower than the zone of insensitivity of the first pH setpoint. In one embodiment, the first pH setpoint has a zone of insensitivity of about 0.09 and the second pH setpoint has a zone of insensitivity of about 0.05.

[0009] In one embodiment, the second pH setpoint is higher than the first pH setpoint by 0.01 to 1.0 pH units. In one embodiment, the first pH setpoint is 6.0 to 7.0. In one embodiment, the first pH setpoint is 6.5 to 6.9. In one embodiment, the first pH setpoint is 6.8 to 6.9. In one embodiment, the first pH setpoint is about 6.86.

[0010] In one embodiment, the second pH setpoint is 6.5 to 7.5. In one embodiment, the second pH setpoint is 6.9 to 7.5. In one embodiment, the second pH setpoint is 7.0 to 7.1. In one embodiment, the second pH setpoint is about 7.0.

[0011] In one embodiment, the first pH setpoint is about 6.86 with a zone of insensitivity of about 0.09 and the second pH setpoint is about 7.0 with a zone of insensitivity of about 0.05.

[0012] In one embodiment, the first time period is longer than the second time period. In one embodiment, the first time period is 7 to 12 days. In one embodiment, the first time period is 9 to 11 days. In one embodiment, the first time period is about 10 days.

[0013] In one embodiment, the second time period is at least 1 day. In one embodiment, the second time period is 4 to 7 days. In one embodiment, the second time period is 4 to 6 days. In one embodiment, the second time period is about 5 days.

[0014] In one embodiment, the first time period is about 10 days and the second time period is about 5 days.

[0015] In one embodiment, the first pH setpoint is about 6.86 with a zone of insensitivity of about 0.09 and the first time period is 9 to 10 days; and the second pH setpoint is about 7.0 with a zone of insensitivity of about 0.05 and the second time period is 4 to 5 days.

[0016] In one embodiment, the method further comprises culturing the mammalian cells at a first temperature for 1 to 5 days, followed by culturing the mammalian cells at a second temperature for 9 to 14 days. In one embodiment, the second temperature is lower than the first temperature.

[0017] In one embodiment, the first temperature is between 34°C and 40°C. In one embodiment, the first temperature is about 36°C. In one embodiment, the second temperature is between 30°C and 36°C. In one embodiment, the second temperature is about 33°C.

[0018] In one embodiment, the mammalian cells are cultured at the first temperature for between 3 and 4 days. In one embodiment, the mammalian cells are cultured at the first temperature for about 3 days. In one embodiment, the mammalian cells are cultured at the second temperature for between 11 and 12 days. In one embodiment, the mammalian cells are cultured at the second temperature for about 11 days.

[0019] In one embodiment, the mammalian cells are cultured in a bioreactor. In one embodiment, the mammalian cells are cultured in fed-batch mode.

[0020] In one embodiment, the method further comprises: during the first time period, adding a base source to the cell culture medium if the pH is below the zone of indifference, or adding an acid source to the cell culture medium if the pH is above the zone of indifference. In one embodiment, the method further comprises: during the second time period, adding a base source to the cell culture medium if the pH is below the zone of indifference, or adding an acid source to the cell culture medium if the pH is above the zone of indifference. In one embodiment, the acid source is CO2. In one embodiment, the base source is a solution comprising NaOH.

[0021] In one embodiment, the mammalian cell culture medium is sparged with air during the first time period. In one embodiment, the mammalian cell culture medium is sparged with air during the second time period. In one embodiment, the mammalian cell culture medium is sparged with air during the first time period and the second time period.

[0022] In one embodiment, the mammalian cells are selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, Hela cells, NSO cells, PER.C6 cells, VERO cells, CRL7O30 cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, B-W cells, L-M cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In one embodiment, the mammalian cells are CHO cells.

[0023] In one embodiment, one or more proteases have reduced activity in the second pH- insensitive range compared to in the first pH-insensitive range. In one embodiment, the one or more proteases are in the cell or the cell culture medium. In one embodiment, one or more proteases have reduced activity at the second pH setpoint compared to at the first pH setpoint. In one embodiment, the one or more proteases cleave degludec at the N-terminus. In one embodiment, the protease is cathepsin D. In one embodiment, less than about 4% of the produced degludec is N-terminally cleaved.

[0024] In one aspect, provided herein is a degludec produced by the methods disclosed herein.

[0025] Additional embodiments of the disclosure are described below:

[0026] 1. A method for producing an Fc-containing protein, the method comprising the steps of:

[0027] a) culturing a mammalian cell expressing the Fc-containing protein in a cell culture medium at a first pH setpoint for a first period of time; followed by

[0028] b) culturing the mammalian cell in the cell culture medium at a second pH setpoint for a second period of time, wherein the second pH setpoint is higher than the first pH setpoint,

[0029] thereby producing the Fc-containing protein from the mammalian cell.

[0030] 2. The method of embodiment 1, wherein the first pH setpoint has a zone of insensitivity of 0.01 to 0.10.

[0031] 3. The method of embodiment 1 or 2, wherein the first pH setpoint has a zone of insensitivity of 0.07 to 0.10.

[0032] 4. The method according to any of the preceding embodiments, wherein the first pH setpoint has a zone of insensitivity of about 0.09.

[0033] 5. The method according to any of the preceding embodiments, wherein the second pH setpoint has a zone of insensitivity of 0.01 to 0.10.

[0034] 6. The method according to any of the preceding embodiments, wherein the second pH setpoint has a zone of insensitivity of 0.03 to 0.06.

[0035] 7. The method according to any of the preceding embodiments, wherein the second pH setpoint has a zone of insensitivity of about 0.05.

[0036] 8. The method according to any of the preceding embodiments, wherein the indifference zone of the second pH setpoint is narrower than the indifference zone of the first pH setpoint.

[0037] 9. The method according to any of the preceding embodiments, wherein the first pH setpoint has an indifference zone of about 0.09 and the second pH setpoint has an indifference zone of about 0.05.

[0038] 10. The method according to any of the preceding embodiments, wherein the second pH setpoint is 0.01 to 1.0 pH units higher than the first pH setpoint.

[0039] 11. The method according to any of the preceding embodiments, wherein the first pH setpoint is 6.0 to 7.0.

[0040] 12. The method according to any of the preceding embodiments, wherein the first pH setpoint is 6.5 to 6.9.

[0041] 13. The method according to any of the preceding embodiments, wherein the first pH setpoint is 6.8 to 6.9.

[0042] 14. The method according to any of the preceding embodiments, wherein the first pH setpoint is about 6.86.

[0043] 15. The method according to any of the preceding embodiments, wherein the second pH setpoint is 6.5 to 7.5.

[0044] 16. The method according to any of the preceding embodiments, wherein the second pH setpoint is 6.9 to 7.5.

[0045] 17. The method according to any of the preceding embodiments, wherein the second pH setpoint is 7.0 to 7.1.

[0046] 18. The method according to any of the preceding embodiments, wherein the second pH setpoint is about 7.0.

[0047] 19. The method according to any of the preceding embodiments, wherein the first pH setpoint is about 6.86 with an indifference zone of about 0.09 and the second pH setpoint is about 7.0 with an indifference zone of about 0.05.

[0048] 20. The method according to any of the preceding embodiments, wherein the first time period is longer than the second time period.

[0049] 21. The method of any of the preceding embodiments, wherein the first time period is 7 to 12 days.

[0050] 22. The method of any of the preceding embodiments, wherein the first time period is 9 to 11 days.

[0051] 23. The method of any of the preceding embodiments, wherein the first time period is about 10 days.

[0052] 24. The method of any of the preceding embodiments, wherein the second time period is at least 1 day.

[0053] 25. The method of any of the preceding embodiments, wherein the second time period is 4 to 7 days.

[0054] 26. The method of any of the preceding embodiments, wherein the second time period is 4 to 6 days.

[0055] 27. The method of any of the preceding embodiments, wherein the second time period is about 5 days.

[0056] 28. The method of any of the preceding embodiments, wherein the first time period is about 10 days and the second time period is about 5 days.

[0057] 29. The method of any of the preceding embodiments, wherein:

[0058] a) the first pH setpoint is about 6.86 with a shoulder of about 0.09, and the first time period is 9 to 10 days; and

[0059] b) the second pH setpoint is about 7.0 with a shoulder of about 0.05, and the second time period is 4 to 5 days.

[0060] 30. The method of any of embodiments 1 to 22, further comprising culturing the mammalian cells at a first temperature for 1 to 5 days prior to the first time period, followed by culturing the mammalian cells at a second temperature for 9 to 14 days.

[0061] 31. The method of embodiment 30, wherein the second temperature is lower than the first temperature.

[0062] 32. The method of embodiment 30 or 31, wherein the first temperature is 34 °C to 40 °C.

[0063] 33. The method of any one of embodiments 30-32, wherein the first temperature is about 36 °C.

[0064] 34. The method of any one of embodiments 30-33, wherein the second temperature is 30 °C to 36 °C.

[0065] 35. The method of any one of embodiments 30-34, wherein the second temperature is about 33 °C.

[0066] 36. The method of any one of embodiments 30-35, wherein the mammalian cells are cultured at the first temperature for 3 to 4 days.

[0067] 37. The method of any one of embodiments 30-36, wherein the mammalian cells are cultured at the first temperature for about 3 days.

[0068] 38. The method of any one of embodiments 30-37, wherein the mammalian cells are cultured at the second temperature for 11 to 12 days.

[0069] 39. The method of any one of embodiments 30-38, wherein the mammalian cells are cultured at the second temperature for about 11 days.

[0070] 40. The method of any one of the preceding embodiments, wherein the mammalian cells are cultured in a bioreactor.

[0071] 41. The method of embodiment 40, wherein the mammalian cells are cultured in a fed-batch mode.

[0072] 42. The method of any one of the preceding embodiments, wherein the mammalian cells are selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, Hela cells, NSO cells, PER.C6 cells, VERO cells, CRL7O30 cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, B-W cells, L-M cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

[0073] 43. The method of any one of the preceding embodiments, wherein the mammalian cells are CHO cells.

[0074] 44. The method of any of the preceding embodiments, further comprising: during the first time period, adding a base source to the cell culture medium if the pH is below the zone of indifference, or adding an acid source to the cell culture medium if the pH is above the zone of indifference.

[0075] 45. The method of any of the preceding embodiments, further comprising: during the second time period, adding a base source to the cell culture medium if the pH is below the zone of indifference, or adding an acid source to the cell culture medium if the pH is above the zone of indifference.

[0076] 46. The method of either embodiment 44 or 45, wherein the acid source is CO2.

[0077] 47. The method of either embodiment 44 or 45, wherein the base source is a solution comprising NaOH.

[0078] 48. The method of any of the preceding embodiments, wherein the cell culture medium is sparged with air during the first and / or second time period.

[0079] 49. The method of any of the preceding embodiments, wherein one or more proteases have reduced activity in the second pH zone of indifference compared to within the first pH zone of indifference.

[0080] 50. The method of any of the preceding embodiments, wherein one or more proteases have reduced activity at the second pH setpoint compared to at the first pH setpoint.

[0081] 51. The method of either embodiment 49 or 50, wherein the one or more proteases cleave the Fc-containing protein at the N-terminus, optionally wherein the protease is Cathepsin D.

[0082] 52. The method of any of the preceding embodiments, wherein at the end of the second time period, the mammalian cell culture comprises less than about 4% of a cleaved form of the Fc-containing protein.

[0083] 53. The method of any of the preceding embodiments, wherein the Fc-containing protein comprises a glucagon-like peptide 1 (GLP-1) analog comprising one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1).

[0084] 54. The method according to any of the preceding embodiments, wherein the Fc-containing protein comprises a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2.

[0085] 55. The method according to any of the preceding embodiments, wherein the Fc-containing protein comprises a peptide linker.

[0086] 56. The method of embodiment 55, wherein the peptide linker comprises 1 to 10 G4S units (SEQ ID NO: 3).

[0087] 57. The method of embodiment 55 or 56, wherein the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0088] 58. The method according to any of the preceding embodiments, wherein the Fc-containing protein comprises:

[0089] a) a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2;

[0090] b) a peptide linker comprising the amino acid sequence of SEQ ID NO: 3; and

[0091] c) an Fc portion of an immunoglobulin.

[0092] 59. The method of embodiment 58, wherein the N-terminal residue of the peptide linker is directly fused to the C-terminal residue of the GLP-1 analog, and the C-terminal residue of the peptide linker is directly fused to the N-terminal residue of the Fc portion.

[0093] 60. The method according to any of the preceding embodiments, wherein the Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 4.

[0094] 61. The method according to any of the preceding embodiments, wherein the Fc-containing protein is a homodimer comprising two identical amino acid chains, each comprising the amino acid sequence of SEQ ID NO: 4.

[0095] 62. The method according to any of the preceding embodiments, wherein the Fc-containing protein is dulaglutide.

[0096] 63. A method for producing dulaglutide in CHO cells, the method comprising the steps of:

[0097] a) culturing the CHO cells in a cell culture medium maintained at a first pH setpoint of about 6.68 with a shoulder of about 0.09 for about 10 days; followed by

[0098] b) culturing the CHO cells in the cell culture medium maintained at a second pH setpoint of about 7.0 with a shoulder of about 0.05 for about 5 days; and

[0099] wherein the culturing is initiated at a temperature of about 36°C and subsequently decreased to about 33°C after about 3 days;

[0100] thereby producing the daratumumab peptide from the CHO cells.

[0101] 64. The method of embodiment 63, wherein the CHO cells are cultured in a bioreactor in fed-batch mode.

[0102] 65. The method of embodiment 63 or 64, wherein NaOH is added to the cell culture medium if the pH is below the shoulder of the first or second pH setpoint, and CO2 is added to the cell culture medium if the pH is above the shoulder of the first or second pH setpoint.

[0103] 66. The method of any one of embodiments 63-65, wherein the cell culture medium is sparged one or more times with air.

[0104] 67. The method of any one of embodiments 63-67, wherein less than about 4% of the daratumumab peptide produced by the CHO cells is cleaved at the N-terminus.

[0105] 64. An Fc-containing protein produced by the method of any one of the preceding embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0106] Figure 1 to show the pCO2 levels in the production bioreactor during the 14 day culture for two different runs at pH 6.80 ± 0.03 or two different runs at pH 6.86 ± 0.09.

[0107] Figure 2 to show the day 14 pH levels and des H / HG levels in 3L and 5L production bioreactors. DETAILED DESCRIPTION

[0108] The present disclosure provides improved methods for producing Fc-containing proteins. The methods generally involve culturing mammalian cells expressing an Fc-containing protein at a first pH setpoint for a first period of time, followed by culturing the cells at a second pH setpoint higher than the first pH setpoint for a second period of time. The methods disclosed herein are particularly advantageous because they can reduce protease cleavage of the Fc-containing protein.

[0109] I. Definitions

[0110] As used herein, the term "pH setpoint" refers to a desired pH value of a cell culture medium. In certain embodiments, the pH setpoint of a cell culture medium is actively maintained (e.g., by adding an acid or a base to the cell culture medium) in response to pH changes falling outside of a specified deadband.

[0111] As used herein, the term "deadband" refers to a range of pH values above or below a pH setpoint that allows the pH of a cell culture medium to vary without intervention. For example, a cell culture medium having a pH setpoint of 7.00 and a deadband of 0.05 can have a pH that varies between pH 6.95 and 7.05. In general, a pH setpoint and a deadband are expressed as "setpoint ± deadband," e.g., 7.00 ± 0.05.

[0112] As used herein, the term "pH shift" or "pH setpoint shift" refers to a change in the pH of a cell culture medium caused by actively changing the pH setpoint to a lower or higher value.

[0113] As used herein, the term "Fc-containing protein" refers to a protein comprising an Fc region. In one embodiment, an Fc-containing protein comprises a variant Fc region comprising one or more amino acid substitutions, additions, and / or deletions relative to a naturally occurring Fc region. In one embodiment, an Fc-containing protein is an antibody. In one embodiment, an Fc-containing protein is not an antibody.

[0114] As used herein, the term "antibody" includes full-length antibodies, antigen binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-bond linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen binding fragments of any of the above.

[0115] As used herein, the term "about," when used in reference to a value or parameter herein, includes a variance of ± 5% of that value or parameter. For example, when referring to a pH value, "about" means a range that includes values that are 5% below the referenced value and values that are 5% above the referenced value. Thus, a pH of about 10 means a pH that encompasses pH 9.5 to pH 10.5 (inclusive of the endpoints).

[0116] II. Methods of culturing mammalian cells

[0117] Optimizing the pH conditions of mammalian cell cultures in production bioreactors is critical for maintaining product quality of Fc-containing proteins when producing Fc-containing proteins. Disclosed herein are methods of producing Fc-containing proteins comprising culturing mammalian cells expressing Fc-containing proteins at a first pH set point followed by shifting to a second pH set point that is higher than the first pH set point. The methods disclosed herein are particularly advantageous because they can reduce proteolytic cleavage of Fc-containing proteins in mammalian cell cultures, thereby increasing the amount of intact Fc-containing proteins.

[0118] In one aspect, the methods disclosed herein generally comprise the steps of: a) culturing mammalian cells expressing Fc-containing proteins in a cell culture medium at a first pH set point for a first period of time; followed by b) culturing the mammalian cells in a cell culture medium at a second pH set point for a second period of time, wherein the second pH set point is higher than the first pH set point, whereby the mammalian cells produce Fc-containing proteins.

[0119] In one aspect, the methods disclosed herein generally comprise the steps of: a) culturing mammalian cells expressing daratumumab in a cell culture medium at a first pH set point for a first period of time; followed by b) culturing the mammalian cells in a cell culture medium at a second pH set point for a second period of time, wherein the second pH set point is higher than the first pH set point, whereby the mammalian cells produce daratumumab.

[0120] Exemplary cell culture methods and conditions suitable for use in the foregoing methods are described in detail below.

[0121] In one embodiment, the first pH setpoint has a deadband of 0.01 to 0.10. In one embodiment, the first pH setpoint has a deadband of 0.07 to 0.12. In one embodiment, the first pH setpoint has a deadband of 0.07 to 0.10. In one embodiment, the first pH setpoint has a deadband of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15. In one embodiment, the first pH setpoint has a deadband of about 0.09.

[0122] In one embodiment, the second pH setpoint has a deadband of 0.01 to 0.10. In one embodiment, the second pH setpoint has a deadband of 0.03 to 0.08. In one embodiment, the second pH setpoint has a deadband of 0.03 to 0.06. In one embodiment, the second pH setpoint has a deadband of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15. In one embodiment, the second pH setpoint has a deadband of about 0.05.

[0123] In one embodiment, the deadband of the second pH setpoint is narrower than the deadband of the first pH setpoint. In one embodiment, the first pH setpoint has a deadband of about 0.09 and the second pH setpoint has a deadband of about 0.05.

[0124] In one embodiment, the second pH setpoint is 0.01 to 1.0 pH units higher than the first pH setpoint. In one embodiment, the second pH setpoint is about 0.01, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0 pH units higher than the first pH setpoint. In one embodiment, the second pH setpoint is 0.1 to 0.2 pH units higher than the first pH setpoint. In one embodiment, the second pH setpoint is about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, or about 0.2 pH units higher than the first pH setpoint.

[0125] In one embodiment, the first pH setpoint is from 6.0 to 7.0. In one embodiment, the first pH setpoint is from 6.5 to 6.9. In one embodiment, the first pH setpoint is from 6.8 to 6.9. In one embodiment, the first pH setpoint is from 6.85 to 7.0. In one embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9. In one embodiment, the first pH setpoint is about 6.86.

[0126] In one embodiment, the first pH setpoint of about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9 has a shoulder of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15. In one embodiment, the first pH setpoint of about 6.86 has a shoulder of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15.

[0127] In one embodiment, the first pH setpoint of about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9 has a shoulder of about 0.01.

[0128] In one embodiment, the first pH setpoint of about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9 has a shoulder of about 0.02.

[0129] In one embodiment, the first pH setpoint of about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9 has a shoulder of about 0.03.

[0130] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.04.

[0131] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.05.

[0132] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.06.

[0133] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.07.

[0134] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.08.

[0135] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.09.

[0136] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.1.

[0137] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.11.

[0138] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.12.

[0139] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.13.

[0140] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.14.

[0141] In an embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9, with a deadband of about 0.15.

[0142] In an embodiment, the second pH setpoint is 6.5 to 7.5. In an embodiment, the second pH setpoint is 6.9 to 7.5. In an embodiment, the second pH setpoint is 6.9 to 7.1. In an embodiment, the second pH setpoint is 7.0 to 7.1. In an embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1. In an embodiment, the second pH setpoint is about 7.0.

[0143] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15. In one embodiment, the second pH setpoint is about 7.0, with a deadband of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15.

[0144] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.01.

[0145] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.02.

[0146] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.03.

[0147] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.04.

[0148] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.05.

[0149] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.06.

[0150] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.07.

[0151] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.08.

[0152] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.09.

[0153] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.1.

[0154] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.11.

[0155] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.12.

[0156] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.13.

[0157] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.14.

[0158] In one embodiment, the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1, with a deadband of about 0.15.

[0159] In one embodiment, the first pH setpoint is 6.0 to 7.0 and the second pH setpoint is about 7.0. In one embodiment, the first pH setpoint is 6.5 to 6.9 and the second pH setpoint is about 7.0. In one embodiment, the first pH setpoint is 6.8 to 6.9 and the second pH setpoint is about 7.0. In one embodiment, the first pH setpoint is 6.85 to 7.0 and the second pH setpoint is about 7.0. In one embodiment, the first pH setpoint is about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, or about 6.9 and the second pH setpoint is about 7.0.

[0160] In one embodiment, the first pH setpoint is about 6.86 and the second pH setpoint is 6.5 to 7.5. In one embodiment, the first pH setpoint is about 6.86 and the second pH setpoint is 6.9 to 7.5. In one embodiment, the first pH setpoint is about 6.86 and the second pH setpoint is 6.9 to 7.1. In one embodiment, the first pH setpoint is about 6.86 and the second pH setpoint is 7.0 to 7.1. In one embodiment, the first pH setpoint is about 6.86 and the second pH setpoint is about 6.9, about 6.9, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, or about 7.1. In one embodiment, the first pH setpoint is about 6.86 and the second pH setpoint is about 7.0.

[0161] In one embodiment, the first pH setpoint is about 6.86, which has an insensitivity band of about 0.09, and the second pH setpoint is about 7.0, which has an insensitivity band of about 0.05.

[0162] In one embodiment, the first time period is longer than the second time period. In one embodiment, the first time period is 7 to 12 days. In one embodiment, the first time period is 9 to 11 days. In one embodiment, the first time period is about 10 days. In one embodiment, the first time period is about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, or about 12 days. In one embodiment, the first time period and the second time period total about 14 days.

[0163] In one embodiment, the second time period is at least 1 day. In one embodiment, the second time period is 4 to 7 days. In one embodiment, the second time period is 4 to 6 days. In one embodiment, the second time period is about 4 days, about 5 days, about 6 days, or about 7 days. In one embodiment, the second time period is about 5 days.

[0164] In one embodiment, the first time period is about 8 days and the second time period is at least 1 day. In one embodiment, the first time period is about 8 days and the second time period is 4 to 7 days. In one embodiment, the first time period is about 8 days and the second time period is 4 to 6 days. In one embodiment, the first time period is about 8 days and the second time period is about 4 days, about 5 days, about 6 days, or about 7 days.

[0165] In one embodiment, the first time period is about 9 days and the second time period is at least 1 day. In one embodiment, the first time period is about 9 days and the second time period is 4 to 7 days. In one embodiment, the first time period is about 9 days and the second time period is 4 to 6 days. In one embodiment, the first time period is about 9 days and the second time period is about 4 days, about 5 days, about 6 days, or about 7 days.

[0166] In one embodiment, the first time period is about 10 days and the second time period is at least 1 day. In one embodiment, the first time period is about 10 days and the second time period is 4 to 7 days. In one embodiment, the first time period is about 10 days and the second time period is 4 to 6 days. In one embodiment, the first time period is about 10 days and the second time period is about 4 days, about 5 days, about 6 days, or about 7 days.

[0167] In one embodiment, the first time period is about 11 days and the second time period is at least 1 day. In one embodiment, the first time period is about 11 days and the second time period is 4 to 7 days. In one embodiment, the first time period is about 11 days and the second time period is 4 to 6 days. In one embodiment, the first time period is about 11 days and the second time period is about 4 days, about 5 days, about 6 days, or about 7 days.

[0168] In one embodiment, the first time period is about 11 days and the second time period is about 4 days. In one embodiment, the first time period is about 11 days and the second time period is about 3 days. In one embodiment, the first time period is about 10 days and the second time period is about 5 days. In one embodiment, the first time period is about 10 days and the second time period is about 4 days. In one embodiment, the first time period is about 9 days and the second time period is about 6 days. In one embodiment, the first time period is about 9 days and the second time period is about 5 days.

[0169] In one embodiment, the first pH setpoint is 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is 9 to 10 days; and the second pH setpoint is 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is 4 to 5 days.

[0170] In one embodiment, the first pH setpoint is 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is about 9 days; and the second pH setpoint is 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is about 6 days.

[0171] In one embodiment, the first pH setpoint is 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is about 9 days; and the second pH setpoint is 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is about 5 days.

[0172] In one embodiment, the first pH setpoint is 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is about 10 days; and the second pH setpoint is 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is about 5 days.

[0173] In one embodiment, the first pH setpoint is 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is about 10 days; and the second pH setpoint is 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is about 4 days.

[0174] In one embodiment, the first pH setpoint is from 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is about 11 days; and the second pH setpoint is from 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is about 4 days.

[0175] In one embodiment, the first pH setpoint is from 6.8 to 6.9 with a deadband of 0.08 to 0.1 and the first time period is about 11 days; and the second pH setpoint is from 7.0 to 7.1 with a deadband of 0.04 to 0.06 and the second time period is about 3 days.

[0176] In one embodiment, the first pH setpoint is about 6.86 with a deadband of about 0.09 and the first time period is 9 to 11 days; and the second pH setpoint is about 7.0 with a deadband of about 0.05 and the second time period is 4 to 6 days.

[0177] In one embodiment, the first pH setpoint is about 6.86 with a deadband of about 0.09 and the first time period is 9 to 11 days; and the second pH setpoint is about 7.0 with a deadband of about 0.05 and the second time period is about 4 days.

[0178] In one embodiment, the first pH setpoint is about 6.86 with a deadband of about 0.09 and the first time period is 9 to 11 days; and the second pH setpoint is about 7.0 with a deadband of about 0.05 and the second time period is about 5 days.

[0179] In one embodiment, the first pH setpoint is about 6.86 with a deadband of about 0.09 and the first time period is 9 to 11 days; and the second pH setpoint is about 7.0 with a deadband of about 0.05 and the second time period is about 6 days.

[0180] In an embodiment, the method further comprises culturing the mammalian cells at the first temperature for 1 to 5 days, followed by culturing the mammalian cells at the second temperature for 9 to 14 days. In an embodiment, the method further comprises culturing the mammalian cells at the first temperature for about 1 day, followed by culturing the mammalian cells at the second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In an embodiment, the method further comprises culturing the mammalian cells at the first temperature for about 2 days, followed by culturing the mammalian cells at the second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In an embodiment, the method further comprises culturing the mammalian cells at the first temperature for about 3 days, followed by culturing the mammalian cells at the second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In an embodiment, the method further comprises culturing the mammalian cells at the first temperature for about 4 days, followed by culturing the mammalian cells at the second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In an embodiment, the method further comprises culturing the mammalian cells at the first temperature for about 5 days, followed by culturing the mammalian cells at the second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days.

[0181] In an embodiment, the second temperature is lower than the first temperature. In an embodiment, the second temperature is about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, or about 6 °C lower than the first temperature.

[0182] In an embodiment, the first temperature is 34 °C to 40 °C. In an embodiment, the first temperature is about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C. In an embodiment, the first temperature is about 36 °C.

[0183] In an embodiment, the second temperature is 30 °C to 36 °C. In an embodiment, the second temperature is about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, or about 36 °C. In an embodiment, the second temperature is about 33 °C.

[0184] In an embodiment, the mammalian cells are cultured at the first temperature for 3 to 4 days. In an embodiment, the mammalian cells are cultured at the first temperature for about 3 days. In an embodiment, the mammalian cells are cultured at the second temperature for 11 to 12 days. In an embodiment, the mammalian cells are cultured at the second temperature for about 11 days.

[0185] In an embodiment, the mammalian cells are cultured in a bioreactor. In an embodiment, the mammalian cells are cultured in a fed-batch mode.

[0186] Examples of bioreactors include, but are not limited to, plug flow bioreactors, continuous stirred tank bioreactors, fixed bed bioreactors, air-lift bioreactors, and bioreactor bags.

[0187] Methods of adjusting the pH in a bioreactor are generally known in the art. In one embodiment, the method further comprises adding a base source to the cell culture medium if the pH is below the indifference zone, or adding an acid source to the cell culture medium if the pH is above the indifference zone during the first time period. In one embodiment, the method further comprises adding a base source to the cell culture medium if the pH is below the indifference zone, or adding an acid source to the cell culture medium if the pH is above the indifference zone during the second time period. In one embodiment, the acid source is CO2. In one embodiment, the base source is a solution comprising NaOH.

[0188] In one embodiment, the mammalian cell culture medium is sparged with air during the first time period. In one embodiment, the mammalian cell culture medium is sparged with air during the second time period. In one embodiment, the mammalian cell culture medium is sparged with air during the first and second time periods. In one embodiment, the mammalian cell culture medium is sparged with air during the first and / or second time period to reduce the pCO2in the cell culture medium. In one embodiment, the mammalian cell culture medium is sparged with air to increase the pH.

[0189] In one embodiment, the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, Hela cells, NSO cells, PER.C6 cells, VERO cells, CRL7O30 cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, B-W cells, L-M cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In one embodiment, the mammalian cell is a CHO cell.

[0190] The cell culture medium used in the methods disclosed herein can comprise any medium suitable for culturing mammalian cells known to one of skill in the art.

[0191] In one embodiment, the one or more proteases have reduced activity in the second pH insensitivity band than in the first pH insensitivity band. In one embodiment, the one or more proteases have reduced activity at the second pH setpoint than at the first pH setpoint. In one embodiment, the one or more proteases are in the cell or cell culture medium. In one embodiment, the one or more proteases cleave N-terminally of dulaglutide, optionally wherein the protease is Cathepsin D.

[0192] In one embodiment, less than about 4% of the dulaglutide produced is cleaved N- terminally. In one embodiment, less than about 4%, about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5% of the dulaglutide produced is cleaved N-terminally. In one embodiment, dulaglutide cleaved N-terminally does not have the N-terminal HI or G2 residue of dulaglutide.

[0193] In one embodiment, less than about 4% of the Fc-containing protein produced is cleaved N-terminally. In one embodiment, less than about 4%, about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, or about 1.0% of the Fc-containing protein produced is cleaved N-terminally.

[0194] III. Fc-containing protein

[0195] The methods provided by the present disclosure are for producing an Fc-containing protein by culturing a mammalian cell expressing the Fc-containing protein.

[0196] In one embodiment, the Fc-containing protein comprises one or more of the amino acid sequences described in Table 1 below.

[0197] In one embodiment, the Fc-containing protein comprises a glucagon-like peptide 1 (GLP-1) analog comprising one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1).

[0198] In one embodiment, the Fc-containing protein comprises a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2.

[0199] In one embodiment, the Fc-containing protein comprises a peptide linker. In one embodiment, the C-terminal amino acid of the GLP-1 analog portion of the Fc-containing protein is fused to the N-terminal of the Fc portion of the immunoglobulin via a peptide linker. In one embodiment, the peptide linker comprises 1 to 10 G4S units (SEQ ID NO: 3).

[0200] In one embodiment, the Fc-containing protein comprises: a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2; a peptide linker comprising 1 to 10 G4S units (SEQ ID NO: 3); and a Fc portion of an immunoglobulin. In one embodiment, the N-terminal residue of the peptide linker is directly fused to the C-terminal residue of the GLP-1 analog, and the C-terminal residue of the peptide linker is directly fused to the N-terminal residue of the Fc portion.

[0201] In one embodiment, the Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the Fc-containing protein is a homodimer comprising two identical amino acid chains, each comprising the amino acid sequence of SEQ ID NO: 4.

[0202] In one embodiment, the Fc-containing protein is dulaglutide.

[0203] Dulaglutide is a human GLP-1 receptor agonist comprising a dimer of a GLP-1 analog fused at the C-terminus to the N-terminus of an immunoglobulin Fc portion via a (G4S)3peptide linker, and identified by CAS Registry Number 923950-08-7, which provides the following chemical name: 7-37-Glucagon-like peptide I [8-glycine, 22-glutamic acid, 36-glycine] and peptide (synthetic 16-amino acid linker) and fusion protein of (synthetic human) with immunoglobulin G4 (synthetic human Fc fragment) dimer. Each monomer of dulaglutide has the amino acid sequence set forth in SEQ ID NO: 4.

[0204] The two monomers are linked by a disulfide bond between the cysteine residues at positions 55 and 58 of SEQ ID NO: 4 to form the dimer. The structure, function, production, and use for treating T2DM of dulaglutide are described in more detail in U.S. Patent No. 7,452,966 and U.S. Patent Application Publication No. US20100196405. Dulaglutide agonizes the GLP-1 receptor, resulting in stimulation of insulin synthesis and secretion, and has been shown to provide improved glycemic control in patients with T2DM.

[0205] As used herein, the term "dulaglutide" refers to any GLP-1 receptor agonist protein dimer of two monomers having the amino acid sequence of SEQ ID NO: 4, including any protein that is the subject of regulatory submission data seeking approval of a GLP-1 receptor agonist product that relies in whole or in part on information related to dulaglutide submitted by Eli Lilly and Company to a regulatory agency, regardless of whether the party seeking approval of the protein actually identifies the protein as dulaglutide or uses some other term.

[0206] Table 1. Sequences included in certain Fc-containing proteins

[0207]

[0208] In one embodiment, the Fc-containing protein is etanercept, alefacept, abatacept, rilonacept, romiplostim, belatacept, aflibercept, conbercept, efmoroctocog alfa, eftrenonacog alfa, asfotase alfa, or luspatercept.

[0209] In one embodiment, the Fc-containing protein is an antibody. In one embodiment, the Fc-containing protein is not an antibody.

[0210] In one aspect, provided herein is an Fc-containing protein produced by any of the methods disclosed herein.

[0211] In one aspect, provided herein is dulaglutide produced by any of the methods disclosed herein.

[0212] Examples

[0213] The following examples are provided by way of illustration and not limitation.

[0214] Example 1: Analysis of elevated pC02 levels and pH setpoint shifts in production bioreactors

[0215] This example describes studies that evaluated initial pH setpoint and deadband changes and implementation of pH setpoint and deadband changes during the production bioreactor phase of degarelix manufacturing to identify parameters that reduce pC02 levels and minimize levels of degarelix clipped forms (e.g., N-terminal clipped species of degarelix, des H / HG, which is a variant of degarelix in which either the H1 residue or both the H1 and G2 residues are clipped from the N-terminus of degarelix) in the product. The initial pH setpoint and deadband in the production bioreactor phase in the degarelix manufacturing process was 6.80 ± 0.03. However, under these conditions, the increased pC02 in the cell culture resulted in increased clipped forms of degarelix and, thus, reduced degarelix titer.

[0216] The initial studies evaluated changing the initial pH setpoint and deadband to 6.86 ± 0.09, indicating that changing the initial pH setpoint and widening the deadband reduced the elevated pC02 levels observed in the production bioreactor phase under the previous culture conditions. Figure 1 ) under the previous culture conditions. Thus, the initial pH of 6.86 ± 0.09 maintained lower pC02 levels under the conditions tested. The small-scale studies described below investigated whether the addition of pH shifts could further optimize the cell culture conditions for this degarelix production phase.

[0217] These small-scale studies were performed using equipment, current degarelix working cell bank (WCB), raw materials, process schedule, and process conditions that best mimic the degarelix commercial manufacturing cell culture process when performed at the laboratory scale. The conditions tested specifically focused on pH control during the production bioreactor phase of degarelix manufacturing.

[0218] The objective of the following studies was to implement a CO2 clamp software in order to simulate elevated pC02 levels and evaluate the effect of a pH shift on day 11 on des H / HG levels. The conditions for this study are described in Table 2 below.

[0219] Table 2. Culture conditions

[0220]

[0221] The CO2pincer software in the Applikon 3L bioreactor system used in these studies was run via a computer internal script that calculated the CO2gas flow based on a manual input of the percentage of total gas flow (air sparge flow + O2sparge flow) and a specific pCO2target increase setpoint. When the process was outside the pH insensitivity zone, the CO2pincer automatically turned off, thereby allowing the pH control (CO2sparging cascade or 2 N NaOH) to maintain the pH. For example, if the baseline pCO2was 30 mmHg without any additional CO2sparging and the CO2pincer software was set to 5 mmHg, the pCO2was expected to increase to 35 mmHg.

[0222] The viable cell density (VCD) was comparable across all test conditions over the 14-day culture period. Cell viability was comparable across conditions until the end of the culture, with the CO2pincer condition resulting in a slight decrease in cell viability. Glucose concentration in the cell culture medium was comparable across conditions over the 14-day culture, with the high pH shift to 7.00 ± 0.05 on day 11 requiring a glucose feed on day 13 to prevent glucose depletion prior to the day 14 harvest. A slight increase in lactate concentration in the cell culture medium was observed with the CO2pincer and pH shift to 6.75 ± 0.02 condition from day 11 to the end of the culture on day 14, otherwise lactate concentrations in the medium were comparable.

[0223] Sodium levels in the medium were comparable across all conditions throughout the 14 days, and the offline pH profiles were highly comparable until the pH shift on day 11. The pCO2(n = 8) under the CO2pincer condition gradually increased after day 6, with an average pCO2stabilizing at approximately 70 mmHg from approximately day 10 until the end of the run. Degarelix titers were comparable, with the exception of the CO2pincer condition resulting in a lower average titer on day 14.

[0224] Of the four conditions tested in this study, the CO2pincer condition resulted in the lowest average titer and the greatest overall variability, which can be attributed to the lactate concentrations observed in this set of bioreactor conditions, i.e., the increase in lactate resulted in a decrease in titer (Table 3). The highest average titer was achieved with the pH setpoint shift to 7.00 on day 11.

[0225] Table 3. Degarelix titer results

[0226]

[0227] CO2 clamp conditions resulted in an average des H / HG value of 3.36%, with several data points exceeding the acceptance criteria of no more than 3.5%. In addition, a shift in the pH setpoint to 6.75 on day 11 (condition 4) resulted in an average des H / HG value of 3.20%, with two data points near or above the acceptance criteria, while a shift in the pH setpoint to 7.00 on day 11 (condition 3) resulted in the lowest average des H / HG of 2.27% (Table 4).

[0228] Table 4. Des H / HG Results

[0229]

[0230] A positive correlation between pCO2 on day 14 and des H / HG was observed. A negative correlation between off-line pH on day 14 and des H / HG was observed.

[0231] The results of this study indicate that elevated pCO2 or a shift to a lower pH setpoint on day 11 is associated with higher des H / HG levels and lower taspoglutide titers, and a shift to a higher pH setpoint on day 11 is associated with lower des H / HG levels and higher taspoglutide titers.

[0232] Example 2: Identification of Optimal pH Conditions in Production Bioreactor

[0233] This study was designed to identify the optimal pH strategy (including pH shift day, pH setpoint, and nongrowth zone shift amplitude) for the production bioreactor stage of the taspoglutide manufacturing process, and the level of pCO2 for process robustness and key quality attributes of the product, particularly the level of the clipped form of taspoglutide (des H / HG). The conditions used in this study are described in Table 5 below. All conditions were performed in a 5 L production bioreactor for a total of 14 days.

[0234] CO2 control in the Sartorius 5 L system used in these studies was manually controlled by using the total gas flow (air sparge flow + O2 sparge flow) to calculate daily the setpoint for the CO2 gas flow. See the following equation:

[0235]

[0236] Table 5. Culture Conditions

[0237]

[0238] The viable cell density and survival levels were comparable across all conditions throughout the 14-day culture, with a slight decrease in survival in the last two days observed in conditions where lactate increased. The glucose concentration in the cell culture medium was comparable until the pH shift at days 8, 9, and 10, and a general increase in glucose consumption in the culture was observed when the pH shift was to 7.07 ± 0.05. An increase in lactate concentration in the medium was observed in all bioreactors with high pC02 until the end of the 14-day culture.

[0239] The results of this study show that the off-line pH profiles and sodium levels were comparable until the different pH shifts were implemented at days 8, 9, and 10. The dulaglutide titers were comparable at day 8, but decreased at day 14. The lowest titer was observed in the bioreactor where high pC02 and elevated lactate were also observed.

[0240] Analysis of the des H / HG levels showed that the pH levels at day 14 of the production bioreactor were negatively correlated with the des H / HG levels in the production bioreactor study Figure 2 ), indicating that shifting the pH to a higher pH setpoint and a narrower zone of indifference for the duration of the production bioreactor phase of dulaglutide production in this study was the optimal pH control strategy.

[0241] Further analysis of the results of this study showed that there was a statistically significant negative correlation between the post-pH shift setpoint and the des H / HG levels (scaled correlation estimate of -0.25, p = 0.0363), indicating that shifting to a higher pH setpoint reduced the amount of protease clipping in the culture. In addition, there was a statistically significant negative correlation between the pC02 and the dulaglutide harvest titer (scaled correlation estimate of -0.154, p = 0.0077), indicating that an increase in pC02 can result in a lower dulaglutide titer in the production bioreactor culture. The results also showed that the day of the pH shift (day 8, 9, or 10) had minimal or the least correlation with the dulaglutide titer or the des H / HG levels.

[0242] In summary, the foregoing studies described in Example 1 and Example 2 show that an initial pH setpoint of 6.86 ± 0.09 and a zone of indifference that slows down the observed increase in pC02 at a pH setpoint of 6.80 ± 0.03 and a zone of indifference. In addition, shifting to a higher pH setpoint and a narrower zone of indifference at days 8, 9, 10, or 11 of the cell culture reduces the des H / HG levels and increases the dulaglutide titer when compared to cell cultures that maintain the initial pH setpoint and zone of indifference throughout the cell culture.

[0243] The scope of the application is not limited to the specific embodiments described herein. Indeed, various modifications thereto in addition to those described previously and shown in the accompanying drawings would become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0244] Other embodiments are within the scope of the following claims.

Claims

1. A method for producing degludec, the method comprising the steps of: a) culturing mammalian cells expressing degludec in a cell culture medium at a first pH setpoint for a first period of time; subsequently b) culturing the mammalian cells in a cell culture medium at a second pH setpoint for a second period of time, wherein the second pH setpoint is higher than the first pH setpoint, whereby the mammalian cells produce degludec.

2. The method according to claim 1, wherein the first pH setpoint has a shoulder of 0.01 to 0.

10.

3. The method according to claim 1 or 2, wherein the first pH setpoint has a shoulder of 0.07 to 0.

10.

4. The method according to any one of the preceding claims, wherein the first pH setpoint has a shoulder of about 0.

09.

5. The method according to any one of the preceding claims, wherein the second pH setpoint has a shoulder of 0.01 to 0.

10.

6. The method according to any one of the preceding claims, wherein the second pH setpoint has a shoulder of 0.03 to 0.

06.

7. The method according to any one of the preceding claims, wherein the second pH setpoint has a shoulder of about 0.

05.

8. The method according to any one of the preceding claims, wherein the shoulder of the second pH setpoint is narrower than the shoulder of the first pH setpoint.

9. The method according to any one of the preceding claims, wherein the first pH setpoint has a shoulder of about 0.09 and the second pH setpoint has a shoulder of about 0.

05.

10. The method according to any one of the preceding claims, wherein the second pH setpoint is 0.01 to 1.0 pH units higher than the first pH setpoint.

11. The method according to any one of the preceding claims, wherein the first pH setpoint is 6.0 to 7.

0.

12. The method according to any one of the preceding claims, wherein the first pH setpoint is 6.5 to 6.

9.

13. The method according to any one of the preceding claims, wherein the first pH setpoint is 6.8 to 6.

9.

14. The method according to any one of the preceding claims, wherein the first pH setpoint is about 6.

86.

15. The method according to any one of the preceding claims, wherein the second pH setpoint is 6.5 to 7.

5.

16. The method according to any one of the preceding claims, wherein the second pH setpoint is 6.9 to 7.

5.

17. The method according to any one of the preceding claims, wherein the second pH setpoint is 7.0 to 7.

1.

18. The method according to any one of the preceding claims, wherein the second pH setpoint is about 7.

0.

19. The method of any of the preceding claims, wherein the first pH setpoint is about 6.86, has a shoulder of about 0.09, and the second pH setpoint is about 7.0, has a shoulder of about 0.

05.

20. The method according to any one of the preceding claims, wherein the first period of time is longer than the second period of time.

21. The method of any one of the preceding claims, wherein the first time period is 7 to 12 days.

22. The method of any one of the preceding claims, wherein the first time period is 9 to 11 days.

23. The method of any one of the preceding claims, wherein the first time period is about 10 days.

24. The method of any one of the preceding claims, wherein the second time period is at least 1 day.

25. The method of any one of the preceding claims, wherein the second time period is 4 to 7 days.

26. The method of any one of the preceding claims, wherein the second time period is 4 to 6 days.

27. The method of any one of the preceding claims, wherein the second time period is about 5 days.

28. The method of any one of the preceding claims, wherein the first time period is about 10 days and the second time period is about 5 days.

29. A method for producing degludec, the method comprising the steps of: a) culturing mammalian cells expressing degludec in a cell culture medium having a pH set point of about 6.86, with a zone of insensitivity of about 0.09, for a first time period, comprising 9 to 10 days; followed by b) culturing the mammalian cells in a cell culture medium having a pH set point of about 7.0, with a zone of insensitivity of about 0.05, for a second time period, comprising 4 to 5 days, whereby the mammalian cells produce degludec.

30. The method of any one of the preceding claims, further comprising culturing the mammalian cells at a first temperature for 1 to 5 days prior to the first time period, followed by culturing the mammalian cells at a second temperature for 9 to 14 days.

31. The method of claim 30, wherein the second temperature is lower than the first temperature.

32. The method of claim 30 or 31, wherein the first temperature is 34 °C to 40 °C.

33. The method of any one of claims 30 to 32, wherein the first temperature is about 36 °C.

34. The method of any one of claims 30 to 33, wherein the second temperature is 30 °C to 36 °C.

35. The method of any one of claims 30 to 34, wherein the second temperature is about 33 °C.

36. The method of any one of claims 30 to 35, wherein the mammalian cells are cultured at the first temperature for 3 to 4 days.

37. The method of any one of claims 30 to 36, wherein the mammalian cells are cultured at the first temperature for about 3 days.

38. The method of any one of claims 30 to 37, wherein the mammalian cells are cultured at the second temperature for 11 to 12 days.

39. The method of any one of claims 30 to 38, wherein the mammalian cells are cultured at the second temperature for about 11 days.

40. The method of any one of the preceding claims, wherein the mammalian cells are cultured in a bioreactor.

41. The method of claim 40, wherein the mammalian cells are cultured in fed-batch mode.

42. The method of any one of the preceding claims, further comprising adding a base source to the cell culture medium if the pH is below the zone of indifference, or adding an acid source to the cell culture medium if the pH is above the zone of indifference during the first time period.

43. The method of any one of the preceding claims, further comprising adding a base source to the cell culture medium if the pH is below the zone of indifference, or adding an acid source to the cell culture medium if the pH is above the zone of indifference during the second time period.

44. The method of claim 42 or 43, wherein the acid source is CO2.

45. The method of any one of claims 42 to 44, wherein the base source is a solution comprising NaOH.

46. The method of any one of the preceding claims, wherein the cell culture medium is sparged with air during the first and / or second time period.

47. The method of any one of the preceding claims, wherein the mammalian cells are selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, Hela cells, NSO cells, PER.C6 cells, VERO cells, CRL7O30 cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, B-W cells, L-M cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

48. The method of any one of the preceding claims, wherein the mammalian cells are CHO cells.

49. The method of any one of the preceding claims, wherein one or more proteases have reduced activity in the second pH zone of indifference than in the first pH zone of indifference.

50. The method of any one of the preceding claims, wherein one or more proteases have reduced activity at the second pH setpoint than at the first pH setpoint.

51. The method of claim 50, wherein the one or more proteases cleave N-terminus of degludec, optionally wherein the protease is cathepsin D.

52. The method of any one of the preceding claims, wherein less than about 4% of the produced degludec is cleaved at the N-terminus.

53. The method of claim 52, wherein the cleaved form of degludec does not have an HG residue at the N-terminus of degludec.

54. A method for producing degludec in CHO cells, the method comprising the steps of: a) culturing CHO cells in a cell culture medium maintained at a first pH setpoint of about 6.68, with a zone of indifference of about 0.09, for about 10 days; followed by b) culturing the CHO cells in a cell culture medium maintained at a second pH setpoint of about 7.0, with a zone of indifference of about 0.05, for about 5 days; and wherein the culturing is initiated at a temperature of about 36°C and subsequently reduced to about 33°C after about 3 days; thereby the CHO cells produce degludec.

55. The method of claim 54, wherein the CHO cells are cultured in a bioreactor in a fed-batch mode.

56. The method of claim 54 or 55, wherein NaOH is added to the cell culture medium if the pH is below the insensitivity zone of the first or second pH setpoint, and CO2 is added to the cell culture medium if the pH is above the insensitivity zone of the first or second pH setpoint.

57. The method of any one of claims 54 to 56, wherein the cell culture medium is sparged with air one or more times.

58. The method of any one of claims 54 to 57, wherein less than about 4% of the degludec produced by the CHO cells is clipped at the N-terminus.

59. Degludec produced by the method of any one of the preceding claims.

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