Protein manufacturing process

Abstract

The present disclosure provides novel methods for controlling the glycosylation profile of proteins during production. The present disclosure also provides novel methods for improving protein yield while controlling the glycosylation profile of proteins.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 066,122, filed on 14 August 2020, which is incorporated herein by reference in its entirety.

[0002] References to electronically submitted sequence listings via EFS-WEB The contents of the sequence listing submitted electronically in the ASCII text file filed with this application (name: 3338_188PC01_Seqlisting_ST25.txt, size: 24,720 bytes; and creation date: August 12, 2021) are incorporated herein by reference in their entirety. [Background technology]

[0003] Background of the Invention The market for protein-based therapeutics is growing rapidly, and the pace of development continues to accelerate. However, maintaining desired quality attributes, such as glycosylation, while keeping volumes, reducing production costs, and providing production flexibility is a challenge for the industry. To meet market demand, efficient, large-scale production of protein-based therapeutics is necessary.

[0004] Glycosylation is one of the most abundant post-translational modifications (PTMs) of proteins. This results from the formation of glycoproteins by adding sugar residues to protein side chains. While mammalian glycoprotein oligosaccharides are generally constructed from a limited number of monosaccharides, their structural diversity is enormous, mainly because they often form complex branching patterns.

[0005] Glycosylation plays a crucial role in many specific biological functions, including immune defense, fertilization, viral replication, parasitic infection, cell proliferation, inflammation, and cell-cell adhesion. For pharmaceutical glycoproteins, glycosylation affects the conformational stability, clearance rate, and protection from proteolysis of the protein, improving its solubility. Because different glycoforms can have different biological activities, the ability to monitor and control glycosylation during production is critical to the quality of biopharmaceutical molecules. [Overview of the project] [Problems that the invention aims to solve]

[0006] However, glycosylation during fermentation occurs naturally with a degree of heterogeneity and can be influenced by many different factors, such as the expression system, process conditions, culture medium composition, feed protocol, purification process, or any combination thereof. As a result, there is a need to improve protein production processes, including culture, while maintaining a consistent protein glycosylation pattern. [Means for solving the problem]

[0007] This disclosure relates to a method for improving the yield of proteins by cells, comprising culturing cells in a bioreactor for a protein induction phase under suitable conditions, wherein the suitable conditions are: a first temperature setting of 36°C, a second temperature setting of 33°C, and a third temperature setting of 31°C; a first pH setting of 7.0 and a second pH setting of 6.9; and 0.15 × 10⁻¹⁵ 6 cells / mL and 0.45 × 10 6 Between cells / mL, for example, 0.30 × 10 6The method comprises an initial viable cell density (VCD) setting of cells / mL; an initial pH of 6.9; an initial CO2 setting of between 10% and 40%; or any combination thereof. In some embodiments, suitable conditions include an initial temperature setting of 36°C, a second temperature setting of 33°C, and a third temperature setting of 31°C; an initial pH setting of 7.0 and a second pH setting of 6.9; and 0.30 × 10 6 This includes the initial viable cell density (VCD) setting, as well as the initial CO2 setting, which is between 15% and 25%.

[0008] This disclosure relates to a method for controlling cell proliferation rate, cell viability, live cell density and / or cell titer in order to produce a protein, comprising culturing cells in a bioreactor for a protein induction phase under a first temperature setting of 36°C, and culturing cells under a second temperature setting of 33°C and a final temperature setting of 31°C.

[0009] The disclosure also relates to a method for improving the yield of proteins by cells, comprising culturing cells in a bioreactor under suitable conditions, wherein the suitable conditions include (i) a first temperature setpoint of 36.0°C and a second temperature setpoint below 36.0°C, (ii) a first temperature setpoint below 36.5°C and a final temperature setpoint of 31.0°C, or (iii) a first temperature setpoint below 36.5°C, a second temperature setpoint of 33.0°C, and a final temperature setpoint below 33.0°C.

[0010] In some embodiments, the final temperature setpoint occurs between approximately 228 and 252 hours. In some embodiments, the final temperature setpoint occurs approximately 228, 234, 240, 246, or 252 hours after the first temperature setpoint. In some embodiments, the final temperature setpoint is 31.0°C and occurs after 240 hours. In some embodiments, the second temperature setpoint occurs between approximately 120 and 168 hours. In some embodiments, the second temperature setpoint occurs approximately 120, 126, 132, 138, 144, 150, 156, 162, or 168 hours. In some embodiments, the second temperature setpoint is 33.0°C and occurs after 144 hours.

[0011] In some embodiments, these conditions improve protein yield by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% compared to methods without suitable conditions. In some embodiments, these conditions reduce the cell proliferation rate. In some embodiments, cell proliferation exhibits an average doubling time of 0–5 days, ranging from approximately 30.0 hours to approximately 40.0 hours. In other embodiments, cell proliferation exhibits an average doubling time of 0–5 days, ranging from approximately 35.1 hours.

[0012] In some embodiments, this method controls cell viability. In some embodiments, the cell viability is approximately 10.0 × 10⁻⁶. 6 Approximately 15.0 × 10 cells / mL 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is approximately 11.2 × 10⁶. 6shows an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is about 0.05×10 9 cells / mL to about 0.11×10 9 cells / mL, which is the 0-14 day average integral viable cell density (IVCD). In some embodiments, the cell viability is about 0.10×10 9 cells / mL, which is the 0-14 day average integral viable cell density (IVCD). In some embodiments, the method controls titer. In some embodiments, the titer exhibits a Day 14 average titer of from about 1.50 g / L to about 3.5 g / L. In some embodiments, the titer exhibits a Day 14 average titer of about 2.87 g / L. In some embodiments, the titer exhibits an average specific productivity of from about 20.0 pg / cell·day to about 40.0 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of about 38.5 pg / cell·day. In some embodiments, the process further comprises modifying an upstream bioreactor parameter, wherein the upstream reactor parameter is selected from the group consisting of (i) feed time, (ii) initial pH, (iii) pH shift, (iv) CO2, (v) initial cell density, or (vi) any combination thereof.

[0013] In some embodiments, the method controls the glycosylation profile of a protein. In some embodiments, the glycosylation profile includes one or more N-linked glycans. In some embodiments, the N-linked glycans include G0F, G1F, G2F, S1G1F, S1G2F, and / or S2G2F. In some embodiments, the method further includes measuring the glycosylation profile after 14 days. In some embodiments, the protein includes a CTLA4 domain. In some embodiments, the protein is a fusion protein. In some embodiments, the fusion protein includes an Fc moiety. In some embodiments, the protein is abatacept. In some embodiments, the protein is the amino acid sequence shown in Sequence ID No. 5. In some embodiments, one or more N-linked glycans are located at one or more residues selected from the group consisting of Asn76(T5), Asn108(T7), and / or Asn207(T14) of abatacept. In some embodiments, G0F accounts for a relative abundance of approximately 7.0% or less, or approximately 6.5% or less. In some embodiments, G1F accounts for a relative abundance of approximately 7.5% or less, or approximately 7%. In some embodiments, G2F accounts for approximately 25% or less, or approximately 1.5% to approximately 23%. In some embodiments, S1G1F accounts for a relative abundance of approximately 13.5% or less, or approximately 12.5%. In some embodiments, S1G2F accounts for approximately 33% or more, or approximately 32% to approximately 49%. In some embodiments, S2G2F accounts for approximately 12% or more, or approximately 14% to approximately 48.5%. In some embodiments, G2F accounts for a relative abundance of approximately 1.5% to approximately 23%, S1G2F accounts for a relative abundance of approximately 32% to approximately 49%, and / or S2G2F accounts for a relative abundance of approximately 14% to approximately 48.5%. In some embodiments, G2F accounts for a relative abundance of approximately 25% or less, S1G2F accounts for a relative abundance of approximately 33% or more, and / or S2G2F accounts for a relative abundance of approximately 12% or more.In some embodiments, G0F accounts for a relative abundance of approximately 6.5% or less, G1F accounts for a relative abundance of approximately 7% or less, G2F accounts for a relative abundance of approximately 1.5% to approximately 23%, S1G1F accounts for a relative abundance of approximately 12.5% ​​or less, S1G2F accounts for a relative abundance of approximately 32% to approximately 49%, and / or S2G2F accounts for a relative abundance of approximately 14% to approximately 48.5%. In some embodiments, G0F accounts for a relative abundance of approximately 7.0% or less. In some embodiments, G1F accounts for a relative abundance of approximately 7.5% or less, G2F accounts for a relative abundance of approximately 25% or less, S1G1F accounts for a relative abundance of approximately 13.5% or less, S1G2F accounts for a relative abundance of approximately 33% or more, and / or S2G2F accounts for a relative abundance of approximately 12% or more.

[0014] In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 2.0% and approximately 10.0%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 2.5% and approximately 10%, between approximately 2.5% and approximately 9.5%, between approximately 2.5% and approximately 9%, between approximately 2.5% and approximately 8.5%, between approximately 2.5% and approximately 8%, between approximately 2.5% and approximately 7.5%, between approximately 2.5% and approximately 7%, between approximately 2.5% and approximately 6.5%, between approximately 3.0% and approximately 10%, between approximately 3.0% and approximately 9.5%, between approximately 3.0% and approximately 9%, between approximately 3.0% and approximately 8.5%, between approximately 3.0% and approximately 8%, between approximately 3.0% and approximately 7.5%, between approximately 3.0% and approximately 7%, or between approximately 3.0% and approximately 6.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) that is between approximately 3.2% and 6.6%, between approximately 3.2% and 4.6%, or between approximately 3.2% and 6.6%. In some embodiments, the relative abundance of G0F is approximately 4.0%.

[0015] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between approximately 1.0% and approximately 6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn108(T7) between approximately 1.0% and approximately 6%, between approximately 1.0% and approximately 5.5%, between approximately 1.0% and approximately 5.0%, between approximately 1.0% and approximately 4.5%, between approximately 1.0% and approximately 4.0%, between approximately 1.5% and approximately 6%, between approximately 1.5% and approximately 5.5%, between approximately 1.5% and approximately 5.0%, between approximately 1.5% and approximately 4.5%, between approximately 1.5% and approximately 4.0%, between approximately 2.0% and approximately 6%, between approximately 2.0% and approximately 5.5%, between approximately 2.0% and approximately 5.0%, between approximately 2.0% and approximately 4.5%, or between approximately 2.0% and approximately 4.0%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn108(T7) between approximately 2.1% and approximately 4.0%, between approximately 1.8% and approximately 3.5%, or between approximately 1.8% and approximately 4.0%. In some embodiments, the relative abundance of G0F is approximately 3.4%.

[0016] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5% and approximately 12%. This includes the relative abundance of G2F between 5%, between approximately 6% and 11%, between approximately 6% and 10.5%, between approximately 6% and 10%, between approximately 6.5% and 12%, between approximately 6.5% and 11.5%, between approximately 6.5% and 11%, between approximately 6.5% and 10.5%, between approximately 6.5% and 10%, between approximately 7% and 12%, between approximately 7% and 11.5%, between approximately 7% and 11%, between approximately 7% and 10.5%, or between approximately 7% and 10%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) that is between approximately 7.2% and approximately 9.8%, between approximately 6.3% and approximately 10.6%, or between approximately 7.2% and approximately 10.6%. In some embodiments, the relative abundance of G2F is approximately 7.8%.

[0017] In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 5% and approximately 21%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and are distributed between approximately 5% and approximately 21%, between approximately 5% and approximately 20.5%, between approximately 5% and approximately 20%, between approximately 5% and approximately 19.5%, between approximately 5% and approximately 19%, between approximately 5.5% and approximately 21%, between approximately 5.5% and approximately 20.5%, between approximately 5.5% and approximately 20%, between approximately 5.5% and approximately 19.5%, between approximately 5.5% and approximately 19%, between approximately 6% and approximately 21%, between approximately 6% and approximately 20.5%, between approximately 6% and approximately 20%, between approximately 6% and approximately 19.5%, between approximately 6% and approximately 19%, between approximately 6.5% and approximately 21%, and between approximately 6.5% and approximately 20.5%. This includes the relative abundance of G2F between % and %, between approximately 6.5% and 20%, between approximately 6.5% and 19.5%, between approximately 6.5% and 19%, between approximately 7% and 21%, between approximately 7% and 20.5%, between approximately 7% and 20%, between approximately 7% and 19.5%, between approximately 7% and 19%, between approximately 7.5% and 21%, between approximately 7.5% and 20.5%, between approximately 7.5% and 20%, between approximately 7.5% and 19.5%, between approximately 7.5% and 19%, between approximately 8% and 21%, between approximately 8% and 20.5%, between approximately 8% and 20%, between approximately 8% and 19.5%, or between approximately 8% and 19%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 8.2% and approximately 14.1%, between approximately 8.0% and approximately 18.6%, or between approximately 8.2% and approximately 14.1%. In some embodiments, the relative abundance of G2F is approximately 12.4%. In some embodiments, G2F further contains a galactose-α-1,3-galactose moiety (G2F-Gal), with G2F-Gal accounting for a relative abundance of approximately 1.4% or less. In some embodiments, G2F-Gal accounting for a relative abundance between approximately 1.0% and approximately 1.4%. In some embodiments, G2F-Gal accounting for a relative abundance between approximately 0.4% and approximately 0.9%.

[0018] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and occupy a relative abundance of S1G2F between approximately 29% and approximately 38%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 31.6% and approximately 35.1%, between approximately 31.3% and approximately 36.5%, between approximately 30% and approximately 37.5%, between approximately 30% and approximately 36.5%, between approximately 31.5% and approximately 38%, between approximately 31.5% and approximately 37.5%, between approximately 31.5% and approximately 37%, and between approximately 31.5% and approximately 36.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 31.6% and approximately 35.1%, between approximately 31.3% and approximately 36.5%, or between approximately 31.3% and approximately 36.5%. In some embodiments, the relative abundance of S1G2F is approximately 33.3%.

[0019] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between 33% and about 45%. This includes the relative abundance of S1G2F between approximately 42.5%, between approximately 34% and 45%, between approximately 34% and 44.5%, between approximately 34% and 44%, between approximately 34% and 43.5%, between approximately 34% and 43%, between approximately 34% and 42.5%, between approximately 34.5% and 45%, between approximately 34.5% and 44.5%, between approximately 34.5% and 44%, between approximately 34.5% and 43.5%, between approximately 34.5% and 43%, or between approximately 34.5% and 42.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34.2% and approximately 37.7%, between approximately 35.5% and approximately 42.3%, or between approximately 34.2% and approximately 42.3%. In some embodiments, the relative abundance of S1G2F is approximately 36.5%. In some embodiments, S1G2F further contains a galactose-α-1,3-galactose moiety (S1G2F-Gal), with S1G2F-Gal accounting for a relative abundance of approximately 4.7% or less. In some embodiments, S1G2F-Gal accounting for a relative abundance between approximately 2.3% and approximately 4.7%. In some embodiments, S1G2F-Gal accounting for a relative abundance between approximately 1.4% and approximately 1.8%.

[0020] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 13% and approximately 25%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and are distributed between approximately 13% and approximately 25%, between approximately 13% and approximately 24.5%, between approximately 13% and approximately 24%, between approximately 13% and approximately 23.5%, between approximately 13% and approximately 23%, between approximately 13.5% and approximately 25%, between approximately 13.5% and approximately 24.5%, between approximately 13.5% and approximately 24%, between approximately 13.5% and approximately 23.5%, between approximately 13.5% and approximately 23%, between approximately 14% and approximately 25%, between approximately 14% and approximately 24.5%, between approximately 14% and approximately 24%, between approximately 14% and approximately 23.5%, and between approximately 14% and approximately 2 This includes the relative abundance of S2G2F between 3%, between approximately 14.5% and 25%, between approximately 14.5% and 24.5%, between approximately 14.5% and 24%, between approximately 14.5% and 23.5%, between approximately 14.5% and 23%, between approximately 15% and 25%, between approximately 15% and 24.5%, between approximately 15% and 24%, between approximately 15% and 23.5%, between approximately 15% and 23%, between approximately 15.5% and 25%, between approximately 15.5% and 24.5%, between approximately 15.5% and 24%, between approximately 15.5% and 23.5%, or between approximately 15.5% and 23%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 18.1% and approximately 22.9%, between approximately 15.4% and approximately 20%, and between approximately 15.4% and approximately 22.9%. In some embodiments, the relative abundance of S2G2F is approximately 18.5%.

[0021] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between about 18% and about 36%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between about 18% and about 36%, between about 18% and about 35.5%, between about 18% and about 35%, between about 18% and about 34.5%, between about 18% and about 34%, between about 18% and about 33.5%, between about 18.5% and about 36%, between about 18.5% and about 3 Between 5.5%, between approximately 18.5% and approximately 35%, between approximately 18.5% and approximately 34.5%, between approximately 18.5% and approximately 34%, between approximately 18.5% and approximately 33.5%, between approximately 19% and approximately 36%, between approximately 19% and approximately 35.5%, between approximately 19% and approximately 35%, between approximately 19% and approximately 34.5%, between approximately 19% and approximately 34%, and between approximately 19% and approximately 33.5% Between % and %; between approximately 19.5% and 36%; between approximately 19.5% and 35.5%; between approximately 19.5% and 35%; between approximately 19.5% and 34.5%; between approximately 19.5% and 34%; between approximately 19.5% and 33.5%; between approximately 20% and 36%; between approximately 20% and 35.5%; between approximately 20% and 35%; between approximately 20% and 34%. The relative abundance of S2G2F is between 5%, between approximately 20% and approximately 34%, between approximately 20% and approximately 33.5%, between approximately 20.5% and approximately 36%, between approximately 20.5% and approximately 35.5%, between approximately 20.5% and approximately 35%, between approximately 20.5% and approximately 34.5%, between approximately 20.5% and approximately 34%, or between approximately 20.5% and approximately 33.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between approximately 23.2% and approximately 33.8%, between approximately 20.8% and approximately 32.6%, or between approximately 20.8% and approximately 33.8%. In some embodiments, the relative abundance of S2G2F is approximately 23.5%.

[0022] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 8%, between about 2% and about 7.5%, between about 2% and about 7%, between about 2% and about 6.5%, between about 2% and about 6%, between about 2% and about 5.5%, between about 2.5% and about 8%, between about 2.5% and about 7.5%, between about 2.5% and about 7%, between about 2.5% and about 6.5%, between about 2.5% and about 6%, between about 2.5% and about 5.5%, between about 3% and about 8%, between about 3% and about 7.5%, This includes the relative abundance of S1G3F between 3% and approximately 7%, between approximately 3% and approximately 6.5%, between approximately 3% and approximately 6%, between approximately 3% and approximately 5.5%, between approximately 3.5% and approximately 8%, between approximately 3.5% and approximately 7.5%, between approximately 3.5% and approximately 7%, between approximately 3.5% and approximately 6.5%, between approximately 3.5% and approximately 6%, between approximately 3.5% and approximately 5.5%, between approximately 4% and approximately 8%, between approximately 4% and approximately 7.5%, between approximately 4% and approximately 7%, between approximately 4% and approximately 6.5%, between approximately 4% and approximately 6%, or between approximately 4% and approximately 5.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) that is between approximately 4.4% and approximately 5.6% or between approximately 4.0% and approximately 5.5%. In some embodiments, the relative abundance of S1G3F is approximately 4.6%.

[0023] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) that is between about 0.5% and about 4%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) that is between about 0.5% and about 4%, between about 0.5% and about 3.5%, between about 0.5% and about 3%, between about 0.5% and about 2.5%, between about 1% and about 4%, between about 1% and about 3.5%, between about 1% and about 3%, or between about 1% and about 2.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) that is between approximately 1.4% and approximately 2.2%, between approximately 1.1% and approximately 1.9%, or between approximately 1.1% and approximately 2.2%. In some embodiments, the relative abundance of S1G3F is approximately 1.8%.

[0024] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) that is between about 0.5% and about 4%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) that is between about 0.5% and about 4%, between about 0.5% and about 3.5%, between about 0.5% and about 3%, between about 0.5% and about 2.5%, between about 1% and about 4%, between about 1% and about 3.5%, between about 1% and about 3%, or between about 1% and about 2.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) that is between approximately 1.9% and approximately 2.4%, between approximately 1.4% and approximately 2.1%, or between approximately 1.4% and approximately 2.4%. In some embodiments, the relative abundance of S2G4F is approximately 2.3%.

[0025] In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8 to about 11. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8.3 to about 11, about 9.5 to about 10.1, or about 8.3 to about 10.1. In some embodiments, the molar ratio of NANA is about 10.0.

[0026] In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of about 0.1 to about 2.0. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of 0.90 to about 1.20 or about 0.3 to about 1.2. In some embodiments, the molar ratio of NGNA is about 1.0.

[0027] In some embodiments, the glycosylation profile is analyzed via N-linked carbohydrate profile release. In some embodiments, the glycosylation profile comprises one or more non-sialylated glycans (domain I), monosialylated glycans (domain II), disialylated glycans (domain III), and / or trisialylated and tetrasialylated glycans (domains IV+V). In some embodiments, the non-sialylated glycan (domain I) has molar ratios of about 28 to about 37, about 29 to about 32, about 28 to about 32, or about 29 to about 37. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 31. In some embodiments, the monosialylated glycan (domain II) has molar ratios of about 26 to about 28, about 27 to about 33, about 26 to about 33, or about 27 to about 28. In some embodiments, the monosialylated glycan (domain II) has a molar ratio of about 27. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 27 to about 28, about 22 to about 31, about 27 to about 31, or about 22 to about 28. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 27.4. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13 to about 16, about 8 to about 16, or about 8 to about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 14.6.

[0028] In some embodiments, the glycosylation profile includes one or more O-linked glycans. In some embodiments, the glycosylation profile does not include multiple galactose-α-1,3-galactose (α-gal) links. In some embodiments, CTLA4 includes C-terminal lysine. In some embodiments, C-terminal lysine accounts for a relative abundance of about 20% to about 25%. In some embodiments, C-terminal lysine accounts for a relative abundance of about 3% to about 10%. In some embodiments, the O-linked glycans are located at residues Ser129, Ser130, Ser136, and / or Ser139. In some embodiments, the bioreactor includes a supply medium containing glucose or galactose. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are Chinese hamster ovary (CHO) cells. In some embodiments, the cells are CHO-K1 cells, CHO-DXB11 cells, or CHO-DG44 cells.

[0029] The method of this disclosure also relates to a method for analyzing the branched glycans of a CTLA4-Fc fusion protein, comprising measuring one or more N-linked glycans bound to one or more asparagine residues in the CTLA4 protein, wherein one of the branched glycans is G2F. In some embodiments, the branched glycans are selected from the group consisting of G0F, G1F, G2F, S1G1F, S1G2F, and / or S2G2F. In some embodiments, the branched glycans are measured via ultra-high performance liquid chromatography using fluorescence detection (UPLC-FLR). In some embodiments, the Fc domain of the CTLA4-Fc fusion protein is cleaved before measurement. In some embodiments, the method further comprises collecting the protein from a bioreactor. In some embodiments, the method further comprises subjecting the protein to a virus inactivation step. In some embodiments, the virus inactivation step is performed using 0.5% Triton X-100.

[0030] In some embodiments, the method further includes adjusting the concentration of the protein. In some embodiments, the method further includes formulating the protein. This disclosure provides, for example, the following: [Section 1] A method for controlling cell proliferation rate, cell viability, live cell density and / or cell titer to produce a protein, comprising culturing cells in a bioreactor for a protein induction phase under a first temperature setting of 36°C, and culturing cells under a second temperature setting of 33°C and a final temperature setting of 31°C. [Section 2] A method for improving the yield of proteins by cells, comprising culturing cells in a bioreactor under suitable conditions, wherein the suitable conditions include (i) a first temperature setpoint of 36.0°C and a second temperature setpoint of less than 36°C, (ii) a first temperature setpoint of less than 36.5°C and a final temperature setpoint of 31°C, or (iii) a first temperature setpoint of less than 36.5°C, a second temperature setpoint of 33°C, and a final temperature setpoint of less than 33°C. [Section 3] The method according to item 1 or 2, wherein suitable conditions further include a first pH setting of 7.0 and a second pH setting of 6.9, or a first pH setting of pH 6.9. [Section 4] The method according to any one of items 1 to 3, wherein suitable conditions further include a pH shift over approximately 96 hours. [Section 5] The suitable conditions are approximately 0.30 × 10 6 The method according to any one of claims 1 to 4, further comprising an initial viable cell density (VCD) setting of cells / mL. [Section 6] The method according to any one of items 1 to 5, further comprising an initial CO2 setting value where suitable conditions are between 15% and 25%. [Section 7] The method according to any one of claims 1 to 6, wherein suitable conditions further include a first supply time of approximately 76 hours. [Section 8] A method for improving the yield of proteins by cells, comprising culturing cells in a bioreactor for a protein induction phase under suitable conditions, wherein the suitable conditions are a. The first temperature setting is 36°C, the second temperature setting is 33°C, and the third temperature setting is 31°C. b. The first pH setting is 7.0 and the second pH setting is 6.9, or the first pH is 6.9. c.0.15×10 6 cells / mL and 0.45 × 10 6 The initial viable cell density (VCD) setting is between cells / mL. d. The initial CO2 setting value is between 10% and 40%, or e. Any combination of them Methods that include... [Section 9] Suitable conditions, a. The first temperature setting is 36°C, the second temperature setting is 33°C, and the third temperature setting is 31°C. b. The first pH setting is 7.0 and the second pH setting is 6.9. c. Approx. 0.30×10 6 The initial viable cell density (VCD) setting is cells / mL, and d. The initial CO2 setting value is between 15% and 25%. The method described in item 1, including the method described in item 1. [Section 10] The method according to any one of items 1 to 9, wherein the last temperature setting occurs within approximately 228 to 252 hours. [Section 11] The method described in item 10, wherein the last temperature setting occurs approximately 228 hours, 234 hours, 240 hours, 246 hours, or 252 hours after the first temperature setting. [Section 12] The method according to any one of items 1 to 9, wherein the last temperature setting is 31°C and occurs after 240 hours. [Section 13] The method according to any one of items 1 to 12, wherein the second temperature setting occurs within approximately 120 to 168 hours. [Section 14] The method according to item 13, wherein the second temperature setting occurs after approximately 120 hours, approximately 126 hours, approximately 132 hours, approximately 138 hours, approximately 144 hours, approximately 150 hours, approximately 156 hours, approximately 162 hours, or approximately 168 hours. [Section 15] The method according to any one of items 1 to 14, wherein the second temperature setting is 33°C and occurs after 144 hours. [Section 16] The method according to any one of claims 1 to 15, wherein the conditions improve the protein yield by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% compared to a method without suitable conditions. [Section 17] The method according to any one of items 1 to 16, which reduces the rate of cell proliferation. [Section 18] The method according to item 17, wherein cell proliferation exhibits an average doubling time of 0 to 5 days, which is approximately 30.0 hours to approximately 40.0 hours. [Section 19] The method according to item 17, wherein cell proliferation exhibits an average doubling time of 0 to 5 days, which is approximately 35.1 hours. [Section 20] A method according to any one of items 1 to 19 for controlling cell viability. [Section 21] The cell viability rate is approximately 10.0 × 10⁻⁶. 6 Approximately 15.0 × 10 cells / mL 6 The method according to item 20, which exhibits an average peak live cell density (VCD) of cells / mL. [Section 22] The cell viability rate is approximately 11.2 × 10⁻⁶. 6 The method according to item 20, which exhibits an average peak live cell density (VCD) of cells / mL. [Section 23] The cell viability rate is approximately 0.05 × 10⁻⁶. 9 Approximately 0.11 × 10¹ / mL 9 The method according to item 20, showing the 0-14 day mean live cell density integral (IVCD) of cells / mL. [Section 24] The cell viability rate is approximately 0.10 × 10 9 The method described in item 21, which shows the 0-14 day mean live cell density integral (IVCD) of cells / mL. [Section 25] The method according to any one of claims 1 to 24, for controlling the titer. [Section 26] The method according to item 25, wherein the potency is expressed as a 14-day average potency ranging from approximately 1.50 g / L to approximately 3.5 g / L. [Section 27] The method according to item 25, wherein the potency is approximately 2.87 g / L, and the average potency is shown on day 14. [Section 28] The method according to item 25, which exhibits an average specific productivity of approximately 20.0 pg / cell·day to approximately 40.0 pg / cell·day. [Section 29] The method according to item 25, which exhibits an average specific productivity with a titer of approximately 38.5 pg / cell·day. [Section 30] The method according to any one of claims 1 to 29, wherein the process further comprises modifying upstream bioreactor parameters, the upstream reactor parameters being selected from the group consisting of (i) supply time, (ii) initial pH, (iii) pH shift, (iv) CO2, (v) initial cell density, or (vi) any combination thereof. [Section 31] A method according to any one of items 1 to 30 for controlling the glycosylation profile of a protein. [Section 32] The method according to claim 31, wherein the glycosylation profile comprises one or more N-linked glycans. [Section 33] The method according to claim 32, wherein the N-linked glycan includes G0F, G1F, G2F, S1G1F, S1G2F, and / or S2G2F. [Section 34] The method according to any one of claims 31 to 33, further comprising measuring the glycosylation profile after 14 days. [Section 35] The method according to any one of items 1 to 34, wherein the protein contains a CTLA4 domain. [Section 36] The method according to any one of items 1 to 28, wherein the protein is a fusion protein. [Section 37] The method according to item 36, wherein the fusion protein includes an Fc moiety. [Section 38] The method according to any one of items 1 to 37, wherein the protein is abatacept. [Section 39] The method according to item 38, wherein the protein has the amino acid sequence shown in SEQ ID NO: 5. [Section 40] The method according to any one of items 33 to 39, wherein G0F accounts for a relative abundance of approximately 7.0% or less, or approximately 6.5% or less. [Section 41] The method according to any one of items 33 to 40, wherein G1F accounts for a relative abundance of approximately 7.5% or less or approximately 7%. [Section 42] The method according to any one of items 33 to 41, wherein G2F accounts for a relative abundance of approximately 25% or less, or between approximately 1.5% and approximately 23%. [Section 43] The method according to any one of terms 33 to 42, wherein S1G1F accounts for a relative abundance of approximately 13.5% or less, or approximately 12.5%. [Section 44] The method according to any one of terms 33 to 43, wherein S1G2F accounts for a relative abundance of approximately 33% or more, or between approximately 32% and approximately 49%. [Section 45] The method according to any one of items 33 to 44, wherein S2G2F accounts for a relative abundance of approximately 12% or more, or between approximately 14% and approximately 48.5%. [Section 46] The method according to any one of terms 33 to 45, wherein G2F accounts for a relative abundance of approximately 1.5% to approximately 23%, S1G2F accounts for a relative abundance of approximately 32% to approximately 49%, and / or S2G2F accounts for a relative abundance of approximately 14% to approximately 48.5%. [Section 47] The method according to any one of items 33 to 45, wherein G2F accounts for a relative abundance of approximately 25% or less, S1G2F accounts for a relative abundance of approximately 33% or more, and / or S2G2F accounts for a relative abundance of approximately 12% or more. [Section 48] The method according to any one of items 33 to 45, wherein G0F accounts for a relative abundance of approximately 6.5% or less, G1F accounts for a relative abundance of approximately 7% or less, G2F accounts for a relative abundance of approximately 1.5% to approximately 23%, S1G1F accounts for a relative abundance of approximately 12.5% ​​or less, S1G2F accounts for a relative abundance of approximately 32% to approximately 49%, and / or S2G2F accounts for a relative abundance of approximately 14% to approximately 48.5%. [Section 49] The method according to any one of items 33 to 45, wherein G0F accounts for a relative abundance of approximately 7.0% or less, G1F accounts for a relative abundance of approximately 7.5% or less, G2F accounts for a relative abundance of approximately 25% or less, S1G1F accounts for a relative abundance of approximately 13.5% or less, S1G2F accounts for a relative abundance of approximately 33% or more, and / or S2G2F accounts for a relative abundance of approximately 12% or more. [Section 50] The method according to any one of claims 39 to 49, wherein one or more N-linked glycans are located at one or more residues selected from the group consisting of Asn76(T5), Asn108(T7), and / or Asn207(T14) of abatacept. [Section 51] The method according to claim 50, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of G0F is between approximately 2.0% and approximately 10.0%. [Section 52] The method according to claim 51, wherein one or more N-linked glycans are located at residue Asn76(T5) and include a relative abundance of G0F between approximately 2.5% and approximately 10%, between approximately 2.5% and approximately 9.5%, between approximately 2.5% and approximately 9%, between approximately 2.5% and approximately 8.5%, between approximately 2.5% and approximately 8%, between approximately 2.5% and approximately 7.5%, between approximately 2.5% and approximately 7%, between approximately 2.5% and approximately 6.5%, between approximately 3.0% and approximately 10%, between approximately 3.0% and approximately 9.5%, between approximately 3.0% and approximately 9%, between approximately 3.0% and approximately 8.5%, between approximately 3.0% and approximately 8%, between approximately 3.0% and approximately 7.5%, between approximately 3.0% and approximately 7%, or between approximately 3.0% and approximately 6.5%. [Section 53] The method according to claim 51, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of G0F is between approximately 3.2% and 6.6%, between approximately 3.2% and 4.6%, or between approximately 3.2% and 6.6%. [Section 54] The method described in item 51, wherein the relative abundance of G0F is approximately 4.0%. [Section 55] The method according to any one of claims 50 to 54, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of G0F is between approximately 1.0% and approximately 6%. [Section 56] One or more N-linked glycans are located at residue Asn108(T7), between approximately 1.0% and 6%, between approximately 1.0% and 5.5%, between approximately 1.0% and 5.0%, between approximately 1.0% and 4.5%, between approximately 1.0% and 4.0%, between approximately 1.5% and 6%, between approximately 1.5% and 5.5%, and between approximately 1.5% and 5.0%. The method according to any one of claims 50 to 54, comprising the relative abundance of G0F being between approximately 1.5% and approximately 4.5%, between approximately 1.5% and approximately 4.0%, between approximately 2.0% and approximately 6%, between approximately 2.0% and approximately 5.5%, between approximately 2.0% and approximately 5.0%, between approximately 2.0% and approximately 4.5%, or between approximately 2.0% and approximately 4.0%. [Section 57] The method according to any one of claims 50 to 54, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of G0F is between about 2.1% and about 4.0%, between about 1.8% and about 3.5%, or between about 1.8% and about 4.0%. [Section 58] The method described in item 57, wherein the relative abundance of G0F is approximately 3.4%. [Section 59] The method according to any one of claims 50 to 58, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of G2F is between approximately 5% and approximately 12%. [Section 60] One large N-linked glycan is located at residue Asn76(T5), between approximately 5% and 12%, between approximately 5% and 11.5%, between approximately 5% and 11%, between approximately 5% and 10.5%, between approximately 5% and 10%, between approximately 5.5% and 12%, between approximately 5.5% and 11.5%, between approximately 5.5% and 11%, between approximately 5.5% and 10.5%, between approximately 5.5% and 10%, between approximately 6% and 12%, between approximately 6% and 11.5%, and between approximately 6% and 11%. The method according to any one of claims 50 to 58, including a relative abundance of G2F between approximately 6% and approximately 10.5%, between approximately 6% and approximately 10%, between approximately 6.5% and approximately 12%, between approximately 6.5% and approximately 11.5%, between approximately 6.5% and approximately 11%, between approximately 6.5% and approximately 10.5%, between approximately 6.5% and approximately 10%, between approximately 7% and approximately 12%, between approximately 7% and approximately 11.5%, between approximately 7% and approximately 11%, between approximately 7% and approximately 10.5%, or between approximately 7% and approximately 10%. [Section 61] The method according to any one of claims 50 to 58, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of G2F is between about 7.2% and about 9.8%, between about 6.3% and about 10.6%, or between about 7.2% and about 10.6%. [Section 62] The method according to item 61, wherein the relative abundance of G2F is approximately 7.8%. [Section 63] The method according to any one of claims 50 to 62, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of G2F is between approximately 5% and approximately 21%. [Section 64] One or more N-linked glycans are located at residue Asn108(T7), between approximately 5% and 21%, between approximately 5% and 20.5%, between approximately 5% and 20%, between approximately 5% and 19.5%, between approximately 5% and 19%, between approximately 5.5% and 21%, between approximately 5.5% and 20.5%, and between approximately 5.5% and 20%. Between % and %, between approximately 5.5% and 19.5%, between approximately 5.5% and 19%, between approximately 6% and 21%, between approximately 6% and 20.5%, between approximately 6% and 20%, between approximately 6% and 19.5%, between approximately 6% and 19%, between approximately 6.5% and 21%, between approximately 6.5% and 20.5%, between approximately 6.5% and 20 The method according to any one of claims 50 to 62, comprising a relative abundance of G2F between %, between approximately 6.5% and approximately 19.5%, between approximately 6.5% and approximately 19%, between approximately 7% and approximately 21%, between approximately 7% and approximately 20.5%, between approximately 7% and approximately 20%, between approximately 7% and approximately 19.5%, between approximately 7% and approximately 19%, between approximately 7.5% and approximately 21%, between approximately 7.5% and approximately 20.5%, between approximately 7.5% and approximately 20%, between approximately 7.5% and approximately 19.5%, between approximately 7.5% and approximately 19%, between approximately 8% and approximately 21%, between approximately 8% and approximately 20.5%, between approximately 8% and approximately 20%, between approximately 8% and approximately 19.5%, or between approximately 8% and approximately 19%. [Section 65] The method according to any one of claims 50 to 62, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of G2F is between about 8.2% and about 14.1%, between about 8.0% and about 18.6%, or between about 8.2% and about 14.1%. [Section 66] The method described in item 65, wherein the relative abundance of G2F is approximately 12.4%. [Section 67] The method according to any one of claims 63 to 66, wherein G2F further contains a galactose-α-1,3-galactose moiety (G2F-Gal), and G2F-Gal accounts for a relative abundance of approximately 1.4% or less. [Section 68] The method according to item 67, wherein G2F-Gal accounts for a relative abundance between approximately 1.0% and approximately 1.4%. [Section 69] The method according to item 67, wherein G2F-Gal accounts for a relative abundance between approximately 0.4% and approximately 0.9%. [Section 70] The method according to any one of claims 50 to 69, wherein one or more N-linked glycans include a relative abundance of S1G2F located at residue Asn76(T5) and being between approximately 29% and approximately 38%. [Section 71] One or more N-linked glycans are located at residue Asn76(T5), between approximately 29% and 38%, between approximately 29% and 37.5%, between approximately 29% and 37%, between approximately 29% and 36.5%, between approximately 29.5% and 38%, between approximately 29.5% and 37.5%, between approximately 29.5% and 37%, between approximately 29.5% and 36.5%, and approximately 3 The method according to any one of items 50 to 69, including the relative abundance of S1G2F between 0% and approximately 38%, between approximately 30% and approximately 37.5%, between approximately 30% and approximately 37%, between approximately 30% and approximately 36.5%, between approximately 31.5% and approximately 38%, between approximately 31.5% and approximately 37.5%, between approximately 31.5% and approximately 37%, and between approximately 31.5% and approximately 36.5%. [Section 72] The method according to any one of claims 50 to 69, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S1G2F is between approximately 31.6% and approximately 35.1%, between approximately 31.3% and approximately 36.5%, or between approximately 31.3% and approximately 36.5%. [Section 73] The method described in item 72, wherein the relative abundance of S1G2F is approximately 33.3%. [Section 74] The method according to any one of claims 50 to 73, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S1G2F is between 33% and about 45%. [Section 75] One or more N-linked glycans are located at residue Asn108(T7), between approximately 33% and 45%, between approximately 33% and 44.5%, between approximately 33% and 44%, between approximately 33% and 43.5%, between approximately 33% and 43%, between approximately 33% and 42.5%, between approximately 33.5% and 45%, between approximately 33.5% and 44.5%, between approximately 33.5% and 44%, between approximately 33.5% and 43.5%, between approximately 33.5% and 42.5%, and approximately 34 The method according to any one of items 50 to 73, including the relative abundance of S1G2F between % and approximately 45%, between approximately 34% and approximately 44.5%, between approximately 34% and approximately 44%, between approximately 34% and approximately 43.5%, between approximately 34% and approximately 42.5%, between approximately 34.5% and approximately 45%, between approximately 34.5% and approximately 44.5%, between approximately 34.5% and approximately 44%, between approximately 34.5% and approximately 43.5%, between approximately 34.5% and approximately 43%, or between approximately 34.5% and approximately 42.5%. [Section 76] The method according to any one of claims 50 to 73, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S1G2F is between approximately 34.2% and approximately 37.7%, between approximately 35.5% and approximately 42.3%, or between approximately 34.2% and approximately 42.3%. [Section 77] The method described in item 76, wherein the relative abundance of S1G2F is approximately 36.5%. [Section 78] The method according to any one of claims 74 to 77, wherein S1G2F further contains a galactose-α-1,3-galactose moiety (S1G2F-Gal), and S1G2F-Gal accounts for a relative abundance of approximately 4.7% or less. [Section 79] The method according to item 78, wherein S1G2F-Gal accounts for a relative abundance between approximately 2.3% and approximately 4.7%. [Section 80] The method according to item 78, wherein S1G2F-Gal accounts for a relative abundance between approximately 1.4% and approximately 1.8%. [Section 81] The method according to any one of claims 50 to 80, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S2G2F is between approximately 13% and approximately 25%. [Section 82] One or more N-linked glycans are located at residue Asn76(T5), between approximately 13% and 25%, between approximately 13% and 24.5%, between approximately 13% and 24%, between approximately 13% and 23.5%, between approximately 13% and 23%, between approximately 13.5% and 25%, between approximately 13.5% and 24.5%, between approximately 13.5% and 24%, between approximately 13.5% and 23.5%, between approximately 13.5% and 23%, between approximately 14% and 25%, between approximately 14% and 24.5%, between approximately 14% and 24%, between approximately 14% and 23.5%, between approximately 14% and 23%, between approximately 14.5% and 2 The method according to any one of items 50 to 80, including the relative abundance of S2G2F between 5%, between approximately 14.5% and approximately 24.5%, between approximately 14.5% and approximately 24%, between approximately 14.5% and approximately 23.5%, between approximately 14.5% and approximately 23%, between approximately 15% and approximately 25%, between approximately 15% and approximately 24.5%, between approximately 15% and approximately 24%, between approximately 15% and approximately 23.5%, between approximately 15% and approximately 23%, between approximately 15.5% and approximately 25%, between approximately 15.5% and approximately 24.5%, between approximately 15.5% and approximately 24%, between approximately 15.5% and approximately 23.5%, or between approximately 15.5% and approximately 23%. [Section 83] The method according to any one of claims 50 to 80, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S2G2F is between approximately 18.1% and approximately 22.9%, between approximately 15.4% and approximately 20%, and between approximately 15.4% and approximately 22.9%. [Section 84] The method described in item 83, wherein the relative abundance of S2G2F is approximately 18.5%. [Section 85] The method according to any one of claims 50 to 84, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S2G2F is between approximately 18% and approximately 36%. [Section 86] One or more N-linked glycans are located at residue Asn108(T7), between approximately 18% and approximately 36%, between approximately 18% and approximately 35.5%, between approximately 18% and approximately 35%, between approximately 18% and approximately 34.5%, between approximately 18% and approximately 34%, between approximately 18% and approximately 33.5%, between approximately 18.5% and approximately 36%, between approximately 18.5% and approximately 35.5%, and approximately 18 Between 0.5% and approximately 35%, between approximately 18.5% and approximately 34.5%, between approximately 18.5% and approximately 34%, between approximately 18.5% and approximately 33.5%, between approximately 19% and approximately 36%, between approximately 19% and approximately 35.5%, between approximately 19% and approximately 35%, between approximately 19% and approximately 34.5%, between approximately 19% and approximately 34%, between approximately 19% and approximately 33.5%, between approximately 19.5% and approximately 3 Between 6%, between approximately 19.5% and approximately 35.5%, between approximately 19.5% and approximately 35%, between approximately 19.5% and approximately 34.5%, between approximately 19.5% and approximately 34%, between approximately 19.5% and approximately 33.5%, between approximately 20% and approximately 36%, between approximately 20% and approximately 35.5%, between approximately 20% and approximately 35%, between approximately 20% and approximately 34.5%, between approximately 20% and approximately 34% The method according to any one of items 50 to 84, including the relative abundance of S2G2F between approximately 20% and approximately 33.5%, between approximately 20.5% and approximately 36%, between approximately 20.5% and approximately 35.5%, between approximately 20.5% and approximately 34.5%, between approximately 20.5% and approximately 34%, or between approximately 20.5% and approximately 33.5%. [Section 87] The method according to any one of claims 50 to 84, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S2G2F is between approximately 23.2% and approximately 33.8%, between approximately 20.8% and approximately 32.6%, or between approximately 20.8% and approximately 33.8%. [Section 88] The method described in item 87, wherein the relative abundance of S2G2F is approximately 23.5%. [Section 89] The method according to any one of claims 50 to 88, wherein one or more N-linked glycans include a relative abundance of S1G3F located at residue Asn76(T5) and being between approximately 2% and approximately 8%. [Section 90] Multiple N-linked glycans are located at residue Asn76(T5), between approximately 2% and 8%, between approximately 2% and 7.5%, between approximately 2% and 7%, between approximately 2% and 6.5%, between approximately 2% and 6%, between approximately 2% and 5.5%, between approximately 2.5% and 8%, between approximately 2.5% and 7.5%, between approximately 2.5% and 7%, between approximately 2.5% and 6.5%, between approximately 2.5% and 6%, between approximately 2.5% and 5.5%, between approximately 3% and 8%, between approximately 3% and 7.5%, between approximately 3% and 7%, between approximately 3% and The method according to any one of claims 50 to 88, comprising a relative abundance of S1G3F between 6.5%, between approximately 3% and approximately 6%, between approximately 3% and approximately 5.5%, between approximately 3.5% and approximately 8%, between approximately 3.5% and approximately 7.5%, between approximately 3.5% and approximately 7%, between approximately 3.5% and approximately 6.5%, between approximately 3.5% and approximately 6%, between approximately 3.5% and approximately 5.5%, between approximately 4% and approximately 8%, between approximately 4% and approximately 7.5%, between approximately 4% and approximately 7%, between approximately 4% and approximately 6.5%, between approximately 4% and approximately 6%, or between approximately 4% and approximately 5.5%. [Section 91] The method according to any one of claims 50 to 88, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S1G3F is between approximately 4.4% and approximately 5.6%, or between approximately 4.0% and approximately 5.5%. [Section 92] The method described in item 91, wherein the relative abundance of S1G3F is approximately 4.6%. [Section 93] The method according to any one of claims 50 to 92, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S1G3F is between approximately 0.5% and approximately 4%. [Section 94] The method according to any one of claims 50 to 92, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S1G3F is between about 0.5% and about 4%, between about 0.5% and about 3.5%, between about 0.5% and about 3%, between about 0.5% and about 2.5%, between about 1% and about 4%, between about 1% and about 3.5%, between about 1% and about 3%, or between about 1% and about 2.5%. [Section 95] The method according to any one of claims 50 to 92, wherein one or more N-linked glycans are located at residue Asn108(T7) and the relative abundance of S1G3F is between about 1.4% and about 2.2%, between about 1.1% and about 1.9%, or between about 1.1% and about 2.2%. [Section 96] The method described in item 95, wherein the relative abundance of S1G3F is approximately 1.8%. [Section 97] The method according to any one of claims 50 to 96, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S2G4F is between approximately 0.5% and approximately 4%. [Section 98] The method according to any one of claims 50 to 96, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S2G4F is between about 0.5% and about 4%, between about 0.5% and about 3.5%, between about 0.5% and about 3%, between about 0.5% and about 2.5%, between about 1% and about 4%, between about 1% and about 3.5%, between about 1% and about 3%, or between about 1% and about 2.5%. [Section 99] The method according to any one of claims 50 to 96, wherein one or more N-linked glycans are located at residue Asn76(T5) and the relative abundance of S2G4F is between about 1.9% and about 2.4%, between about 1.4% and about 2.1%, or between about 1.4% and about 2.4%. [Section 100] The method described in item 99, wherein the relative abundance of S2G4F is approximately 2.3%. [Section 101] The method according to any one of claims 32 to 100, wherein one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8 to about 11. [Section 102] The method according to any one of claims 32 to 100, wherein one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8.3 to about 11, about 9.5 to about 10.1, or about 8.3 to about 10.1. [Section 103] The method described in item 102, wherein the molar ratio of NANA is approximately 10.0. [Section 104] The method according to any one of claims 32 to 103, wherein one or more N-linked glycans are sialic acid and have a molar ratio of NGNAs of about 0.1 to about 2.0. [Section 105] The method according to any one of claims 32 to 103, wherein one or more N-linked glycans are sialic acid and have a molar ratio of NGNAs of 0.90 to about 1.20, or about 0.3 to about 1.2. [Section 106] The method according to item 105, wherein the molar ratio of NGNA is approximately 1.0. [Section 107] The method according to any one of claims 32 to 106, wherein the glycosylation profile is analyzed via an N-linked carbohydrate profile release method. [Section 108] The method according to item 107, wherein the glycosylation profile comprises one or more non-sialylated glycans (domain I), monosialylated glycans (domain II), disialylated glycans (domain III), and / or trisialylated and tetrasialylated glycans (domains IV+V). [Section 109] The method according to item 108, wherein the non-sialylated glycan (domain I) has a molar ratio of about 28 to about 37, about 29 to about 32, about 28 to about 32, or about 29 to about 37. [Section 110] The method according to item 109, wherein the non-sialylated glycan (domain I) has a molar ratio of about 31. [Section 111] The method according to any one of claims 108 to 110, wherein the monosially glycan (domain II) has molar ratios of about 26 to about 28, about 27 to about 33, about 26 to about 33, and about 27 to about 28. [Section 112] The method according to item 111, wherein the monosialylated glycan (domain II) has a molar ratio of approximately 27. [Section 113] The method according to any one of claims 108 to 112, wherein the diciallylated glycan (domain III) has a molar ratio of about 27 to about 28, about 22 to about 31, about 27 to about 31, or about 22 to about 28. [Section 114] The method according to item 113, wherein the diciallylated glycan (domain III) has a molar ratio of approximately 27.4. [Section 115] The method according to any one of claims 108 to 114, wherein the trisiallylated and tetrasiallylated glycans (domains IV and V) have a molar ratio of about 13 to about 16, about 8 to about 16, or about 8 to about 16. [Section 116] The method according to item 115, wherein the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 14.6. [Section 117] The method according to any one of claims 31 to 116, wherein the glycosylation profile comprises one or more O-linked glycans. [Section 118] The method according to any one of claims 31 to 117, wherein the glycosylation profile does not contain multiple galactose-α-1,3-galactose (α-gal) bonds. [Section 119] The method according to any one of claims 35 to 118, wherein CTLA4 contains C-terminal lysine. [Section 120] The method according to item 119, wherein C-terminal lysine accounts for a relative abundance of approximately 20% to approximately 25%. [Section 121] The method according to item 119, wherein C-terminal lysine accounts for a relative abundance of approximately 3% to approximately 10%. [Section 122] The method according to item 117, wherein the O-linked glycan is located at residues Ser129, Ser130, Ser136, and / or Ser139. [Section 123] The method according to any one of claims 1 to 122, wherein the bioreactor comprises a feed medium containing glucose or galactose. [Section 124] The method according to any one of items 1 to 123, wherein the cells are mammalian cells. [Section 125] The method according to item 124, wherein the cells are Chinese hamster ovary (CHO) cells. [Section 126] The method according to item 125, wherein the cells are CHO-K1 cells, CHO-DXB11 cells, or CHO-DG44 cells. [Section 127] A method for analyzing the branched glycans of a CTLA4-Fc fusion protein, comprising measuring one or more N-linked glycans bound to one or more asparagine residues in the CTLA4 protein, wherein one of the branched glycans is G2F. [Section 128] The method according to item 127, wherein the branched glycan is selected from the group consisting of G0F, G1F, G2F, S1G1F, S1G2F, and / or S2G2F. [Section 129] The method according to section 127 or 128, wherein branched glycans are measured via ultra-high performance liquid chromatography (UPLC-FLR) with fluorescence detection. [Section 130] The method according to any one of claims 127 to 129, wherein branched glycans are measured via the HILIC N-linked glycan profiling method. [Section 131] The method according to any one of sections 127 to 129, wherein the Fc domain of the CTLA4-Fc fusion protein is cleaved before measurement. [Section 132] A method for analyzing the branched glycans of CTLA4-Fc fusion proteins, comprising performing isoelectric focusing of the CTLA4-Fc fusion proteins. [Section 133] The method according to item 132, wherein isoelectric focusing is imaged capillary isoelectric focusing. [Section 134] The method according to section 132 or 133, wherein the CTLA4-Fc fusion proteins subjected to isoelectric focusing form groups I, II, and III. [Section 135] The method according to paragraph 134, wherein Group I accounts for 4% or less of the total, Group II accounts for 87% or more of the total, and / or Group III accounts for 10% or less of the total. [Section 136] Cells produced by the method described in any one of items 1 to 131. [Section 137] A mammalian cell, as described in item 136. [Section 138] Chinese hamster ovary (CHO) cells, as described in item 137. [Section 139] The cells described in item 138, which are CHO-K1 cells, CHO-DXB11 cells, or CHO-DG44 cells. [Brief explanation of the drawing]

[0031] Figure 1A shows the overall structure of a typical N-linked glycan. GlcNAc (square) is N-acetylglucosamine. Man (circle) is mannose. Gal (black triangle) is galactose. NeuAc or Neu5Ac (star) is N-acetylneuraminic acid. Fuc (bullet) is fucose. The core structure is shown within the gray frame. Figures 1B and 1C show exemplary structures of G0F (mannose-3-N-acetylglucosamine-4-fucose), G1F (mannose-3-N-acetylglucosamine-4-galactose-1-fucose), G2F (mannose-3-N-acetylglucosamine-4-galactose-2-fucose), S1G1F (monosialylated mannose-3-N-acetylglucosamine-4-galactose-1-fucose), S1G2F (monosialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose), S1G3F (monosialylated mannose-3-N-acetylglucosamine-4-galactose-3-fucose), and S2G2F (dicialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose). Figure 1D shows the specific N-linked glycosylation sites on the abatacept molecule (T5 on Asn76, T7 on Asn108, and T14 on Asn207). The protease and amino acid sequences are also shown.

[0032] Figure 2A is a diagram of mean viable cell density (VCD) profiles after treatment under various conditions in a 5L bioreactor. The conditions tested included: a CO2 setpoint of 22.4%, first supply after 96 hours, initial pH of 6.9, and 0.15 × 10⁻⁶. 6 Initial viable cell density of cells / mL, 0.45 × 10⁶ 6 The initial viable cell density is cells / mL, the overall temperature shift is -1°C (three temperature settings: 36°C, 33°C, and 31°C), the timing of the pH shift is 72 hours, the temperature shift at the second setting (37°C, 33°C, and 32°C), and the temperature shift at the third setting (37°C, 34°C, and 31°C).

[0033] Figure 2B shows the average peak VCD (10) after experiments with various factors. 6 (units / mL), Dunnett test p-value for peak VCD, mean doubling time from 0 to 5 days (hours), Dunnett test p-value for doubling time, mean IVCD from 0 to 14 days (10 9 The table shows the values ​​(individuals / day / mL), Dunnett's test p-value for IVCD, and VCD profile values: High CO2 represents a CO2 setpoint of 22.4%. Slow first supply is the first supply at 96 hours. Low first pH is the derived pH of 6.9. Low first VCD is 0.15 × 10⁻⁶. 6 The induction density is 10¹ / mL. The first high VCD is 0.45 × 10¹⁶ 6 The induction density is 1 / mL. The low overall temperature is a temperature shift profile using three setpoints: 36°C as the first temperature setpoint, 33°C as the second temperature setpoint, and 31°C as the last temperature setpoint. Low temperature shift 1 is a temperature shift profile using three setpoints: 37°C as the first temperature setpoint, 33°C as the second temperature setpoint, and 32°C as the last temperature setpoint. Low temperature shift 2 is a temperature shift profile using three setpoints: 37°C as the first temperature setpoint, 34°C as the second temperature setpoint, and 31°C as the last temperature setpoint.

[0034] Figure 3 shows the average production bioreactor survival profile for each treatment group. The conditions tested included: a CO2 setpoint of 22.4%, first supply after 96 hours, initial pH of 6.9, and 0.15 × 10⁻⁶. 6 Initial viable cell density of cells / mL, 0.45 × 10⁶ 6 The initial viable cell density is cells / mL, the overall temperature shift is -1°C, the pH shift timing is 72 hours, the temperature shift at the second setpoint (37°C, 33°C, and 32°C), and the temperature shift at the third setpoint (37°C, 34°C, and 31°C).

[0035] Figures 4A–4H show various glycosylation data collected on day 14 of bioreactor production operation for abatacept. Figure 4A shows a statistical comparison of sialic acid and N-linked domains (domains I, I, III, and IV+V) on day 14. Figure 4B shows a comparison of titer and specific productivity between test groups. High CO2 represents a CO2 setpoint of 22.4%. Slow first feed is the first feed at 96 hours. Low first pH is the derived pH of 6.9. Low first VCD is 0.15 × 10⁻⁶. 6 The induction density is 10¹ / mL. The first high VCD is 0.45 × 10¹⁶ 6 The induction density is 1 / mL. The low overall temperature is a temperature shift profile using three setpoints: 36°C as the first temperature setpoint, 33°C as the second temperature setpoint, and 31°C as the last temperature setpoint. Low temperature shift 1 is a temperature shift profile using three setpoints (37°C, 33°C, and 32°C). Low temperature shift 2 is a temperature shift profile using three setpoints (37°C, 34°C, and 31°C). Figure 4C shows the analysis of G0F glycans present at sites T5 and T7 on day 14 of 5L bioreactor production operation using each test condition. Figure 4D shows the analysis of G2F glycans present at sites T5 and T7 on day 14 of 5L bioreactor production operation using each test condition. Figure 4E shows the analysis of S1G2F glycans present at sites T5 and T7 on day 14 of 5L bioreactor production operation using each test condition. Figure 4F shows the analysis of S2G2F glycans present at sites T5 and T7 on day 14 of the 5L bioreactor production run using each test condition. Figure 4G shows the analysis of S1G3F glycans present at sites T5 and T7 on day 14 of the 5L bioreactor production run using each test condition. Figure 4H shows the analysis of S2G4F glycans present at sites T5 and T7 on day 14 of the 5L bioreactor production run using each test condition.

[0036] Figure 5 shows the data collected on day 16 for N-linked glycosylation (domains I, II, III, and IV+V), high molecular weight contaminants, host cell proteins, and ribosomal DNA (rDNA) contamination. The Dunnett test p-values ​​for N-linked glycosylation, HMW, HCP, and rDNA prior to day 16 are also presented.

[0037] Figure 6A shows a chart of process parameters tested for a 5L bioreactor. Figures 6B–6C show the glycosylation profiles of proteins produced during operation of several sample bioreactors, along with contaminants such as HMW, HCP, and DNA.

[0038] Figure 7 shows an overview of the process attributes affected by modifications to process parameters.

[0039] Figure 8A shows the relative abundances of various glycoforms present on the T5 glycosylation site during the process (J) used in this disclosure and an alternative process (F). Figure 8B shows a representative mass spectrometry showing the identification of peaks representing these glycoforms. The alternative process refers to a controlled process that does not use any of the conditions disclosed herein.

[0040] Figure 9A shows the relative abundances of various glycoforms present on the T5 glycosylation site during the process (J) used in this disclosure and an alternative process (F). Figure 9B shows a representative mass spectrometry showing the identification of peaks representing these glycoforms.

[0041] Figures 10A–10F show a statistical comparison of various sialylated N-linked glycans between the process used in this disclosure (J) and an alternative process (F). Glycan analysis includes domain I (Figure 10A), domain II (Figure 10B), domain III (Figure 10C), domain IV+V (Figure 10D), NANA (Figure 10E), and NGNA (Figure 10F). [Modes for carrying out the invention]

[0042] This disclosure relates to methods for improving the yield of a cell-mediated protein while maintaining a desired protein property, such as the glycosylation pattern. In some embodiments, the method for improving the yield includes culturing cells in a bioreactor for a protein induction phase under preferred conditions, including but not limited to adjusting to multiple temperatures, setting a pH, using a specific live cell density, setting a CO2 concentrate, or any combination thereof.

[0043] I. Definition As used herein, the term "and / or" is construed to mean that each of the two specified features or components is specifically disclosed, with or without the other. Accordingly, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0044] Whenever an aspect is described herein using the term "contains," it is understood that other aspects similar in other respects are also provided, which are described using "consisting of" and / or "essentially consisting of."

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry And Molecular Biology*, Revised, 2000, Oxford University Press provide general dictionaries of many of the terms used herein.

[0046] Units, prefixes, and symbols are expressed in the form recognized by the International System of Units (SI). Numerical ranges include the numerical values ​​that define the range. The headings presented herein are not intended to limit the various aspects of this disclosure that can be obtained by referring to the entire specification. Thus, the terms defined immediately below are more fully defined by referring to the entire specification.

[0047] The use of alternative options (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternative options. Where used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of the mentioned or enumerated components.

[0048] The terms “approximately” or “essentially including” refer to a value or composition that falls within the tolerance range of a particular value or composition as determined by those skilled in the art, and this is considered to depend in part on how that value or composition is measured or determined, i.e., on the limits of the measuring system. For example, “approximately” or “essentially including” may mean within one standard deviation or within one standard deviation by convention in the art. Alternatively, “approximately” or “essentially including” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, this term may mean a value of up to one order of magnitude, or up to five times. If a particular value or composition is presented in the application and claims, unless otherwise stated, the meaning of “approximately” or “essentially including” should be assumed to be within the tolerance range of that particular value or composition.

[0049] Where used herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the range mentioned, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise indicated.

[0050] As used herein, the term “CTLA4 extracellular domain” refers to a protein domain that binds to B7-1(CD80) and / or B7-2(CD86) and contains all or part of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the CTLA4 extracellular domain may include a polypeptide having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The CTLA4 extracellular domain is represented by the following sequence: Sequence ID 1: MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD

[0051] As used herein, the terms “CTLA4-Ig” or “CTLA4-Ig molecule” or “CTLA4Ig molecule” or “CTLA4-Ig protein” or “CTLA4Ig protein” or “CTLA4-Fc” are used interchangeably and refer to a protein molecule comprising a CTLA4-Ig polypeptide having at least a CTLA4 extracellular domain and an immunoglobulin constant region or a portion thereof. In some embodiments, for example, the CTLA4-Ig polypeptide comprises at least the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the CTLA4 extracellular domain and the immunoglobulin constant region or a portion thereof may be wild-type, mutant, or modified. A mutant CTLA4-Ig polypeptide is a CTLA4-Ig polypeptide comprising a mutant CTLA4 extracellular domain. A mutant CTLA4Ig molecule comprises at least a mutant CTLA4-Ig polypeptide. In some embodiments, the CTLA4 extracellular domain and the immunoglobulin constant region or a portion thereof may be mammalian, including human or mouse. In some embodiments, the mutant CTLA4 extracellular domain may have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CTLA4 extracellular domain shown in any one or more of SEQ ID NOs: 2, 3, 4, 5, 6, 7, or 8. The polypeptide may further contain additional protein domains. The CTLA4-Ig molecule may refer to the monomer of the CTLA4-Ig polypeptide, or it may refer to the multimeric form of the polypeptide, e.g., dimers, tetramers, and hexamers (or other high molecular weight species). The CTLA4-Ig molecule may also bind to CD80 and / or CD86. Examples of CTLA4-Ig and fragments (e.g., abatacept) are shown in SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. In some embodiments, abatacept is a combination of sequence numbers 2, 3, 4, 5, 6, 7, and 8. Sequence ID 2 [CTLA4-Ig amino acid sequence] MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3 [Amino acids 25-383 of SEQ ID NO: 2] MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4 [Amino acids 26-383 of SEQ ID NO: 2] AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 5 [Amino acids 27-383 of SEQ ID NO: 2] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 6 [Amino acids 25-382 of SEQ ID NO: 2] MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 7 [Amino acids 26-382 of SEQ ID NO: 2] AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 8 [Amino acids 27-382 of SEQ ID NO: 2] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0052] As used herein, the term “soluble CTLA4” means a molecule that can circulate in vivo, or CTLA4 that is not bound to the cell membrane. For example, soluble CTLA4 may include CTLA4-Ig, which contains the extracellular region of CTLA4 bound to Ig.

[0053] As used herein, the term “dimer” refers to a CTLA4-Ig protein or CTLA4-Ig molecule composed of two CTLA4-Ig polypeptides or monomers linked together or joined into a single molecule. The linkage between monomers or dimers may be non-covalent, covalent (e.g., one or more disulfide bonds), or both. A CTLA4-Ig protein or CTLA4-Ig molecule composed of two identical monomers is a homodimer. CTLA4-Ig homodimers also include molecules containing two monomers with slightly different sequences. Homodimers include dimers in which the joined monomers have substantially the same sequence. The monomers constituting a homodimer have a fairly high degree of structural homology. For example, sequence differences may be due to N-terminal processing modifications of the monomers.

[0054] As used herein, the terms “glutamic acid” and “glutamic acid” are to be used interchangeably.

[0055] As used herein, "T5," "T7," and "T15" refer to specific glycosylation sites present on the abatacept molecule. These labels correspond to asparagine 76, asparagine 108, and asparagine 207, respectively, and to the residues (in bold) in Sequence ID No. 5. The peptide sequences of T5, T7, and T14 are shown in Figure 1C. The relative abundances of the major glycoforms were calculated from the extracted ion chromatograms of the glycopeptides.

[0056] The N-linked glycosylation sites have been identified as Asn76, Asn108 (CTLA4 region), and Asn207 (Fc region) for SEQ ID NO: 5 by LC-MS trypsin peptide mapping using mass spectrometry detection. Five-letter codes, e.g., P2100, P2120, P2121, P3131, and P4142, are used to define the structural classes of the carbohydrates. In this labeling scheme, the first letter (P) of the code defines the released carbohydrate as an N-linked structure containing a trimannosyl core structure. The second letter represents the number of N-acetylglucosamine (GlcNAc) units bound to the core. The third letter (0 or 1) indicates whether or not there is fucose (Fuc) bound to the first GlcNAc in the core. The fourth letter represents the number of galactose (Gal) sugars bound to the core. The fifth letter represents the number of SA (N-acetylneuraminic acid or N-glycolylneuraminic acid) molecules bound to the carbohydrate. P2100 can also be written as G0F. P2110 can also be written as G1F. P2120 can also be written as G2F. P2121 can also be written as S1G2F. P2122 can also be written as S2G2F. P3131 can also be written as S1G3F. P4142 can also be written as S2G4F. Representative diagrams of these glycans can be seen in Figures 1A and 1B.

[0057] As used herein, “Domain I” refers to non-sialylated glycans (such as G0F, G1F, and G2F), “Domain II” refers to monosialylated glycans (such as S1G1F and S1G2F), “Domain III” refers to diciallylated glycans (such as S2G2F), and “Domain IV” and “Domain V” refer to trisialylated and tetrasialylated glycans.

[0058] As used herein, the term “purified” means a composition containing CTLA4-Ig molecules or a selected population of CTLA4-Ig molecules that have been removed from their natural environment (e.g., isolated) and are free from at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of other components, such as naturally associated cell material or culture medium. “Purified” may also mean a composition containing CTLA4-Ig molecules or a selected population of CTLA4-Ig molecules that have been removed from their natural environment and are free from at least 60%, 65%, 70%, 75%, 80%, or 85% of other components, such as naturally bound cell material or culture medium. For example, with respect to recombinantly produced CTLA4-Ig protein molecules, the term “purified” may also refer to a composition containing CTLA4-Ig protein molecules that have been removed from the production environment such that the protein molecules do not contain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of protein molecules that are not the target SEQ ID NO: 2 polypeptide or the mutant polypeptide of SEQ ID NO: 2. “Purified” does not exclude mixtures of CTLA4-Ig molecules (such as dimers) and other CTLA4-Ig molecules (such as tetramers). “Purified” does not exclude pharmaceutically acceptable excipients or carriers combined with CTLA4-Ig molecules from which the CTLA4-Ig molecules have been removed from their original environment.

[0059] As used herein, the term “large-scale process” is used interchangeably with the term “industrial-scale process.” The term “culture vessel” is used interchangeably with “bioreactor,” “reactor,” and “tank.” Bioreactors used on an industrial scale may be at least 2,000 L, at least 5,000 L, at least 10,000 L, at least 15,000 L, at least 20,000 L, at least 25,000 L, or any size suitable for the large-scale production required to produce industrial supplies.

[0060] A "liquid culture" refers to cells that grow on a support (e.g., bacteria, plants, insects, yeast, animal cells) or cells that float and grow in a liquid nutrient medium.

[0061] A "seed culture" refers to a cell culture that has been cultured for inoculation into a larger volume of culture medium. Seed cultures can be used to inoculate larger volumes of culture medium in order to increase the number of cells that grow in the culture (for example, cells grown in a suspension).

[0062] As used herein, the terms “culture medium,” “cell culture medium,” “supply medium,” and “fermentation medium” refer to nutrient solutions used for the growth and maintenance of cells, particularly mammalian cells. These solutions typically provide, but are not limited to, at least one component from one or more of the following categories: (1) an energy source, usually in the form of carbohydrates such as glucose; (2) all essential amino acids, usually the basic set of 20 amino acids plus cysteine; (3) vitamins and / or other organic compounds required in low concentrations; (4) free fatty acids or lipids, e.g., linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements required in very low concentrations, usually in the range of micromolar concentrations. Nutrient solutions can be effectively supplemented with one or more components from any of the following categories: (1) hormones and other growth factors, e.g., serum, insulin, transferrin, and epidermal growth factor; (2) salts, e.g., magnesium, calcium, and phosphates; (3) buffers, e.g., HEPES; (4) nucleosides and bases, e.g., adenosine, thymidine, and hypoxanthine; (5) protein and tissue hydrolysates, e.g., purified gelatin, peptone or peptone mixtures obtained from plant materials or animal by-products; (6) antibiotics, e.g., gentamicin; (7) cytoprotective agents, e.g., pluronic polyols; and (8) galactose. Commercially available culture media, e.g., Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), (Sigma)) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979) or Barnes et al., Anal. Biochem. 102:255 (1980) can be used as a culture medium for host cells. Any other necessary nutritional supplements may also be included in suitable concentrations.

[0063] As used herein, “culture” refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culture conditions that may be defined include temperature, gas mixture, time, and medium formulation.

[0064] As used herein, “increase” means culturing one or more cells in vitro for the purpose of obtaining a larger number of cells in a culture.

[0065] As used herein, “temperature setpoint” refers to the temperature setting of a bioreactor or other upstream processing vessel used to grow cells and / or produce protein products. The temperature setpoint may be determined at the start of cell culture and may also be referred to as the “initial temperature setpoint.” Subsequent changes in temperature during cell culture after the initial temperature setpoint may be referred to using an ordinal number, namely the second temperature setpoint, and thereafter the third temperature setpoint. The final temperature setpoint before downstream processing may also be referred to as the “final temperature setpoint.” In some cases, the process may include an initial temperature setpoint, a second temperature setpoint, and a third (and final) temperature setpoint.

[0066] As used herein, "CO2 setpoint" or "CO2 setpoint" refers to the concentration of CO2 present in the upstream production process. The CO2 setpoint is expressed as a percentage (%) of volume.

[0067] As used herein, “population” refers to a group of two or more molecules (“population of molecules”) or cells (“population of cells”) characterized by the presence or absence of one or more measurable or detectable properties. In a homogeneous population, molecules or cells within the population are characterized by the same or substantially the same properties (e.g., cells in a clonal cell line). In a heterogeneous population, molecules or cells within the population are characterized by the same or substantially the same at least one property, and cells or molecules may also exhibit properties that are not the same (e.g., a population of CTLA4-Ig molecules having substantially similar average sialic acid content but dissimilar mannose content).

[0068] As used herein, “high molecular weight aggregate” is used interchangeably with “high molecular weight species” or “HMW” to refer to a CTLA4-Ig molecule containing at least three CTLA4-Ig monomers. For example, a high molecular weight aggregate may be a tetramer, pentamer, or hexamer.

[0069] As used herein, “protein A” refers to a protein of approximately 42 kDa that binds very strongly to the Fc portion of immunoglobulins, and its use in antibody purification is well known in the art. Protein A has been widely used in the art for purification (Boyle et al., 1993; Hou et al. 1991). When applied to protein A, the term “persistent” or “rPA” refers to any persistent protein A present in a mixture for its use in the purification of the protein of interest or further upstream in the manufacturing process of antibodies.

[0070] "Percent yield (%)" is the value obtained by dividing the actual yield by the theoretical yield and multiplying the result by 100. Actual yield can be obtained in grams or moles (e.g., molar yield). Theoretical yield can be obtained as an ideal yield or a mathematically calculated yield.

[0071] As used herein, "glycosylation content" refers to the amount of N-linked or O-linked sugar residues covalently attached to a protein molecule, such as a glycoprotein like the CTLA4-Ig molecule.

[0072] As used herein, “glycosylation” refers to the addition of a complex oligosaccharide structure to a protein at a specific site within a polypeptide chain. Protein glycosylation and subsequent processing of the added carbohydrates can affect protein folding and structure, protein stability including protein half-life, and protein functional properties. Protein glycosylation can be divided into two classes, O-linked glycosylation and N-linked glycosylation, depending on the sequence situation in which the modification occurs. O-linked polysaccharides are linked to a hydroxyl group, usually to a hydroxyl group of either a serine or threonine residue. O-glycans are not added to all serine and threonine residues. O-linked oligosaccharides are usually monobranched or bibranched, meaning they have one or at most two branches, and contain one to four different types of sugar residues, which are added one at a time. N-linked polysaccharides are linked to the amide nitrogen of asparagine. Only asparagine, which is part of one of two tripeptide sequences, asparagine-X-serine or asparagine-X-threonine (where X is any amino acid other than proline), is a target for glycosylation. N-linked oligosaccharides can have one to four branches and are referred to as monobranched, bibranched, tribranched, or tetrabranched. The structures of the sugar residues found in N-linked and O-linked oligosaccharide structures are different. Despite this difference, the terminal residues on each branch of N-linked and O-linked polysaccharides are modified by sialic acid molecules, a modification called sialic acid capping. Sialic acid is the common name for a family of unique 9-carbon monosaccharides that can be linked with other oligosaccharides. Two family members are N-acetylneuraminic acid, abbreviated as Neu5Ac, NeuAc, or NANA, and N-glycolylneuraminic acid, abbreviated as Neu5Gc or NGNA. The most common form of sialic acid in humans is NANA. N-acetylneuraminic acid (NANA) is the major sialic acid species present in the CTLA4-Ig molecule. However, it should be noted that the CTLA4-Ig molecule also contains trace amounts of N-glycolylneuraminic acid (NGNA) at detectable levels.Furthermore, the methods described herein can be used to determine the number of moles of sialic acid for both NANA and NGNA, and thus the levels of both NANA and NGNA are determined and reported for the CTLA4-Ig molecule. N-linked oligosaccharides and O-linked oligosaccharides differ in the number of branches, and therefore offer different numbers of potential binding sites for sialic acid molecules. N-linked oligosaccharides can offer up to four sialic acid binding sites, while O-linked oligosaccharides can offer up to two.

[0073] As used herein, the term “molar ratio of sialic acid to protein” or “MR” is calculated and given as the number of moles of sialic acid molecules per mole of protein (CTLA4-Ig molecule) or dimer.

[0074] As used herein, the term "glycoprotein" refers to a protein modified by the addition of one or more carbohydrates, including the addition of one or more sugar residues.

[0075] As used herein, the term “sialylation” refers to the addition of a sialic acid residue to a protein, including glycoproteins.

[0076] As used herein, the term “glycoprotein isoform” refers to a molecule characterized by its carbohydrate and sialic acid content, determined by isoelectric focusing (IEF) gel electrophoresis or other preferred methods for identifying different proteins in a mixture by their molecular weight, charge, and / or other properties. For example, each individual band observed on an IEF gel represents a molecule having a specific isoelectric point (pI) and therefore having the same net overall charge. A glycoprotein isoform is an individual band observed on an IEF gel, where each band may be a collection of molecules having a specific pI.

[0077] As used herein, imaging capillary isoelectric focusing (iCIEF) refers to a method used for the separation of proteins by their isoelectric focus (pI). In this method, the sample is prepared to a final concentration of approximately 1 mg / mL with water, methylcellulose, amphoteric electrolytes, and a pI marker, and then injected into an imaging capillary isoelectric focusing system (iCIEF) by an autosampler. Electrophoresis separates the sample through a pH gradient within fluorocarbon (FC) coated capillaries based on the charge variations of the isoforms.

[0078] "Immune tolerance" refers to a state in which a person does not react to a specific antigen or group of antigens to which they normally would (for example, a state in which T cells become unable to react to an antigen).

[0079] "Efficacy" refers to a measure of the response as a function of ligand concentration. For example, the efficacy of an agonist is the ligand concentration (EC2) at which half of the maximum effect is produced. 50 It is quantified as (). While not limited to, the pharmacological definition of potency includes elements of affinity and efficacy, where efficacy is the ability of a drug to elicit a response once bound. Potency is related to affinity, but potency and affinity are different measures of pharmacokinetics.

[0080] As used herein, “pharmaceutically acceptable carrier” refers to a medium for a pharmacologically active drug. The carrier facilitates the delivery of the active drug to a target site without terminating the drug’s function. Non-limiting examples of preferred forms of carriers include solutions, creams, gels, gel emulsions, jellies, pastes, lotions, ointments, sprays, powders, solid mixtures, aerosols, emulsions (e.g., water-in-oil or oil-in-water), gel aqueous solutions, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical administration.

[0081] As used herein, the phrase “pharmaceutically acceptable composition” (or “pharmaceutical composition”) means a composition that is acceptable for pharmaceutical administration, such as for administration to humans. Such a composition may contain substances in which impurities do not exceed acceptable levels for pharmaceutical administration (including levels in which such impurities are not present), and may also contain, in addition to any active agent, pharmaceutically acceptable excipients, media, carriers, and other inactive components, for example, to formulate such a composition for the purpose of facilitating administration. For example, a pharmaceutically acceptable CTLA4-Ig composition may contain MCP-1 or DNA, but only if those substances are in acceptable levels for administration to humans.

[0082] The term "API" refers to the active pharmaceutical ingredient contained in a pharmaceutical composition. The term "API" includes the active pharmaceutical ingredient in solution and / or buffered form. "Formulation" refers to a pharmaceutical composition containing an API formulated for pharmaceutical administration. The following are exemplary APIs and / or formulations that can be assayed for the purposes of the assays contained in the examples and elsewhere in this specification, which may refer to APIs and / or formulations:

[0083] Exemplary formulations of the CTLA4Ig molecule include the following:

[0084] [Table 1]

[0085] As used herein, the term “inoculation” refers to the addition of cells to a culture medium to initiate a culture.

[0086] As used herein, the terms “induction,” “induction phase,” or “proliferation phase” of a cell culture refer to the initial seeding into the bioreactor at the start of the upstream cell culture, which includes a period of exponential cell proliferation (e.g., logarithmic phase) in which cells are primarily rapidly dividing. During this period, the rate of increase in the density of viable cells is higher than at any other point in time.

[0087] As used herein, the term “productive phase” of a cell culture refers to the period during which cell proliferation is in a steady state or maintained at a nearly constant level. The density of viable cells is approximately constant over a given period. Logarithmic cell proliferation has ended, and protein production is the primary activity of the productive phase. At this point, the culture medium is generally replenished to support continued protein production and achieve the desired glycoprotein product.

[0088] As used herein, the terms “expression” or “express” are used to refer to transcription and translation occurring within a cell. The expression level of a product gene in a host cell can be determined based on either the amount of corresponding mRNA present in the cell, or the amount of protein encoded by the product gene produced by the cell, or both.

[0089] As used herein, “N-linked glycan” refers to a protein modification in which a glycan is linked to a complex carbohydrate via a nitrogen bond. The glycan acceptors are selected asparagine residues of the polypeptide chain that have entered the periplasm or lumen of the ER, respectively. Oligosaccharide transferase, the central enzyme in the N-glycosylation pathway, catalyzes the formation of an N-glycosidic bond of the oligosaccharide to the side-chain amide of the asparagine residue identified by the consensus sequence NXS / T. All eukaryotic N-glycans share a common core sequence, Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr, and are classified into three types: (1) oligomannose, in which only Man residues elongate the core; (2) complex, in which a "branching" initiated by GlcNAc elongates the core; and (3) hybrid, in which Man elongates the Manα1-6 arms of the core, and one or two GlcNAc elongates the Manα1-3 arms.

[0090] As used herein, "N-linked glycosylation" refers to the linkage of an oligosaccharide to a nitrogen atom, typically N4 of an asparagine residue. N-glycosylation can occur on secretory or membrane-bound proteins, primarily in eukaryotes and archaea. A detailed review of the biosynthetic pathways and enzymes used to produce N-linked glycans (e.g., high-mannose oligosaccharides) is provided in Stanley et al., "N-Glycans" in Essentials of Glycobiology, Ed. Varki, Cummings, and Eskho, Cold Spring Harbor Press, 2009.

[0091] As used herein, the term “mass spectrometry” refers to a highly sensitive technique used to detect, identify, and quantify molecules based on their mass-to-charge ratio (m / z). An electric field is used to separate ions according to their mass-to-charge ratio (m / z), i.e., the ratio of mass to an integer of charge (z), and the ions pass along the central axis of parallel and equidistant poles or rods, such as a quadrupole containing four poles or rods. Two voltages are applied to each rod, one of which is a fixed DC and the other is a periodically changing AC superimposed with high frequencies. The magnitude of the applied electric field can be directed before detection so that only ions with a specific m / z ratio can pass through the quadrupole. Ions with all other m / z values ​​are deflected to trajectories such as colliding with the quadrupole rods and discharging, or being ejected from the field of the mass spectrometer and removed through the vacuum. A quadrupole is often referred to as an exclusive detector because only ions with a specific m / z are always stable within the quadrupole. Ions with stable orbitals are often described as having non-colliding, resonant, or stable orbitals.

[0092] Generally, in a triple quadrupole mass spectrometer experiment, the first quadrupole (Q1) is set to allow only ions of a specified m / z (precursor ion) of the expected chemical species in the sample to pass through. The second quadrupole (i.e., Q2 or collision cell) is used to fragment the ions passing through Q1. The third quadrupole (Q3) is set to pass only ions of a specified m / z (fragment ions) corresponding to the expected fragmentation product of the expected chemical species to the detector. In some embodiments, the sample is ionized in the mass spectrometer, producing one or more protonated or deprotonated molecular ions. In some embodiments, one or more protonated or deprotonated molecules have single, double, triple, or more charges. In some embodiments, the mass spectrometer is a triple quadrupole mass spectrometer. The resolution used for Q1 and Q3 is, in some embodiments, unit resolution. In other embodiments, the resolutions used for Q1 and Q3 are different. In other embodiments, the resolution used for Q1 is higher than the unit resolution of Q3.

[0093] As used herein, the term “fluorescent phore” refers to a fluorescent compound that can re-emit light upon photoexcitation. Fluorescent phores typically consist of several bonded aromatic groups or planar or cyclic molecules having several π bonds. Two commonly used fluorescent phores are 2-AB (2-aminobenzamide) and 2-AA (anthranilic acid or 2-aminobenzoic acid). Other fluorescent phores include PA (2-aminopyridine), AMAC (2-aminoacridone), ANDS (7-amino-1,3-naphthalenedisulfonic acid), ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid), APTS (9-aminopyrene-1,4,6-trisulfonic acid), and 3-(acetylamino)-6-aminoacridine.

[0094] As used in this disclosure, “glycan profile” should be understood as any defined set of quantitative results regarding glycans that can be used for comparison with reference values ​​or profiles obtained from other samples or groups of samples. For example, the glycan profile of a sample derived from a protein sample may differ significantly from the glycan profile of a sample from an alternative source. Glycan profiles can be used to predict or forecast the pharmacodynamic (PD) or pharmacokinetic (PK) therapeutic effect of a protein by comparing the profile to a reference or standard profile. Reference and sample glycan profiles can be generated by any analytical instrument capable of detecting glycans, such as mass spectrometry. One or more N-glycans may be galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (Glc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynononic acid (Kdn), fucose (Fuc), glucuronic acid (GlcA), iduronic acid (IdoA), galacturonic acid (GalA), mannuronic acid (ManA), or a combination thereof.

[0095] As used herein, the term “working solution” refers to a solution used in a particular method. Non-limiting examples of working solutions include buffer solutions.

[0096] As used herein, “reference substance” refers to a substance used as a standard in a particular method. For example, a reference substance can be used as a standard for comparing experimental samples.

[0097] The absence of a substance is assumed when no lower limit is specified for the range of quantities of such a substance.

[0098] Where used herein, temperature referred to in relation to cell culture refers to the temperature setting on the bioreactor temperature control device. Naturally, the temperature of the liquid culture itself is taken as the temperature set on the bioreactor temperature control device. When temperature refers to cell cultures maintained on shelves within an incubator, temperature refers to the temperature of the incubator shelf.

[0099] II. Methods to improve yield This disclosure provides a method for improving the yield of proteins by cells, comprising culturing cells in a bioreactor for a protein induction phase under suitable conditions, wherein suitable conditions include, but are not limited to, adjustments of an initial temperature setpoint, a second temperature setpoint, a final temperature setpoint, a supply time, an initial pH, a pH shift, a CO2 concentration, an initial cell density, or any combination thereof.

[0100] The method disclosed herein is useful for setting reactor conditions, which are reactor conditions that improve protein yield. In some embodiments, adjusting the temperature settings, for example, to the first, second, and last temperature setting values, such as 36°C, 33°C, and 31°C, improves the protein yield by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, or at least about 400% compared to a method without suitable conditions. In some embodiments, this condition improves protein yield by at least 150%. In some embodiments, this condition improves protein yield by at least 160%. In some embodiments, this condition improves protein yield by at least 170%. In some embodiments, this condition improves protein yield by at least 180%. In some embodiments, this condition improves protein yield by at least 190%. In some embodiments, this condition improves protein yield by at least 200%. In some embodiments, this condition improves protein yield by at least 210%. In some embodiments, this condition improves protein yield by at least 220%. In some embodiments, this condition improves protein yield by at least 230%. In some embodiments, this condition improves protein yield by at least 240%. In some embodiments, this condition improves protein yield by at least 250%. In some embodiments, this condition improves protein yield by at least 260%. In some embodiments, this condition improves protein yield by at least 270%. In some embodiments, this condition improves protein yield by at least 280%. In some embodiments, this condition improves protein yield by at least 290%.In some embodiments, this condition improves protein yield by at least 300%. In some embodiments, this condition improves protein yield by at least 310%. In some embodiments, this condition improves protein yield by at least 320%. In some embodiments, this condition improves protein yield by at least 330%. In some embodiments, this condition improves protein yield by at least 340%. In some embodiments, this condition improves protein yield by at least 350%. In some embodiments, this condition improves protein yield by at least 360%. In some embodiments, this condition improves protein yield by at least 370%. In some embodiments, this condition improves protein yield by at least 380%. In some embodiments, this condition improves protein yield by at least 390%. In some embodiments, this condition improves protein yield by at least 400%.

[0101] In some embodiments, the method improves protein yield by at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, at least approximately 7 times, at least approximately 8 times, at least approximately 9 times, or at least approximately 10 times compared to a method without suitable conditions. In some embodiments, the method improves protein yield by about 2 times to about 3 times. In some embodiments, the method improves protein yield by about 3 times to about 4 times. In some embodiments, the method improves protein yield by about 4 times to about 5 times. In some embodiments, the method improves protein yield by about 5 times to about 6 times. In some embodiments, the method improves protein yield by about 6 times to about 7 times. In some embodiments, the method improves protein yield by about 7 times to about 8 times. In some embodiments, this method improves protein yield by approximately 8 to 9 times.

[0102] The methods of this disclosure are also useful for increasing total protein production as a percentage of total host cell protein so that a higher protein yield per batch is achieved. In some embodiments, the methods of this disclosure are also useful for increasing total protein production with a desired glycosylation pattern based on changes in the residence time of the protein in the Golgi and exposure to glycosylation enzymes. In some embodiments, the conditions are adjusted, for example, to temperature setpoints of the first, second, and last temperature setpoints, e.g., 36°C, 33°C, and 31°C, to increase the protein yield of the protein with the desired glycosylation profile by at least 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, and at least about 220%, compared to a method without suitable conditions. , improve by at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, or at least approximately 400%.

[0103] The methods of this disclosure are useful for reducing the growth rate and / or steady-state cell density of a culture in order to improve overall yield and / or control the protein glycosylation profile. In some embodiments, the methods reduce the cell growth rate. In some embodiments, cell growth exhibits a 0-5 day average doubling time of about 30.0 hours to about 45.0 hours, about 30.0 hours to about 40.0 hours, about 31 hours to about 44 hours, about 32 hours to about 43 hours, or about 33 hours to about 42 hours. In some embodiments, cell growth exhibits a 0-5 day average doubling time of about 30 hours to about 42 hours, for example, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, or 42 hours. In some embodiments, cell growth exhibits a 0-5 day average doubling time of about 32 hours to about 38 hours. In some embodiments, cell proliferation exhibits an average doubling time of approximately 33 to 37 hours over 0 to 5 days. In other embodiments, cell proliferation exhibits an average doubling time of approximately 34 to 36 hours over 0 to 5 days.

[0104] In some embodiments, the cell proliferation rate exhibits an average doubling time of approximately 30 hours between 0 and 5 days. In some embodiments, the cell proliferation rate exhibits an average doubling time of approximately 32 hours between 0 and 5 days. In some embodiments, the cell proliferation rate exhibits an average doubling time of approximately 34 hours between 0 and 5 days. In some embodiments, the cell proliferation rate exhibits an average doubling time of approximately 38 hours between 0 and 5 days. In some embodiments, the cell proliferation rate exhibits an average doubling time of approximately 40 hours between 0 and 5 days.

[0105] The methods disclosed herein are also useful for controlling the viable cell density or peak viable cell density during protein production. In some embodiments, cell viability is approximately 5 × 10⁻⁶. 6 Approximately 21 x 10 cells / mL 6 It exhibits an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 6 × 10⁻⁶. 6 Approximately 20 x 10 cells / mL 6 It shows an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 7 × 10⁻⁶. 6 Approximately 19 x 10 cells / mL 6It exhibits an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 8 × 10⁻⁶. 6 Approximately 18 x 10 cells / mL 6 It exhibits an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 9 × 10⁻⁶. 6 Approximately 17 x 10 cells / mL 6 It shows an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 10 × 10 6 Approximately 16 x 10 cells / mL 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is approximately 11 × 10⁻⁶. 6 Approximately 15 x 10 cells / mL 6 It shows an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 12 × 10⁻¹⁶. 6 Approximately 14 x 10 cells / mL 6 This shows the mean peak viable cell density (VCD) of cells / mL.

[0106] In some embodiments, the cell viability is 6 × 10 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 6 × 10⁻⁶ 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 10 × 10 6 It exhibits an average peak viable cell density (VCD) of cells / mL. In some embodiments, the cell viability is approximately 10.5 × 10⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 11 × 10⁻¹⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 11.5 × 10⁻⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 12 × 10⁻¹⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 12.5 × 10⁻⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 13 × 10⁻¹⁶. 6The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 13.5 × 10⁻⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 14 × 10⁻⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 14.5 × 10⁻⁶. 6 The mean peak viable cell density (VCD) is shown as cells / mL. In some embodiments, the cell viability is 15 × 10⁻⁶. 6 This shows the mean peak viable cell density (VCD) of cells / mL.

[0107] The methods of this disclosure are also useful for improving or controlling cell viability by the 0-14 day mean viable cell density integral (IVCD). The IVCD measure is an alternative method for measuring upstream process production, in contrast to immediate measures of viable cell density. Since each cell has a variable protein production lifetime and various cell culture conditions affect the viability rate over time, IVCD is useful for estimating the total production of protein products over time across the entire process. In some embodiments, cell viability is approximately 0.05 × 10⁻⁶ 9 Approximately 0.2 × 10¹ / mL 9 The average intracellular viability density integral (IVCD) for 0-14 days is shown as cells / mL. In some embodiments, the cell viability is approximately 0.1 × 10⁻⁶. 9 Approximately 0.15 × 10¹ / mL 9 The average intracellular viability density (IVCD) for 0-14 days is shown as cells / mL. In some embodiments, the cell viability is approximately 0.05 × 10⁻⁶. 9 Approximately 0.15 × 10¹ / mL 9 The average intracellular viability density (IVCD) for 0-14 days is shown as cells / mL. In some embodiments, the cell viability is approximately 0.05 × 10⁻⁶. 9 Approximately 0.1 × 10⁻¹⁴ cells / mL 9 The average intracellular viability density integral (IVCD) for 0-14 days is shown as cells / mL. In some embodiments, the cell viability is approximately 0.09 × 10⁻⁶. 9 Approximately 0.13 × 10¹ / mL 9The average intracellular viability density integral (IVCD) for 0-14 days is shown as cells / mL. In some embodiments, the cell viability is approximately 0.09 × 10⁻⁶. 9 Approximately 0.11 × 10¹ / mL 9 This shows the average intravitage cell density (IVCD) from 0 to 14 days, expressed as cells / mL.

[0108] The method of this disclosure is also useful for controlling protein titer. In some embodiments, the titer is measured after a period of about 14 days. In some embodiments, the titer exhibits a day 14 average titer of about 1.5 g / L to about 3.5 g / L. In some embodiments, the titer exhibits a day 14 average titer of about 1.5 g / L to about 3 g / L. In some embodiments, the titer exhibits a day 14 average titer of about 2 g / L to about 3 g / L. In some embodiments, the titer exhibits a day 14 average titer of about 2 g / L to about 2.5 g / L. In some embodiments, the titer exhibits a day 14 average titer of about 2.5 g / L to about 3 g / L. In some embodiments, the titer exhibits a day 14 average titer of about 2 g / L. In some embodiments, the titer exhibits a day 14 average titer of about 2.5 g / L. In some embodiments, the potency exhibits a 14-day average potency of approximately 2.8 g / L. In some embodiments, the potency exhibits a 14-day average potency of approximately 2.87 g / L. In some embodiments, the potency exhibits a 14-day average potency of approximately 2.9 g / L. In some embodiments, the potency exhibits a 14-day average potency of approximately 3 g / L. In some embodiments, the potency exhibits a 14-day average potency of approximately 3.5 g / L.

[0109] The method of this disclosure is also useful for controlling cellular protein production, which is measured by the output of individual cells. Measuring output through the output of individual cells is useful for monitoring cellular health as a function of reactor conditions. In some embodiments, the titer shows an average specific productivity of about 20 pg / cell·day to about 45 pg / cell·day. In some embodiments, the titer shows an average specific productivity of about 20 pg / cell·day to about 40 pg / cell·day. In some embodiments, the titer shows an average specific productivity of about 30 pg / cell·day to about 45 pg / cell·day. In some embodiments, the titer shows an average specific productivity of about 20 pg / cell·day to about 35 pg / cell·day. In some embodiments, the titer shows an average specific productivity of about 25 pg / cell·day to about 35 pg / cell·day. In some embodiments, the titer shows an average specific productivity of about 25 pg / cell·day to about 30 pg / cell·day. In some embodiments, the titer shows an average specific productivity of approximately 30 pg / cell / day to approximately 40 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 30 pg / cell / day to approximately 35 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 20 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 25 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 30 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 31 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 32 pg / cell / day. In some embodiments, the titer shows an average specific productivity of approximately 33 pg / cell / day.

[0110] In some embodiments, the titer exhibits an average specific productivity of approximately 34 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 35 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 36 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 37 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 38 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 39 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 40 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 41 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 42 pg / cell·day. In some embodiments, the titer exhibits an average specific productivity of approximately 45 pg / cell·day.

[0111] The methods of this disclosure can be utilized through the proliferation of cells in a container using an energy source. In some embodiments, the container is a bioreactor and contains a supply medium comprising glucose or galactose. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are selected from Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), Maidin Darby dog ​​kidney cells (MDBK), and any combination thereof. In one embodiment, the cells are Chinese hamster ovary cells. In some embodiments, the cells are insect cells, e.g., fall armyworm (Spodoptera frugiperda) cells. In other embodiments, the cells are mammalian cells. Examples of such mammalian cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0, CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, the CHO cell is CHO-DG44, CHOZN, CHO / dhfr-, CHOK1SV GS-KO, or CHO-S. In some embodiments, the CHO cell is CHO-DG4. In some embodiments, the CHO cell is CHOZN. Other suitable CHO cell lines disclosed herein include CHO-K (e.g., CHO K1), CHO pro3-, CHO P12, CHO-K1 / SF, DUXB11, CHO DUKX, PA-DUKX, CHO pro5, DUK-BII, or derivatives thereof.

[0112] IIA. Temperature Because bioreactor temperature plays a role in cell proliferation, viable cell density, cell lifespan, and / or glycosylation activity of intracellular glycosylation enzymes, the temperature of production vessels such as bioreactors can be a critical aspect of biological production. Temperature changes can significantly affect the rate of intracellular enzymatic reactions, denature proteins, and / or have other effects on cell cultures. Cells can be cultured at an initial temperature setting, such as 37°C, to promote maximum viable cell density, and then the temperature can be changed to another temperature setting (i.e., a second or final temperature setting) to extend cell lifespan or enhance desired intracellular glycosylation activity. One or more temperature setting values ​​can be used at various stages of the upstream production process to improve overall cell density, protein yield, or the protein glycosylation profile of the target protein. In some embodiments, this method addresses one or more temperature adjustments during protein production. Temperature adjustment may be a decrease in the operating temperature during the production process. Alternatively, temperature adjustment may be an increase in the operating temperature during the production process. In some embodiments, the method of the present disclosure uses at least one, at least two, at least three, or at least four temperature adjustments during the manufacturing process.

[0113] The methods of the present disclosure also relate to controlling cell growth rate, cell viability, viable cell density, and / or cell titer for protein production. The initial temperature setpoint is important for setting reactor conditions that lead to cell growth and proliferation during the logarithmic growth phase. After the initial logarithmic phase, a second temperature setpoint lower than the initial setpoint is used to reduce cell growth conditions and prevent overgrowth of the cell culture, which is thought to lead to undesirable cell density and subsequent decreases in total cell viability. In some embodiments, the methods of the present disclosure involve culturing cells in a bioreactor at an initial temperature setpoint of 36°C for the induction phase, then culturing the cells at a second temperature setpoint of 33°C, and finally culturing the cells at a final temperature setpoint of 31°C. In some embodiments, the methods of the present disclosure use at least two, at least three, at least four, or at least five temperature setpoints, e.g., the initial temperature setpoint, the final setpoint, or more setpoints after the initial temperature setpoint but before the final setpoint. In some embodiments, the method of the present disclosure uses at least three temperature setpoints, namely a first temperature setpoint, a second temperature setpoint, and a last temperature setpoint.

[0114] In some embodiments, the initial temperature setpoint according to this method is approximately 37°C, and the second temperature setpoint is less than approximately 36°C. In some embodiments, the initial temperature setpoint is approximately 36°C, and the second temperature setpoint is lower than the first temperature setpoint, for example, approximately 35°C, approximately 34°C, approximately 33°C, approximately 32°C, or approximately 31°C. In some embodiments, the initial temperature setpoint is approximately 37°C, and the second temperature setpoint is less than approximately 34°C. In some embodiments, the initial temperature setpoint is approximately 36°C, and the second temperature setpoint is less than approximately 35°C, approximately 34°C, or approximately 33°C. In some embodiments, the initial temperature setpoint is less than approximately 36.5°C, and the final temperature setpoint is approximately 31°C. In some embodiments, the initial temperature setpoint is approximately 36.0°C, and the final temperature setpoint is approximately 31°C. In some embodiments, the initial temperature setpoint is less than approximately 35.5°C, and the final temperature setpoint is approximately 31°C. In some embodiments, the initial temperature setting is less than approximately 35.0°C and the final temperature setting is approximately 31°C. In some embodiments, the initial temperature setting is less than approximately 36.5°C, the second temperature setting is approximately 33°C, and the final temperature setting is approximately 33°C or less than approximately 32°C. In some embodiments, the initial temperature setting is approximately 36.0°C, the second temperature setting is approximately 33°C, and the final temperature setting is less than approximately 33°C or approximately 32°C. In some embodiments, the initial temperature setting is approximately 36.0°C, the second temperature setting is approximately 33°C, and the final temperature setting is less than approximately 32°C. In some embodiments, the initial temperature setting is approximately 36.0°C, the second temperature setting is approximately 33°C, and the final temperature setting is approximately 31°C.

[0115] The methods of the present disclosure may include a first temperature setpoint, a second temperature setpoint, and a third and / or final temperature setpoint. The first, second, and third and / or final temperature setpoints are used to further control the steady-state cell density during the manufacturing process, to control the percentage of viable cells, to manage cell cycle division of the culture, and / or to modify the glycosylation rate and glycosylation profile of the protein produced. In some embodiments, the third temperature setpoint is lower than the second temperature setpoint. In some embodiments, the first temperature setpoint is a temperature between 37°C and 34°C, e.g., 37°C, 36°C, 35°C, or 34°C; the second temperature setpoint is a temperature between 34°C and 32°C, e.g., 34°C, 33°C, or 32°C; and the final temperature setpoint is a temperature between 32°C and 30°C, e.g., 32°C, 31°C, or 30°C, where the second temperature setpoint is lower than the first temperature setpoint and the final temperature setpoint is lower than the second temperature setpoint. In some embodiments, the first temperature setpoint is a temperature between 37°C and 34°C, e.g., 37°C, 36°C, 35°C, or 34°C; the second temperature setpoint is a temperature between 34°C and 32°C, e.g., 34°C, 33°C, or 32°C; and the last temperature setpoint is a temperature between 32°C and 30°C, e.g., 32°C, 31°C, or 30°C, where the first temperature setpoint is not 37°C, the second temperature setpoint is not 34°C, and / or the last temperature setpoint is not 32°C.

[0116] The method of the present disclosure may include a first temperature setpoint, a second temperature setpoint, a third temperature setpoint, and optionally a fourth temperature setpoint, optionally a fifth temperature setpoint, and optionally a sixth temperature setpoint. These fourth, fifth, and sixth temperature setpoints are used to further control the steady-state cell density during the manufacturing process, to control the percentage of viable cells, to manage cell cycle division of the culture, and / or to modify the glycosylation rate and glycosylation profile of the protein produced. In some embodiments, the method further includes optionally setting a fourth temperature setpoint, optionally a fifth temperature setpoint, and optionally a sixth temperature setpoint, where optionally the fourth, fifth, and / or sixth temperature setpoints are lower than the third temperature setpoint. In some embodiments, the method further includes setting a fourth temperature setpoint, a fifth temperature setpoint, and a sixth temperature setpoint as appropriate, wherein the fourth, fifth, and / or sixth temperature setpoints are higher than the third temperature setpoint. In some embodiments, the fourth, fifth, or sixth temperature setpoint, the fifth temperature setpoint, and / or the sixth temperature setpoint are approximately 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, the fourth, fifth, or sixth temperature setpoint, the fifth temperature setpoint, and / or the sixth temperature setpoint are approximately 30°C. In some embodiments, the fourth, fifth, or sixth temperature setpoint is approximately 31°C. In some embodiments, the fourth, fifth, or sixth temperature setpoint is approximately 32°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 33°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 34°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 35°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 36°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 37°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 38°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 39°C. In some embodiments, the fourth, fifth, or sixth temperature setting is approximately 40°C.

[0117] Apart from the temperature setpoint, the methods of the present disclosure also include changing the timing of the temperature setpoint to perform a temperature shift within a specific time frame. The timing of the temperature shift is important for controlling the cell density, cell proliferation, and protein production characteristics of the cell culture during the upstream process. In some embodiments, the methods of the present disclosure are methods for improving the protein yield by cells, comprising culturing cells in a bioreactor under suitable conditions, wherein the suitable conditions include (i) a first temperature setpoint of 36.0°C and a second temperature setpoint below 36°C, (ii) a first temperature setpoint below 36.5°C and a final temperature setpoint of 31°C; or (iii) a first temperature setpoint below 36.5°C, a second temperature setpoint of 33°C, and a final temperature setpoint below 33°C. In some embodiments, the method of the present disclosure is a method for improving the yield of proteins by cells, comprising culturing cells in a bioreactor under suitable conditions, wherein the suitable conditions include (i) a first temperature setpoint of 36.0°C and a second temperature setpoint of less than 34°C, (ii) a first temperature setpoint of less than 36.5°C and a final temperature setpoint of 31°C; or (iii) a first temperature setpoint of less than 36.5°C, a second temperature setpoint of 32°C, and a final temperature setpoint of less than 32°C.

[0118] In some embodiments, cells are cultured in a bioreactor under suitable conditions, including (i) a first temperature setpoint higher than 35°C but less than 37°C and a second temperature setpoint higher than 32°C but less than 34°C, (ii) a first temperature setpoint higher than 35.5°C but less than 37.5°C and a second temperature setpoint higher than 32°C but less than 34°C, and (iii) a first temperature setpoint higher than 35°C but less than 37°C, a second temperature setpoint higher than 32°C but less than 34°C, and a final temperature setpoint higher than 30°C but less than 32°C.

[0119] The method of the present disclosure is also useful for improving the yield of proteins by cells by adjusting the temperature setpoint in the bioreactor after the initial temperature setpoint. In some embodiments, the initial temperature setpoint is higher than about 35°C but less than 37°C, for example, about 36°C; the second temperature setpoint is higher than about 32°C but less than about 34°C, for example, about 33°C; and the third temperature setpoint is higher than about 30°C but less than about 32°C, for example, about 31°C, where the second temperature setpoint occurs about 120 hours to about 168 hours after the initial temperature setpoint, for example, about 5 days, about 6 days, or about 7 days. In some embodiments, the first temperature setpoint is higher than approximately 35°C but less than 37°C, for example, approximately 36°C; the second temperature setpoint is higher than approximately 32°C but less than approximately 34°C, for example, approximately 33°C; and the third temperature setpoint is higher than approximately 30°C but less than 32°C, for example, approximately 31°C, where the second temperature setpoint occurs approximately 120 hours, approximately 126 hours, approximately 132 hours, approximately 138 hours, approximately 144 hours, approximately 150 hours, approximately 156 hours, approximately 162 hours, or approximately 168 hours after the first temperature setpoint.

[0120] In some embodiments, the first temperature setpoint is higher than approximately 35°C but less than 37°C, for example, approximately 36°C; the second temperature setpoint is higher than approximately 32°C but less than approximately 34°C, for example, approximately 33°C; and the third temperature setpoint is higher than approximately 30°C but less than approximately 32°C, for example, approximately 31°C, where the second temperature setpoint occurs approximately 120 hours, or 5 days, after the first temperature setpoint. In some embodiments, the first temperature setpoint is higher than approximately 35°C but less than 37°C, for example, approximately 36°C; the second temperature setpoint is higher than approximately 32°C but less than approximately 34°C, for example, approximately 33°C; and the third temperature setpoint is higher than approximately 30°C but less than approximately 32°C, for example, approximately 31°C, where the second temperature setpoint occurs approximately 144 hours, or 6 days, after the first temperature setpoint. In some embodiments, the first temperature setpoint is higher than approximately 35°C but less than 37°C, for example, approximately 36°C; the second temperature setpoint is higher than approximately 32°C but less than approximately 34°C, for example, approximately 33°C; and the third temperature setpoint is higher than approximately 30°C but less than approximately 32°C, for example, approximately 31°C, where the second temperature setpoint occurs approximately 168 hours, or 7 days, after the first temperature setpoint. In some embodiments, the second temperature setpoint occurs approximately 192 hours, or 8 days, after the first temperature setpoint.

[0121] The method of this disclosure can also be used to control the timing of transition to the final temperature setpoint. In some embodiments, the first temperature setpoint is higher than about 35°C and lower than 37°C, for example, about 36°C; the second temperature setpoint is higher than about 32°C and lower than about 34°C, for example, about 33°C; and the final temperature setpoint is higher than about 30°C and lower than about 32°C, for example, about 31°C, where the final temperature setpoint occurs about 168 hours to about 312 hours, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, or about 13 days after the first temperature setpoint. In some embodiments, the first temperature setpoint is higher than approximately 35°C but lower than 37°C, for example, approximately 36°C; the second temperature setpoint is higher than approximately 32°C but lower than approximately 34°C, for example, approximately 33°C; and the final temperature setpoint is higher than approximately 30°C but lower than approximately 32°C, for example, approximately 31°C, where the final temperature setpoint occurs between approximately 192 hours and approximately 300 hours. In some embodiments, the final temperature setpoint occurs between approximately 216 hours and approximately 288 hours. In some embodiments, the final temperature setpoint occurs between approximately 216 hours and approximately 264 hours.

[0122] IIB.pH In one embodiment, the method of the present disclosure involves improving or controlling protein production by altering the pH of a process. Regulation of intracellular pH is a fundamental physiological process of great significance for cell proliferation and metabolism. Cells expend considerable energy regulating cytoplasmic pH because it has far-reaching consequences for nutrient and hormone transport, as well as for intracellular enzymatic reactions. Furthermore, pH also plays a role in the rate and profile of glycosylation of proteins produced by cells.

[0123] In some embodiments, pH modification can be performed by using multiple pH settings, more than two pH settings, more than three pH settings, more than four pH settings, or more than five pH settings during the culture process. In some embodiments, pH modification uses the first pH setting and a second pH setting. In some embodiments, pH modification uses the first pH setting, a second pH setting, and a third pH setting. In some embodiments, pH modification includes lowering the pH during the culture process. In some embodiments, pH modification includes lowering the pH by at least about 0.5, at least about 1.0, at least about 1.5, at least about 2.0, at least about 0.1, at least about 0.2, at least about 0.3, or at least about 0.4. In some embodiments, pH modification includes lowering the pH by at least about 0.1. In some embodiments, pH modification includes lowering the pH by at least about 0.2. In some embodiments, pH modification includes lowering the pH by at least about 0.5. In some embodiments, the pH modification includes lowering the pH by at least about 1. In some embodiments, the pH modification includes lowering the pH by at least about 1.5. In some embodiments, the pH modification includes lowering the pH by at least about 2.

[0124] In some embodiments, the initial pH setting is pH 7.0 and the second pH setting is 6.9. In some embodiments, the initial pH is 7.0. In some embodiments, the initial pH setting is 6. In some embodiments, the initial pH setting is 6.1. In some embodiments, the initial pH setting is 6.2. In some embodiments, the initial pH setting is 6.3. In some embodiments, the initial pH setting is 6.4. In some embodiments, the initial pH setting is 6.5. In some embodiments, the initial pH setting is 6.6. In some embodiments, the initial pH setting is 6.7. In some embodiments, the initial pH setting is 6.8. In some embodiments, the initial pH setting is 6.9. In some embodiments, the initial pH setting is 7. In some embodiments, the second pH setting is 6. In some embodiments, the second pH setting is 6.1. In some embodiments, the second pH setting is 6.2. In some embodiments, the second pH setting is 6.3. In some embodiments, the second pH setting is 6.4. In some embodiments, the second pH setting is 6.5. In some embodiments, the second pH setting is 6.6. In some embodiments, the second pH setting is 6.7. In some embodiments, the second pH setting is 6.8. In some embodiments, the second pH setting is 6.9. In some embodiments, the second pH setting is 7. In some embodiments, the third pH setting is 6. In some embodiments, the third pH setting is 6.1. In some embodiments, the third pH setting is 6.2. In some embodiments, the third pH setting is 6.3. In some embodiments, the third pH setting is 6.4. In some embodiments, the third pH setting is 6.5. In some embodiments, the third pH setting is 6.6. In some embodiments, the third pH setting is 6.7. In some embodiments, the third pH setting is 6.8. In some embodiments, the third pH setting is 6.9. In some embodiments, the third pH setting is 7. In some embodiments, the initial pH is 7.0 and the second pH is 6.9. In some embodiments, the initial pH is 7.1 and the second pH is 6.8. In some embodiments, the initial pH is 7.1 and the second pH is 6.9. In some embodiments, the initial pH is 7.1 and the second pH is 7.0.In some embodiments, the initial pH is 6.9 and the second pH is 6.8. In some embodiments, the initial pH is 6.9 and the second pH is 6.7.

[0125] Not only is the pH of the upstream production process important, but the timing of any pH shifts during production also plays a significant role in the process. pH shifts during production lead to changes in process performance, which are mainly due to their effects on cell proliferation and metabolism. The methods of this disclosure relate to shifting the pH during protein production in order to control protein glycosylation. In some embodiments, the pH is shifted to a pH of approximately 6.0. In some embodiments, the pH is shifted to a pH of approximately 6.1. In some embodiments, the pH is shifted to a pH of approximately 6.2. In some embodiments, the pH is shifted to a pH of approximately 6.3. In some embodiments, the pH is shifted to a pH of approximately 6.4. In some embodiments, the pH is shifted to a pH of approximately 6.5. In some embodiments, the pH is shifted to a pH of approximately 6.6. In some embodiments, the pH is shifted to a pH of approximately 6.7. In some embodiments, the pH is shifted to a pH of approximately 6.8. In some embodiments, the pH is shifted to a pH of approximately 6.9. In some embodiments, the pH is shifted to approximately 7.0. In some embodiments, the pH is shifted to approximately 7.1. In some embodiments, the pH is shifted to approximately 7.2. In some embodiments, the pH is shifted to approximately 7.3. In some embodiments, the pH is shifted to approximately 7.4. In some embodiments, the pH is shifted to approximately 7.5.

[0126] The time after culture induction during which pH is adjusted during production (hereinafter referred to herein as "pH shift" or "pH timing") also leads to process changes due to its effects on cell proliferation and metabolism. The method of this disclosure relates to shifting the pH during protein production in order to control protein glycosylation. In some embodiments, the pH shift is about 50 to 150 hours after induction. In some embodiments, the pH shift is about 72 to 120 hours after induction. In some embodiments, the pH shift is about 80 to 100 hours after induction. In some embodiments, the pH shift is about 90 hours after induction. In some embodiments, the pH shift is about 100 hours after induction. In some embodiments, the pH shift is about 96 hours after induction.

[0127] IIC. Viable cell density The initial viable cell density (VCD) or seeding density at the start of the bioreactor process is a critical aspect of the upstream growth process because the initial VCD affects the steady state and / or maximum viable cell density of the cell culture during production. A higher initial cell density results in a much larger number of cells being generated during the initial growth phase of the upstream process, which can ultimately lead to faster cell death and shorter bioreactor uptime. While the bioreactor production process can be shortened, shorter bioreactor uptime can, in some cases, lead to alterations in protein residence times in the Golgi apparatus, and therefore alterations in post-translational modifications, such as changes in the protein glycosylation pattern, thus potentially affecting the post-translational modifications applied to the proteins produced by the process. Since the initial cell density can broadly affect the overall protein yield and quality outcomes, it is a critical parameter for upstream protein production.

[0128] In some embodiments, the method of the present disclosure involves seeding a bioreactor at an initial viable cell density (VCD). In some embodiments, the initial viable cell density (VCD) setting is approximately 0.05 × 10⁻⁶ 6 cells / mL and approximately 0.95 × 10⁻⁶ 6cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.05×10 6 cells / mL to about 0.9×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.05×10 6 cells / mL to about 0.85×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.05×10 6 cells / mL to about 0.8×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.05×10 6 cells / mL to about 0.75×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.05×10 6 cells / mL to about 0.7×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.05×10 6 cells / mL to about 0.65×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.1×10 6 cells / mL to about 0.6×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.15×10 6 cells / mL to about 0.6×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.15×10 6 cells / mL to about 0.55×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.15×10 6 cells / mL to about 0.5×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.15×10 6 cells / mL to about 0.45×10 6 cells / mL. In some embodiments, the initial viable cell density (VCD) set point is from about 0.15×10 6 cells / mL to about 0.4×10 6It is between cells / mL. In some embodiments, the initial viable cell density (VCD) setting is approximately 0.2 × 10⁻⁶. 6 cells / mL and approximately 0.4 × 10 6 It is between cells / mL. In some embodiments, the initial viable cell density (VCD) setting is approximately 0.2 × 10⁻⁶. 6 cells / mL and approximately 0.35 × 10 6 It is between cells / mL. In some embodiments, the initial viable cell density (VCD) setting is approximately 0.25 × 10⁻⁶. 6 cells / mL and approximately 0.35 × 10 6 It is between cells / mL.

[0129] IID.Cell supply time The methods of this disclosure can also be achieved by altering the cell supply time to influence or control the cell proliferation conditions. Timing of the supply process is crucial for producing desired proliferation characteristics in the cell culture production process, such as cell density. The optimal supply strategy in a bioreactor depends on the structure of the reaction dynamics and the interactions between different reactions, such as protein synthesis and the post-translational modification (i.e., glycosylation) of those proteins. Both oversupply and undersupply to the cell population are detrimental to cell proliferation and product formation; therefore, timing of supply is crucial to ensure maximum product yield. Undersupply to the culture can lead to nutrient depletion and cell death, while oversupply can lead to nutrient excess, increased osmotic pressure and cellular stress in a dense cellular environment, as well as undesirable post-translational modification of the target protein.

[0130] In some embodiments, the cell supply time is approximately 24 to 100 hours after induction. In some embodiments, the cell supply time is approximately 48 to 100 hours after induction. In some embodiments, the cell supply time is approximately 48 to 72 hours after induction. In some embodiments, the cell supply time is approximately 48 to 96 hours after induction. In some embodiments, the cell supply time is approximately 72 to 96 hours after induction. In some embodiments, the cell supply time is approximately 24 hours after induction. In some embodiments, the cell supply time is approximately 48 hours after induction. In some embodiments, the cell supply time is approximately 72 hours after induction. In some embodiments, the cell supply time is approximately 96 hours after induction.

[0131] IIE.CO2 The methods described herein also relate to the initial CO2 setting. The CO2 concentration in the upstream bioreactor process is a critical factor in controlling cell growth and pH. High CO2 concentrations lead to an increase in dissolved CO2 in the cell culture medium, which is thought to lower the pH of the cell culture medium. Furthermore, since carbon dioxide is an inevitable product of the respiration process and is therefore always present in aerobic biological processes, the CO2 concentration is likely to change during the upstream biomanufacturing process. High CO2 concentrations during manufacturing can lead to a decrease in the growth rate and / or protein production of cell cultures. Also, since changes in the protein production rate can affect the protein residence time in the Golgi apparatus, the glycosylation pattern of proteins is also affected by changes in CO2 concentration.

[0132] In some embodiments, the initial CO2 setting is between approximately 5% and 50%, between approximately 10% and 45%, between approximately 10% and 40%, between approximately 10% and 35%, between approximately 10% and 30%, between approximately 15% and 30%, between approximately 15% and 25%, or between approximately 20% and 25%. In some embodiments, the initial CO2 setting is between approximately 0% and 75%. In some embodiments, the initial CO2 setting is between approximately 5% and 50%. In some embodiments, the initial CO2 setting is between approximately 10% and 45%. In some embodiments, the initial CO2 setting is between approximately 10% and 40%. In some embodiments, the initial CO2 setting is between approximately 10% and 35%. In some embodiments, the initial CO2 setting is between approximately 10% and 30%. In some embodiments, the initial CO2 setting is between approximately 15% and approximately 30%. In some embodiments, the initial CO2 setting is between approximately 15% and approximately 25%. In some embodiments, the initial CO2 setting is between approximately 10% and approximately 25%. In some embodiments, the initial CO2 setting is approximately 15%, approximately 16%, approximately 17% (e.g., 16.8%), approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, or approximately 25%. In some embodiments, the initial CO2 setting is approximately 17% (e.g., 16.8%). In some embodiments, the initial CO2 setting is approximately 22%, for example, 22.4%. In some embodiments, the initial CO2 setting is approximately 23%.

[0133] In some embodiments, the initial CO2 concentration is monitored and maintained throughout the cell culture process. In other embodiments, the initial CO2 concentration is set but not monitored throughout the cell culture process.

[0134] IIF. Combination of conditions In some embodiments, the method of the Disclosure includes any combination of the conditions listed above. In some embodiments, the method includes conditions selected from two or more of the following: (i) a first temperature setting between approximately 35°C and approximately 37°C, e.g., approximately 36°C; a second temperature setting between approximately 32°C and approximately 34°C, e.g., approximately 33°C; and a third temperature setting between approximately 30°C and approximately 32°C, e.g., approximately 31°C; (ii) a first pH setting of pH 7.0 and a second pH setting of 6.9; and (iii) approximately 0.15 × 10 6 cells / mL and approximately 0.45 × 10 6 (iv) an initial viable cell density (VCD) setting between cells / mL, (v) an initial pH of 6.9, (v) a pH shift at approximately 96 hours, and (vi) an initial CO2 setting between approximately 15% and approximately 25%, for example, approximately 16.8%.

[0135] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; and (ii) a first pH setpoint of pH 7.0 and a second pH setpoint of pH 6.9.

[0136] In some embodiments, the method includes (i) a first temperature setpoint of 36°C, a second temperature setpoint of 33°C, and a third temperature setpoint of 31°C, and (ii) approximately 0.15 × 10 6 Approximately 0.45 x 10 from each individual 6 pieces (for example, 0.30 × 10 6 This includes the initial viable cell density (VCD) setting of (1).

[0137] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; and (ii) an initial CO2 setpoint between approximately 15% and approximately 25%, for example, approximately 15%, approximately 16%, approximately 16.8%, approximately 17%, approximately 22%, or approximately 22.4%.

[0138] In some embodiments, the method includes (i) a first temperature setting between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setting between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setting between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) a first pH setting of pH 7.0 and a second pH setting of 6.9; and (iii) approximately 0.15 × 10 6 Approximately 0.45 x 10 from each individual 6 pieces (for example, 0.30 × 10 6 This includes the initial viable cell density (VCD) setting of (1).

[0139] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; and (ii) an initial pH setpoint of pH 6.9.

[0140] In some embodiments, the method includes (i) a first temperature setting between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setting between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setting between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) an initial pH setting of pH 6.9; and (iii) approximately 0.15 × 10 6 Approximately 0.45 x 10 from each individual 6 pieces (for example, 0.30 × 10 6 This includes the initial viable cell density (VCD) setting of (1).

[0141] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) a first pH setpoint of pH 7.0 and a second pH setpoint of pH 6.9, or an initial pH setpoint of pH 6.9; and (iii) an initial CO2 setpoint between approximately 15% and approximately 25%, for example, approximately 15%, approximately 16%, approximately 16.8%, approximately 17%, approximately 22%, or approximately 22.4%.

[0142] In some embodiments, the method includes (i) a first temperature setting between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setting between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setting between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) a first pH setting of pH 7.0 and a second pH setting of pH 6.9, or a first pH setting of pH 6.9; and (iii) approximately 0.15 × 10 6 Approximately 0.45 x 10 from each individual 6 pieces (for example, 0.30 × 10 6 (iv) an initial viable cell density (VCD) setpoint of (1) and an initial CO2 setpoint of (iv) between approximately 15% and approximately 25%, for example, approximately 15%, approximately 16%, approximately 16.8%, approximately 17%, approximately 22%, or approximately 22.4%.

[0143] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) a first pH setpoint of pH 7.0 and a second pH setpoint of pH 6.9, or an initial pH setpoint of pH 6.9; (iii) a first CO2 setpoint between approximately 15% and approximately 25%, for example, approximately 15%, approximately 16%, approximately 16.8%, approximately 17%, approximately 22%, or approximately 22.4%; and (iv) approximately 0.15 × 10 6 Approximately 0.45 x 10 from each individual 6pieces (for example, 0.30 × 10 6 This includes the initial viable cell density (VCD) setting of (1).

[0144] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) a first pH setpoint of pH 7.0 and a second pH setpoint of pH 6.9, or a first pH setpoint of pH 6.9, where the shift from the first pH setpoint to the second pH setpoint is at a time of approximately 96 hours; and (iii) a first CO2 setpoint between approximately 15% and approximately 25%, for example, approximately 15%, approximately 16% (e.g., approximately 16.8%), approximately 17%, approximately 22%, or approximately 22.4%.

[0145] In some embodiments, the method includes (i) a first temperature setpoint between approximately 35°C and approximately 37°C, for example, approximately 36°C; a second temperature setpoint between approximately 32°C and approximately 34°C, for example, approximately 33°C; and a third temperature setpoint between approximately 30°C and approximately 32°C, for example, approximately 31°C; (ii) a first pH setpoint of pH 7.0 and a second pH setpoint of pH 6.9, where the shift from the first pH setpoint to the second pH setpoint occurs at approximately 96 hours; and (iii) a first CO2 setpoint between approximately 15% and approximately 25%, for example, approximately 15%, approximately 16%, approximately 16.8%, approximately 17%, approximately 22%, or approximately 22.4%; and (iv) approximately 0.15 × 10 6 Approximately 0.45 x 10 from each individual 6 pieces (for example, 0.30 × 10 6 This includes the initial viable cell density (VCD) setting of (1).

[0146] III. Glycosylation Profile The methods and conditions of the present disclosure affect the glycosylation profile of a protein produced by the methods, and are therefore useful for maintaining, controlling, and / or modifying said glycosylation profile. In some aspects, the methods control the glycosylation profile of the protein. In some aspects, the glycosylation profile of the protein comprises one or more N-linked glycans.

[0147] The methods of the present disclosure can be achieved or demonstrated through glycan analysis using various methods including glycan release assays. The first step in glycan analysis of glycoconjugates such as glycoproteins is the release of glycans from the molecule to which the glycans are attached. N-linked glycans on glycoproteins are released by amidases such as peptide-N-glycosidase F (PNGase F). Most methods for the analysis of biologically derived oligosaccharides require a step of glycan derivatization. Glycans can be derivatized to introduce chromophores or fluorophores, which facilitates detection after separation by chromatography or electrophoresis. Derivatization can also be applied to link a charged or hydrophobic group to the reducing end to enhance glycan separation and mass spectrometric detection. Furthermore, derivatization steps such as complete methylation are aimed at stabilizing sialic acid residues, improving the sensitivity of mass spectrometry, and supporting detailed structural characterization by (tandem) mass spectrometry.

[0148] Mass spectrometry ("MS" or "mass-spec") is an analytical technique used to measure mass-to-charge ratio ions. This is achieved by ionizing a sample, separating ions of different masses, and recording their relative abundance by measuring the intensity of the ion flux. A typical mass spectrometer consists of three components: an ion source, a mass analyzer, and a detector system. The ion source is the part of the mass spectrometer that ionizes the substance to be analyzed (analyte). Ions are then transported to a mass analyzer, which separates ions according to their mass-to-charge ratio (m / z) by means of magnetic or electric fields. Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS / MS). The detector records the induced charge or generated current when ions pass near or strike a surface. A mass spectrum is the result of measuring the signal generated at the detector when an m / z ion is scanned by a mass analyzer.

[0149] In some aspects, N-linked glycans can be analyzed by hydrophobic interaction liquid chromatography (HILIC). Glycans are cleaved from CTLA4, fluorescently labeled, and separated by HILIC for analysis. Tagging of glycans with a fluorescent label (e.g., 2-aminobenzamide) allows detection of glycans at the femtomole level. In some aspects, CTLA4 N-linked glycans are analyzed by HILIC coupled with mass spectrometry.

[0150] Various N-linked glycans may be present in the glycosylation profile of a protein. In some embodiments, the N-linked glycans include G0F, G1F, G2F, S1G1F, S1G2F, S2G2F, S1G3F, and / or S2G4F. Representative schematic diagrams of G0F, G1F, G2F, S1G1F, S2G2F, S1G3F, and S2G4F can be seen in Figures 1B-1C. In some embodiments, the method of this disclosure involves measuring the glycosylation profile after day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or day 21. In some embodiments, the method of the disclosure involves measuring the glycosylation profile after day 7. In some embodiments, the method of the disclosure further includes measuring the glycosylation profile after day 14. In some embodiments, the method of the disclosure involves measuring the glycosylation profile after day 21.

[0151] In some embodiments, the glycosylated protein is the CTLA4 protein. The CTLA4 molecule or the CTLA4 extracellular domain can be fused with Fc, and this molecule is referred to as CTLA4-Fc or CTLA4-Ig. The “Fc region” (fragment crystallization region), “Fc domain,” or “Fc” refers to the C-terminal region of the antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region includes the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains. In IgM and IgE, the Fc region contains three heavy chain constant domains (CH domains 2-4) within each polypeptide chain. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In the case of IgG, the Fc region contains the immunoglobulin domains CH2 and CH3, as well as the hinge between the CH1 and CH2 domains. While there may be various definitions of the boundary of the Fc region of an immunoglobulin heavy chain, as defined herein, the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxyl terminus of the heavy chain, with numbering according to the Kabat numbering scheme. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain within the Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG.As used herein, the Fc region may be a native sequence Fc containing any allotype variant, or a mutant Fc (e.g., an Fc that does not exist in nature). The methods of this disclosure are also useful for producing proteins containing a CTLA4 domain. The methods of this disclosure are also useful for producing proteins in which the CTLA4 domain is fused with an Fc moiety. In some embodiments, the protein is a fusion protein. In some embodiments, the fusion protein contains an Fc moiety. In some embodiments, the protein is abatacept. In some embodiments, the protein contains a sequence selected from the group consisting of SEQ ID NOs: 1 to 8. In some embodiments, the protein contains SEQ ID NOs: 1. In some embodiments, the protein contains SEQ ID NOs: 2. In some embodiments, the protein contains SEQ ID NOs: 3. In some embodiments, the protein contains SEQ ID NOs: 4. In some embodiments, the protein contains SEQ ID NOs: 5. In some embodiments, the protein contains SEQ ID NOs: 6. In some embodiments, the protein contains SEQ ID NOs: 7. In some embodiments, the protein contains SEQ ID NOs: 8.

[0152] CTLA4-Ig fusion proteins may contain one or more mutations. In some embodiments, the CTLA4-Ig fusion protein is (a) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO: 8 (methionine at amino acid position 27 and glycine at amino acid position 382), (b) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO: 5 (methionine at amino acid position 27 and lysine at amino acid position 383), (c) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO: 7 (alanine at amino acid position 26 and glycine at amino acid position 382), (d) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO: 4 (alanine at amino acid position 26 and lysine at amino acid position 383), (e) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO: 6 (methionine at amino acid position 25 and glycine at amino acid position 382), or (f) a CTLA4-Ig fusion protein having the amino acid sequence of SEQ ID NO: 3 (methionine at amino acid position 25 and lysine at amino acid position 383). In some embodiments, the CTLA4-Ig fusion protein comprises (a) approximately 90% CTLA4-Ig polypeptide containing the amino acid sequence of SEQ ID NO: 2 beginning with methionine at residue 27, (b) approximately 10% CTLA4-Ig polypeptide containing the amino acid sequence of SEQ ID NO: 2 beginning with alanine at residue 26, (c) approximately 4% CTLA4-Ig polypeptide containing the amino acid sequence of SEQ ID NO: 2 ending with lysine at residue 383, (d) approximately 96% CTLA4-Ig polypeptide containing the amino acid sequence of SEQ ID NO: 2 ending with glycine at residue 382, ​​and optionally, (e) less than 1% CTLA4-Ig polypeptide containing the amino acid sequence of SEQ ID NO: 2 beginning with methionine at residue 25.

[0153] The proteins of this disclosure have glycosylation sites. Glycosylation is a process involving the addition of complex oligosaccharide structures to a protein at specific sites within a polypeptide chain. Protein glycosylation and subsequent processing of the added carbohydrates can affect protein folding and structure, protein stability including protein half-life, and protein functional properties. Protein glycosylation can be divided into two classes, O-linked glycosylation and N-linked glycosylation, depending on the sequence situation in which the modification occurs. O-linked polysaccharides are linked to a hydroxyl group, usually to a hydroxyl group of either a serine or threonine residue. O-glycans are not added to all serine and threonine residues. O-linked oligosaccharides are usually monobranched or bibranched, meaning they contain one or at most two branches, and contain one to four different types of sugar residues, which are added one at a time. N-linked polysaccharides are linked to the amide nitrogen of asparagine. Only asparagine, which is part of one of two tripeptide sequences, asparagine-X-serine or asparagine-X-threonine (where X is any amino acid other than proline), is a target for glycosylation. N-linked oligosaccharides can have one to four branches and are referred to as monobranched, bibranched, tribranched, or tetrabranched. In some embodiments, one or more N-linked glycans are located at one or more asparagine residues selected from the group consisting of Asn76(T5), Asn108(T7), and / or Asn207(T14) of abatacept.

[0154] In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 2.0% and approximately 10.0%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 2.5% and approximately 10%, between approximately 2.5% and approximately 9.5%, between approximately 2.5% and approximately 9%, between approximately 2.5% and approximately 8.5%, between approximately 2.5% and approximately 8%, between approximately 2.5% and approximately 7.5%, between approximately 2.5% and approximately 7%, between approximately 2.5% and approximately 6.5%, between approximately 3.0% and approximately 10%, between approximately 3.0%, between approximately 9.5%, between approximately 3.0% and approximately 9%, between approximately 3.0% and approximately 8.5%, between approximately 3.0% and approximately 8%, between approximately 3.0% and approximately 7.5%, between approximately 3.0% and approximately 7%, or between approximately 3.0% and approximately 6.5%.

[0155] In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 3.2% and approximately 6.6%, between approximately 3.2% and approximately 4.6%, or between approximately 3.2% and approximately 5.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 3.2% and approximately 4.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 3.2% and approximately 6.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) between approximately 3.2% and approximately 5.6%.

[0156] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between approximately 1.0% and approximately 6%.

[0157] In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn108(T7) between approximately 1.0% and approximately 6%, between approximately 1.0% and approximately 5.5%, between approximately 1.0% and approximately 5.0%, between approximately 1.0% and approximately 4.5%, between approximately 1.0% and approximately 4.0%, between approximately 1.5% and approximately 6%, between approximately 1.5% and approximately 5.5%, between approximately 1.5% and approximately 5.0%, between approximately 1.5% and approximately 4.5%, between approximately 1.5% and approximately 4.0%, between approximately 2.0% and approximately 6%, between approximately 2.0% and approximately 5.5%, between approximately 2.0% and approximately 5.0%, between approximately 2.0% and approximately 4.5%, or between approximately 2.0% and approximately 4.0%. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn108(T7) between approximately 2.1% and approximately 4.0%, between approximately 1.8% and approximately 3.5%, or between approximately 1.8% and approximately 4.0%.

[0158] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.0% and about 6%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.0% and about 5.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.0% and about 5.0%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.0% and about 4.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.0% and about 4.0%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.5% and about 6%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.5% and about 5.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.5% and about 5.0%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.5% and about 4.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G0F between about 1.5% and about 4.0%.

[0159] The methods of this disclosure are useful for controlling or maintaining the G2F content of a protein. The G2F content of a protein is related to the rate of protein elimination from the human body, and therefore the methods also cover controlling the elimination half-life of a protein by controlling its glycan content, including the G2F content. In some embodiments, the methods of this disclosure are useful for extending the cyclic half-life of a protein by reducing its elimination rate. In some embodiments, the protein is recombinant (e.g., abatacept). In some embodiments, the elimination half-life of the protein is shortened. In some embodiments, the cyclic half-life of the protein is extended.

[0160] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5% and approximately 12%. This includes the relative abundance of G2F between 5%, between approximately 6% and 11%, between approximately 6% and 10.5%, between approximately 6% and 10%, between approximately 6.5% and 12%, between approximately 6.5% and 11.5%, between approximately 6.5% and 11%, between approximately 6.5% and 10.5%, between approximately 6.5% and 10%, between approximately 7% and 12%, between approximately 7% and 11.5%, between approximately 7% and 11%, between approximately 7% and 10.5%, or between approximately 7% and 10%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 7.2% and approximately 9.8%, between approximately 6.3% and approximately 10.6%, or between approximately 7.2% and approximately 10.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 5% and approximately 21%. In some embodiments, the method comprises culturing a protein under any one of the conditions disclosed herein, for example, a first temperature setting between about 35°C and about 37°C, for example, about 36°C; a second temperature setting between 32°C and about 34°C, for example, about 33°C; and a third temperature setting between about 30°C and about 32°C, for example, about 31°C, wherein the protein has an N-linked glycan, for example, G2F, at residue Asn76(T5), and the relative abundance of G2F is between about 5% and about 12%.

[0161] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5% and approximately 11.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5% and approximately 11%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5% and approximately 10.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5% and approximately 10%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of G2F between approximately 5.5% and approximately 12%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 5.5% and approximately 11.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 5.5% and approximately 11%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 5.5% and approximately 10.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 5.5% and approximately 10%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 7.2% and approximately 9.8%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 6.3% and approximately 10.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) between approximately 5% and approximately 21%.

[0162] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and are distributed between approximately 5% and approximately 21%, between approximately 5% and approximately 20.5%, between approximately 5% and approximately 20%, between approximately 5% and approximately 19.5%, between approximately 5% and approximately 19%, between approximately 5.5% and approximately 21%, between approximately 5.5% and approximately 20.5%, between approximately 5.5% and approximately 20%, between approximately 5.5% and approximately 19.5%, between approximately 5.5% and approximately 19%, between approximately 6% and approximately 21%, between approximately 6% and approximately 20.5%, between approximately 6% and approximately 20%, between approximately 6% and approximately 19.5%, between approximately 6% and approximately 19%, between approximately 6.5% and approximately 21%, and between approximately 6.5% and approximately 20.5%. This includes the relative abundance of G2F between % and %, between approximately 6.5% and 20%, between approximately 6.5% and 19.5%, between approximately 6.5% and 19%, between approximately 7% and 21%, between approximately 7% and 20.5%, between approximately 7% and 20%, between approximately 7% and 19.5%, between approximately 7% and 19%, between approximately 7.5% and 21%, between approximately 7.5% and 20.5%, between approximately 7.5% and 20%, between approximately 7.5% and 19.5%, between approximately 7.5% and 19%, between approximately 8% and 21%, between approximately 8% and 20.5%, between approximately 8% and 20%, between approximately 8% and 19.5%, or between approximately 8% and 19%.

[0163] In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 8.2% and approximately 14.1%, between approximately 8.0% and approximately 18.6%, or between approximately 8.2% and approximately 14.1%. In some embodiments, the G2F further contains a galactose-α-1,3-galactose moiety (G2F-Gal), with G2F-Gal accounting for a relative abundance of approximately 1.4% or less. In some embodiments, G2F-Gal accounting for a relative abundance between approximately 1.0% and approximately 1.4%. In some embodiments, G2F-Gal accounting for a relative abundance between approximately 0.4% and approximately 0.9%.

[0164] In some embodiments, the glycosylation profile does not contain multiple galactose-α-1,3-galactose (α-gal) bonds.

[0165] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G2F between approximately 5% and approximately 21%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G2F between approximately 5% and approximately 20.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G2F between approximately 5% and approximately 20%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G2F between approximately 5.5% and approximately 21%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of G2F between approximately 5.5% and approximately 21%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 5.5% and approximately 20.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 8.2% and approximately 14.1%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 8.0% and approximately 18.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn108(T7) between approximately 8.2% and approximately 14.1%.

[0166] In some embodiments, the method comprises culturing a protein under any one of the conditions disclosed herein, for example, a first temperature setting between about 35°C and about 37°C, for example, about 36°C; a second temperature setting between 32°C and about 34°C, for example, about 33°C; and a third temperature setting between about 30°C and about 32°C, for example, about 31°C, wherein the protein has an N-linked glycan, for example, G2F, at residue Asn108(T7), and the relative abundance of G2F is between about 5% and about 21%.

[0167] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 29% and approximately 38%. This includes the relative abundances of S1G2F between % and approximately 36.5%, between approximately 30% and approximately 38%, between approximately 30% and approximately 37.5%, between approximately 30% and approximately 37%, between approximately 30% and approximately 36.5%, between approximately 31.5% and approximately 38%, between approximately 31.5% and approximately 37.5%, between approximately 31.5% and approximately 37%, and between approximately 31.5% and approximately 36.5%.

[0168] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 29.5% and approximately 38%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 29.5% and approximately 37.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 29.5% and approximately 37%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 29.5% and approximately 36.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 29% and approximately 38%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 29.5% and approximately 37.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 29.5% and approximately 37%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 29.5% and approximately 36.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G2F between approximately 30% and approximately 38%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 30% and approximately 37.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 30% and approximately 37%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) between approximately 30% and approximately 36.5%.In some aspects, the one or more N-linked glycans are located at residue Asn76(T5) and comprise a relative abundance of S1G2F that is between about 31.6% and about 35.1%, between about 31.3% and about 36.5%, or between about 31.3% and about 36.5%. In some aspects, the one or more N-linked glycans are located at residue Asn76(T5) and comprise a relative abundance of S1G2F that is between about 31.6% and about 35.1%. In some aspects, the one or more N-linked glycans are located at residue Asn76(T5) and comprise a relative abundance of S1G2F that is between about 31.3% and about 36.5%. In some aspects, the one or more N-linked glycans are located at residue Asn76(T5) and comprise a relative abundance of S1G2F that is between about 31.3% and about 36.5%.

[0169] In some aspects, the one or more N-linked glycans are located at residue Asn108(T7) and comprise a relative abundance of S1G2F that is between 33% and about 45%. In some aspects, the one or more N-linked glycans are located at residue Asn108(T7) and comprise a relative abundance of S1G2F that is between about 33% and about 45%, between about 33% and about 44.5%, between about 33% and about 44%, between about 33% and about 43.5%, between about 33% and about 43%, between about 33% and about 42.5%, between about 33.5% and about 45%, between about 33.5% and about 44.5%, between about 33.5% and about 44%, between about 33.5% and about 43.5%, between about 33.5% and about 43%, between about 33.5% and about 42.5%, between about 34% and about 45%, between about 34% and about 44.5%, between about 34% and about 44%, between about 34% and about 43.5%, between about 34% and about 43%, between about 34% and about 42.5%, between about 34.5% and about 45%, between about 34.5% and about 44.5%, between about 34.5% and about 44%, between about 34.5% and about 43.5%, between about 34.5% and about 43%, or between about 34.5% and about 42.5%.

[0170] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33% and approximately 45%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33% and approximately 44.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33% and approximately 44%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33% and approximately 43.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33% and approximately 43%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33% and approximately 43.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33% and approximately 43%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33% and approximately 42.5%.

[0171] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33% and approximately 45%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33.5% and approximately 44.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33.5% and approximately 44%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 33.5% and approximately 43.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33.5% and approximately 43%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33.5% and approximately 43.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33.5% and approximately 43%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 33.5% and approximately 42.5%.

[0172] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 34% and approximately 45%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 34% and approximately 44.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 34% and approximately 44%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 34% and approximately 43.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S1G2F between approximately 34% and approximately 43%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34% and approximately 43.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34% and approximately 43%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34% and approximately 42.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34.2% and approximately 37.7%, between approximately 35.5% and approximately 42.3%, or between approximately 34.2% and approximately 42.3%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34.2% and approximately 42.3%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 35.2% and approximately 42.3%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn108(T7) between approximately 34.2% and approximately 37.7%.In some embodiments, S1G2F further contains a galactose-α-1,3-galactose moiety (S1G2F-Gal), with S1G2F-Gal accounting for a relative abundance of approximately 4.7% or less. In some embodiments, S1G2F-Gal accounting for a relative abundance between approximately 2.3% and approximately 4.7%. In some embodiments, S1G2F-Gal accounting for a relative abundance between approximately 1.4% and approximately 1.8%.

[0173] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 13% and approximately 25%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and are distributed between approximately 13% and approximately 25%, between approximately 13% and approximately 24.5%, between approximately 13% and approximately 24%, between approximately 13% and approximately 23.5%, between approximately 13% and approximately 23%, between approximately 13.5% and approximately 25%, between approximately 13.5% and approximately 24.5%, between approximately 13.5% and approximately 24%, between approximately 13.5% and approximately 23.5%, between approximately 13.5% and approximately 23%, between approximately 14% and approximately 25%, between approximately 14% and approximately 24.5%, between approximately 14% and approximately 24%, between approximately 14% and approximately 23.5%, and between approximately 14% and approximately 2 This includes the relative abundance of S2G2F between 3%, between approximately 14.5% and 25%, between approximately 14.5% and 24.5%, between approximately 14.5% and 24%, between approximately 14.5% and 23.5%, between approximately 14.5% and 23%, between approximately 15% and 25%, between approximately 15% and 24.5%, between approximately 15% and 24%, between approximately 15% and 23.5%, between approximately 15% and 23%, between approximately 15.5% and 25%, between approximately 15.5% and 24.5%, between approximately 15.5% and 24%, between approximately 15.5% and 23.5%, or between approximately 15.5% and 23%.

[0174] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 13% and approximately 25%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 13% and approximately 24.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 13% and approximately 24%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 13% and approximately 23.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 13% and approximately 23.5%.

[0175] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 13.5% and approximately 25%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 13.5% and approximately 24.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 13.5% and approximately 24%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 13.5% and approximately 23.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 13.5% and approximately 23.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 14% and approximately 25%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 14% and approximately 24.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 14% and approximately 24%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G2F between approximately 14% and approximately 23.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 14% and approximately 23.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 15% and approximately 25%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 15% and approximately 24.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 15% and approximately 24%.In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 15% and approximately 23.5%.

[0176] In some embodiments, one or more N-linked glycans contain relative abundances of S2G2F located at residue Asn76(T5) between approximately 18.1% and approximately 22.9%, between approximately 15.4% and approximately 20%, and between approximately 15.4% and approximately 22.9%. In some embodiments, one or more N-linked glycans contain relative abundances of S2G2F located at residue Asn76(T5) between approximately 18% and approximately 24%, between approximately 18% and approximately 23%, or between approximately 19% and approximately 23%. In some embodiments, one or more N-linked glycans contain relative abundances of S2G2F located at residue Asn76(T5) between approximately 18.1% and approximately 22.9%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 15.4% and approximately 20%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 15.4% and approximately 22.9%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 18% and approximately 24%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 18% and approximately 23%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn76(T5) between approximately 19% and approximately 23%.

[0177] In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between about 18% and about 36%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between about 18% and about 36%, between about 18% and about 35.5%, between about 18% and about 35%, between about 18% and about 34.5%, between about 18% and about 34%, between about 18% and about 33.5%, between about 18.5% and about 36%, between about 18.5% and about 3 Between 5.5%, between approximately 18.5% and approximately 35%, between approximately 18.5% and approximately 34.5%, between approximately 18.5% and approximately 34%, between approximately 18.5% and approximately 33.5%, between approximately 19% and approximately 36%, between approximately 19% and approximately 35.5%, between approximately 19% and approximately 35%, between approximately 19% and approximately 34.5%, between approximately 19% and approximately 34%, and between approximately 19% and approximately 33.5% Between % and %; between approximately 19.5% and 36%; between approximately 19.5% and 35.5%; between approximately 19.5% and 35%; between approximately 19.5% and 34.5%; between approximately 19.5% and 34%; between approximately 19.5% and 33.5%; between approximately 20% and 36%; between approximately 20% and 35.5%; between approximately 20% and 35%; between approximately 20% and 34%. The relative abundance of S2G2F is between 5%, between approximately 20% and approximately 34%, between approximately 20% and approximately 33.5%, between approximately 20.5% and approximately 36%, between approximately 20.5% and approximately 35.5%, between approximately 20.5% and approximately 35%, between approximately 20.5% and approximately 34.5%, between approximately 20.5% and approximately 34%, or between approximately 20.5% and approximately 33.5%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between approximately 18% and approximately 36%. In some embodiments, one or more N-linked glycans are located at residue Asn108(T7) and contain a relative abundance of S2G2F between approximately 18% and approximately 35.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18% and approximately 35%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18% and approximately 34.5%.

[0178] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18% and approximately 34%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18% and approximately 33.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18.5% and approximately 36%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18.5% and approximately 35.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18.5% and approximately 35%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18.5% and approximately 34.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18.5% and approximately 34%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 18.5% and approximately 33.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19% and approximately 36%.

[0179] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19% and approximately 35.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19% and approximately 35%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19% and approximately 34.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19% and approximately 34%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19% and approximately 33.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19.5% and approximately 36%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19.5% and approximately 35.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19.5% and approximately 35%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19.5% and approximately 34.5%.

[0180] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19.5% and approximately 34%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 19.5% and approximately 33.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20% and approximately 36%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20% and approximately 35.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20% and approximately 35%.

[0181] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20% and approximately 34.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20% and approximately 34%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20% and approximately 33.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 23.2% and approximately 33.8%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20.8% and approximately 32.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 20.8% and approximately 33.8%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G2F located at residue Asn108(T7) between approximately 23.2% and approximately 33.8%, between approximately 20.8% and approximately 32.6%, or between approximately 20.8% and approximately 33.8%.

[0182] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 8%, between about 2% and about 7.5%, between about 2% and about 7%, between about 2% and about 6.5%, between about 2% and about 6%, between about 2% and about 5.5%, between about 2.5% and about 8%, between about 2.5% and about 7.5%, between about 2.5% and about 7%, between about 2.5% and about 6.5%, between about 2.5% and about 6%, between about 2.5% and about 5.5%, between about 3% and about 8%, between about 3% and about 7.5%, This includes the relative abundance of S1G3F between 3% and approximately 7%, between approximately 3% and approximately 6.5%, between approximately 3% and approximately 6%, between approximately 3% and approximately 5.5%, between approximately 3.5% and approximately 8%, between approximately 3.5% and approximately 7.5%, between approximately 3.5% and approximately 7%, between approximately 3.5% and approximately 6.5%, between approximately 3.5% and approximately 6%, between approximately 3.5% and approximately 5.5%, between approximately 4% and approximately 8%, between approximately 4% and approximately 7.5%, between approximately 4% and approximately 7%, between approximately 4% and approximately 6.5%, between approximately 4% and approximately 6%, or between approximately 4% and approximately 5.5%.

[0183] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 7.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 7%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 6.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between about 2% and about 6%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 2% and approximately 5.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 2.5% and approximately 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 2.5% and approximately 7.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 2.5% and approximately 7%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 2.5% and approximately 6.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 2.5% and approximately 6%.

[0184] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 2.5% and approximately 5.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3% and approximately 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3% and approximately 7.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3% and approximately 7%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3% and approximately 6.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 3% and approximately 6%.

[0185] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3% and approximately 5.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3.5% and approximately 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3.5% and approximately 7.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3.5% and approximately 7%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3.5% and approximately 6.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3.5% and approximately 6%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 3.5% and approximately 5.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 4% and approximately 8%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 4% and approximately 7.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 4% and approximately 7%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 4% and approximately 6.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 4% and approximately 6%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 4% and approximately 5.5%.In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 4.4% and approximately 5.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 4% and approximately 5.6%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 4.4% and approximately 5.6%, or between approximately 4.0% and approximately 5.5%.

[0186] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) that is between about 0.5% and about 4%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) that is between about 0.5% and about 4%, between about 0.5% and about 3.5%, between about 0.5% and about 3%, between about 0.5% and about 2.5%, between about 1% and about 4%, between about 1% and about 3.5%, between about 1% and about 3%, or between about 1% and about 2.5%.

[0187] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) between approximately 1.4% and approximately 2.2%, between approximately 1.1% and approximately 1.9%, or between approximately 1.1% and approximately 2.2%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn108(T7) between approximately 1% and approximately 3%, between approximately 1% and approximately 2%, or between approximately 1% and approximately 1.5%.

[0188] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 0.5% and approximately 4%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 0.5% and approximately 3.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 0.5% and approximately 3%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 0.5% and approximately 2.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 1% and approximately 4%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 1% and approximately 3.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 1% and approximately 3%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 1% and approximately 2.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 1.1% and approximately 1.9%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S1G3F between approximately 1.1% and approximately 2.2%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 1.4% and approximately 2.2%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 1% and approximately 3%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 1% and approximately 2%.In some embodiments, one or more N-linked glycans contain a relative abundance of S1G3F located at residue Asn76(T5) between approximately 1% and approximately 1.5%.

[0189] In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) that is between about 0.5% and about 4%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) that is between about 0.5% and about 4%, between about 0.5% and about 3.5%, between about 0.5% and about 3%, between about 0.5% and about 2.5%, between about 1% and about 4%, between about 1% and about 3.5%, between about 1% and about 3%, or between about 1% and about 2.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) between approximately 1.9% and approximately 2.4%, between approximately 1.4% and approximately 2.1%, or between approximately 1.4% and approximately 2.4%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) between approximately 1% and approximately 2%, between approximately 1% and approximately 2%, or between approximately 1.5% and approximately 2.5%.

[0190] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 0.5% and approximately 4%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 0.5% and approximately 3.4%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 0.5% and approximately 3%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 0.5% and approximately 2.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 1% and approximately 4%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 1% and approximately 3.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 1% and approximately 3%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 1% and approximately 2.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 1.9% and approximately 2.4%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) and contain a relative abundance of S2G4F between approximately 1.4% and approximately 2.1%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) between approximately 1.4% and approximately 2.4%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) between approximately 1% and approximately 2%. In some embodiments, one or more N-linked glycans contain a relative abundance of S2G4F located at residue Asn76(T5) between approximately 1.5% and approximately 2.5%.

[0191] In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 7.0% or less, or about 6.5% or less. In some embodiments, one or more N-linked glycans contain a relative abundance of G0F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 6.0% or less, about 5.5% or less, about 5.0% or less, about 4.5% or less, about 4.0% or less, about 3.5% or less, about 3.0% or less, about 2.5% or less, about 2.0% or less, about 1.5% or less, about 1.0% or less, about 0.5% or less, or about 0.0%.

[0192] In some embodiments, one or more N-linked glycans contain a relative abundance of G1F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 7.5% or less or 7.0%. In some embodiments, one or more N-linked glycans contain a relative abundance of G1F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 6.5% or less, about 6.0% or less, about 5.5% or less, about 5.0% or less, about 4.5% or less, about 4.0% or less, about 3.5% or less, about 3.0% or less, about 2.5% or less, about 2.0% or less, about 1.5% or less, about 1.0% or less, about 0.5% or less, or about 0.0%.

[0193] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, and contain a relative abundance of G2F of about 25% or less, or from about 1.5% to about 23%. In some embodiments, one or more N-linked glycans contain a relative abundance of G2F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is approximately 24% or less, approximately 23% or less, approximately 22% or less, approximately 21% or less, approximately 20% or less, approximately 19% or less, approximately 18% or less, approximately 17% or less, approximately 16% or less, approximately 15% or less, approximately 14% or less, approximately 13% or less, approximately 12% or less, approximately 11% or less, approximately 10% or less, approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, or approximately 1% or less. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, in amounts of approximately 2.0% to approximately 23%, approximately 3% to approximately 23%, approximately 4% to approximately 23%, approximately 5% to approximately 23%, approximately 6% to approximately 23%, approximately 7% to approximately 23%, approximately 8% to approximately 23%, and approximately 9% to approximately 2%. This includes the relative abundances of G2F at 3%, approximately 10% to 23%, approximately 11% to 23%, approximately 12% to 23%, approximately 13% to 23%, approximately 14% to 23%, approximately 15% to 23%, approximately 16% to 23%, approximately 17% to 23%, approximately 18% to 23%, approximately 19% to 23%, approximately 20% to 23%, approximately 21% to 23%, or approximately 22% to 23%.

[0194] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G1F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is 13.5% or less or about 12.5%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G1F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0%.

[0195] In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 33% or more, or about 32% to about 49%. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, which is about 34% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, about 46% or more, about 47% or more, about 48% or more, or about 49% or more. In some embodiments, one or more N-linked glycans contain a relative abundance of S1G2F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, in the following proportions: about 33% to about 49%, about 34% to about 49%, about 35% to about 49%, about 36% to about 49%, about 37% to about 49%, about 38% to about 49%, about 39% to about 49%, about 40% to about 49%, about 41% to about 49%, about 42% to about 49%, about 43% to about 49%, about 44% to about 49%, about 45% to about 49%, about 46% to about 49%, about 47% to about 49%, or about 48% to about 49%.

[0196] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, and contain a relative abundance of S2G2F of about 12% or more, or from about 14% to about 48.5%. In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, in approximately 15% to approximately 45.5%, approximately 15% to approximately 45.5%, approximately 16% to approximately 45.5%, approximately 16% to approximately 45.5%, approximately 17% to approximately 45.5%, approximately 18% to approximately 45.5%, approximately 19% to approximately 45.5%, approximately 20% to approximately 45.5%, approximately 21% to approximately 45.5%, approximately 22% to approximately 45.5%, approximately 23% to approximately 45.5%, approximately 24% to approximately 45.5%, approximately 25% to approximately 45.5%, approximately 26% to approximately 45.5%, approximately 27% to approximately 45.5%, approximately 28% to approximately 45.5%, approximately 29% to approximately 45.5%, approximately 30% to approximately 45.5%, approximately 31% to approximately 45.5%, approximately 32% to approximately 45.5%, approximately 33% to approximately 45.5%, approximately 34% to approximately 45.5%, approximately 35% to approximately 45.5%, approximately 36% to approximately 45.5%, approximately 37% to approximately 45.5%, approximately This includes the relative abundances of S2G2F in the following ranges: 38% to approximately 45.5%, approximately 39% to approximately 45.5%, approximately 40% to approximately 45.5%, approximately 41% to approximately 45.5%, approximately 42% to approximately 45.5%, approximately 43% to approximately 45.5%, approximately 44% to approximately 45.5%, approximately 44% to approximately 45.5%, or approximately 45% to approximately 45.5%.

[0197] In some embodiments, one or more N-linked glycans contain G2F located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, with a relative abundance of about 1.5% to about 23%, S1G2F with a relative abundance of about 32% to about 49%, and / or S2G2F with a relative abundance of about 14% to about 48.5%.

[0198] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, and contain G2F with a relative abundance of about 25% or less, S1G2F with a relative abundance of about 33% or more, and / or S2G2F with a relative abundance of about 12% or more.

[0199] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, and include G0F with a relative abundance of about 6.5% or less, G1F with a relative abundance of about 7% or less, G2F with a relative abundance of about 1.5% to about 23%, S1G1F with a relative abundance of about 12.5% ​​or less, S1G2F with a relative abundance of about 32% to about 49%, and / or S2G2F with a relative abundance of about 14% to about 48.5%.

[0200] In some embodiments, one or more N-linked glycans are located at residue Asn76(T5) alone, Asn108(T7) alone, Asn207(T14) alone, or any combination thereof, and include G0F with a relative abundance of about 7.0% or less, G1F with a relative abundance of about 7.5% or less, G2F with a relative abundance of about 25% or less, S1G1F with a relative abundance of about 13.5% or less, S1G2F with a relative abundance of about 33% or more, and / or S2G2F with a relative abundance of about 12% or more.

[0201] The methods of this disclosure are also useful for characterizing, analyzing, or controlling the sialic acid content of proteins. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8 to about 11. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8.3 to about 11, about 9.5 to about 10.1, or about 8.3 to about 10.1. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8.3 to about 11. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 9.5 to about 10.1. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8.3 to about 10.1. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8 to about 11. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 8 to about 10. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NANA of about 9 to about 10.

[0202] In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of about 0.1 to about 2.0. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of 0.90 to about 1.20, or about 0.3 to about 1.2. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of 0.80 to about 1.20. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of 0.80 to about 1.30. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of 0.80 to about 1.40. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of 0.70 to about 1.40. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from 0.70 to about 1.50. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from about 0.1 to about 2. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from about 0.9 to about 1.2. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from about 0.3 to about 1.2. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from about 0.8 to about 1.2. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from about 0.8 to about 1.3. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA from about 0.8 to about 1.4. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of about 0.7 to about 1.4. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of about 0.7 to about 1.5. In some embodiments, one or more N-linked glycans are sialic acid and have a molar ratio of NGNA of about 0.8 to about 1.5.

[0203] The methods of this disclosure are also useful for analyzing the sialylation profiles of glycans present on proteins. In some embodiments, the glycosylation profiles include one or more non-sialylated glycans (domain I), monosialylated glycans (domain II), disialylated glycans (domain III), and / or trisialylated and tetrasialylated glycans (domains IV+V). These domains can be analyzed via imaging capillary isoelectric focusing (iCIEF), a method used to separate proteins by their isoelectric points (pI). In this method, the sample is prepared to a final concentration of approximately 1 mg / mL with water, methylcellulose, amphoteric electrolytes, and a pI marker, and then injected into an imaging capillary isoelectric focusing system (iCIEF) by an autosampler. Electrophoresis separates the sample through a pH gradient in a fluorocarbon (FC) coated capillary based on the charge variation of the isoforms. The results are then compared with a reference separation to compare and characterize groups of constituents.

[0204] In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 28 to about 37, about 29 to about 32, about 28 to about 32, or about 29 to about 37. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 28 to about 37. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 26 to about 40. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 28 to about 39. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 29 to about 39. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 29 to about 38. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 29 to about 37. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 30 to about 37.

[0205] In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 25. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 25.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 26. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 26.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 27. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 27.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 28. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 28.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 29. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 29.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 30. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 30.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 31. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 31.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 32. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 32.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 33. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 33.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 34. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 34.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 35. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 35.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 36. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 36.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of about 37.In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 37.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 38. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 38.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 39. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 39.5. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 40. In some embodiments, the non-sialylated glycan (domain I) has a molar ratio of approximately 31.

[0206] In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 26 to approximately 28, approximately 27 to approximately 33, approximately 26 to approximately 33, and approximately 27 to approximately 28. In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 26 to approximately 33. In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 26 to approximately 28. In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 27 to approximately 28. In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 27 to approximately 29. In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 27 to approximately 30. In some embodiments, the monosially glycan (domain II) has molar ratios of approximately 27 to approximately 31. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 27 to about 32. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 27 to about 33.

[0207] In some embodiments, the monosially glycan (domain II) has a molar ratio of about 20. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 20.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 21. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 21.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 22. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 22.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 23. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 23.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of about 24. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 24.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 25. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 25.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 26. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 26.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 27. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 27.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 28. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 28.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 29. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 29.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 30. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 30.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 31.In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 31.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 32. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 32.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 33. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 33.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 34. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 34.5. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 35. In some embodiments, the monosially glycan (domain II) has a molar ratio of approximately 27.

[0208] In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 27 to about 28, about 22 to about 31, about 27 to about 31, or about 22 to about 28. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 22 to about 28. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 22 to about 31. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 24 to about 31. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 25 to about 31. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 27 to about 28. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 27 to about 35.

[0209] In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 20. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 20.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 21. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 21.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 22. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 22.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 23. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 23.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of about 24. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 24.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 25. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 25.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 26. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 26.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 27. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 27.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 28. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 28.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 29. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 29.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 30. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 30.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 31.In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 31.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 32. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 32.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 33. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 33.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 34. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 34.5. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 35. In some embodiments, the diciallylated glycan (domain III) has a molar ratio of approximately 27.4.

[0210] In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13 to about 16, about 8 to about 16, or about 8 to about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 8 to about 20. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 8 to about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 10 to about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 12 to about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13 to about 20. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13 to about 18. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13 to about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13 to about 16.

[0211] In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 5.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 6. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 6.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 7. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 7.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 8. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 8.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 9. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 9.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 10. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 10.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 11. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 11.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 12. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 12.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 13.5.In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 14. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 14.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 15. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 15.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 16. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 16.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of about 17. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 17.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 18. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 18.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 19. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 19.5. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 20. In some embodiments, the trisialylated and tetrasialylated glycans (domains IV+V) have a molar ratio of approximately 14.6.

[0212] The method of this disclosure is also useful for analyzing O-linked glycans. In some embodiments, the glycosylation profile includes one or more O-linked glycans. In some embodiments, the O-linked glycans are located at residues Ser129, Ser130, Ser136, and / or Ser139.

[0213] In some embodiments, the CTLA4-Fc fusion proteins described herein contain C-terminal lysine. In some embodiments, the C-terminal lysine accounts for a relative abundance of about 20% to about 25%. In some embodiments, the C-terminal lysine accounts for a relative abundance of about 3% to about 10%.

[0214] The method of this disclosure is also useful for analyzing the branched glycans of a CTLA4-Fc fusion protein, comprising measuring one or more N-linked glycans bound to one or more asparagine residues in the CTLA4 protein, wherein one of the branched glycans is G2F. In some embodiments, one or more N-linked glycans bound to one or more asparagine residues in the CTLA4 protein are measured, wherein one of the branched glycans is G0F. In some embodiments, the branched glycans are selected from the group consisting of G0F, G1F, G2F, S1G1F, S1G2F, and / or S2G2F. Liquid chromatography can be used to analyze the glycans of this disclosure. Certain glycoproteins may exhibit carbohydrate heterogeneity. Heterogeneity can be seen at several levels, ranging from fully occupied to unoccupied glycosylation sites, and many different oligosaccharide structures may be present at any particular site, each structure of which may be modified by a sialic acid molecule, e.g., NANA or NGNA.

[0215] In some embodiments, the disclosure provides a method for analyzing the branched glycans of CTLA4-Fc fusion proteins, comprising performing isoelectric focusing of the CTLA4-Fc fusion proteins. In some embodiments, the isoelectric focusing is imaging capillary isoelectric focusing. In some embodiments, the CTLA4-Fc fusion proteins subjected to isoelectric focusing form groups I, II, and III. In some embodiments, group I accounts for 4% or less of the total, group II accounts for 87% or more of the total, and / or group III accounts for 10% or less of the total.

[0216] The carbohydrate content of the proteins of this disclosure can be analyzed by methods known in the art, including the methods described in the examples herein. Several methods for glycosylation analysis are known in the art and are useful in the context of this disclosure. These methods provide information about the entities and composition of oligosaccharides bound to the produced peptides. Useful methods for carbohydrate analysis in relation to this disclosure include, but are not limited to, lectin chromatography, high-performance anion exchange chromatography combined with pulsed amperometric detection (HPAEC-PAD) to separate oligosaccharides based on charge using high-pH anion exchange chromatography, NMR, mass spectrometry, HPLC, and porous graphitized carbon (GPC) chromatography.

[0217] Methods for releasing oligosaccharides include: 1) enzymatic methods commonly performed using peptide-N-glycosidase F / endo-α-galactosidase; 2) β-elimination methods that primarily release O-linked structures using a harsh alkaline environment; and 3) chemical methods that release both N-linked and O-linked oligosaccharides using anhydrous hydrazine. The analytical method may include one or more of the following steps: 1. Dialysis of the sample against deionized water to remove all buffer salts, followed by freeze-drying; 2. Release of intact oligosaccharide chains with anhydrous hydrazine; 3. Treatment of intact oligosaccharide chains with anhydrous methanolic HCl to liberate individual monosaccharides as O-methyl derivatives; 4. N-acetylation of any primary amino group; 5. Derivatization to obtain per-O-trimethylsilylmethyl glycoside; 6. Separation of derivatives by capillary gas-liquid chromatography (GLC) on a CP-SIL8 column; 7. Identification of individual glycoside derivatives by retention time and mass spectrometry by GLC compared with known standards; and 8. Quantification of individual derivatives by FID using an internal standard (13-O-methyl-D-glucose). In some embodiments, branched glycans are measured via ultra-high performance liquid chromatography using fluorescence detection (UPLC-FLR). In some embodiments, the Fc domain of the CTLA4-Fc fusion protein is cleaved before measurement. In some embodiments, the protein is subjected to a viral inactivation step. In some embodiments, the viral inactivation step is performed using 0.5% Triton X-100.

[0218] IV. Pharmaceutical Compositions The compositions prepared by the methods of this disclosure are also intended to be pharmaceutical formulations. A composition acceptable for pharmaceutical administration, such a composition may contain substances that are impurities in levels not exceeding levels acceptable for pharmaceutical administration (including levels in which such impurities are not present), and in addition to any active agent, may contain pharmaceutically acceptable excipients, media, carriers and other inactive components, for example, for the purpose of formulating such a composition to facilitate administration. For example, a pharmaceutically acceptable CTLA4-Ig composition may contain MCP-1 or DNA, insofar as those substances are in levels acceptable for administration to humans.

[0219] This disclosure also provides any of the described CTLA4-Ig molecules as a lyophilized mixture. A formulation containing lyophilized CTLA4-Ig may further comprise three basic components: (1) additional active ingredients, including other proteins or small molecules (e.g., immunosuppressants); (2) excipients; and (3) a solvent. Excipients may include pharmaceutically acceptable reagents (bulkers) that provide good lyophilized cake properties, as well as those that provide lyophilized and / or cryoprotection of proteins ("stabilizers"), pH maintenance (buffers), and proper conformation of proteins during storage, thereby maintaining substantial retention of biological activity (including the stability of the active ingredient, e.g., protein stability). With respect to excipients, an example formulation may comprise one or more of buffers, bulkers, protein stabilizers, and antimicrobial agents. Sugars or polyols may be used as nonspecific protein stabilizers in solution and during freeze-thaw and lyophilization. Polymers can be used to stabilize proteins in solution and during freeze-thaw and freeze-drying. One common polymer is serum albumin, which is used as both a cryoprotectant and a freeze-drying protectant. In one embodiment, the present disclosure provides an albumin-free formulation. Various salts can be used as extenders. Exemplary salt extenders include, for example, NaCl, MgCl2, and CaCl2.

[0220] Certain amino acids can be used as cryoprotectants and / or lyophilization protectants and / or bulking agents. Possible amino acids include, but are not limited to, glycine, proline, 4-hydroxyproline, L-serine, monosodium glutamate, alanine, arginine, and lysine hydrochloride. Many buffers covering a wide pH range can be selected for the formulation. Examples of buffers include acetates, citrates, glycine, histidine, phosphates (sodium or potassium), diethanolamine, and Tris. Buffers include agents that maintain the solution pH within an acceptable range before lyophilization. In one embodiment, the disclosure provides a lyophilized CTLA4-Ig mixture comprising at least 90%, 95%, 99%, or 99.5% CTLA4-Ig dimers containing any sequence from any one of SEQ ID NOs: 1-8. In one embodiment, the disclosure provides a lyophilized CTLA4-Ig mixture comprising at least 90%, 95%, 99%, or 99.5% of CTLA4-Ig dimers and 5%, 4%, 3%, 2%, or 1% or less of CTLA4-Ig tetramers. In another embodiment, the disclosure provides a lyophilized CTLA4-Ig mixture comprising at least 90%, 95%, 99%, or 99.5% of CTLA4-Ig dimers, 5%, 4%, 3%, 2%, or 1% or less of CTLA4-Ig tetramers, and 2%, 1.5%, 1.0%, 0.8%, 0.5%, or 0.3% or less of CTLA4-Ig monomers. In a further embodiment, the present disclosure provides a lyophilized CTLA4-Ig mixture containing at least 8.0 moles of sialic acid per mole of CTLA4-Ig dimers or CTLA4-Ig molecules.In another embodiment, the Disclosure provides a lyophilized CTLA4-Ig mixture comprising about 15 to about 35 moles of GlcNac per mole of CTLAIg molecules or dimers, about 1 to about 5 moles of GalNac per mole of CTLA4-Ig dimers or CTLA4-Ig molecules, about 5 to about 20 moles of galactose per mole of CTLA4-Ig dimers or CTLA4-Ig molecules, about 2 to about 10 moles of fucose per mole of CTLA4-Ig dimers or CTLA4-Ig molecules, and / or about 5 to 15 moles of mannose per mole of CTLA4-Ig dimers or CTLA4-Ig molecules.

[0221] This disclosure also provides any of the described CTLA4-Ig molecules as subcutaneous (SC) formulations. IV formulations are inconvenient for patients requiring frequent, long-term treatment. Patients must frequently visit the hospital to receive the drug via IV infusions that can last up to an hour. Therefore, SC formulations that can be self-administered at home are considered highly beneficial. For subcutaneous administration, dosage forms with high protein concentrations are desirable. Treatment with high doses exceeding 1 mg / kg (more than 100 mg per dose) requires the development of formulations at concentrations exceeding 100 mg / ml due to the small volume that can be administered via the SC route (less than 1.5 ml). The SC formulations of this disclosure contain, in an aqueous carrier, the CTLA4Ig molecule in combination with a stabilizing level of sugar at a protein concentration of at least 100 mg / ml, preferably at a protein concentration of at least 125 mg / ml in combination with a stabilizing level of sugar. Preferably, the weight ratio of the sugar to the protein is at least 1:1.1. The stabilizer is preferably used in amounts that do not result in undesirable or inappropriate viscosity for administration via the SC syringe. The sugar is preferably a disaccharide, most preferably sucrose. The SC formulation may also contain one or more components selected from a list consisting of buffers, surfactants, and preservatives.

[0222] V. Treatment Methods The compositions prepared by the methods of this disclosure are useful for treating various diseases. This disclosure provides a method for inhibiting the proliferation (or activation) of T cells, comprising contacting T cells with an effective amount of the CTLA4-Ig composition of this disclosure. This disclosure provides a method for inhibiting an immune response in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject that requires it. This disclosure provides a method for inducing immune tolerance to an antigen in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject that requires it. This disclosure provides a method for treating inflammation in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject that requires it. This disclosure provides a method for treating rheumatoid arthritis, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject that requires it.

[0223] This disclosure provides a method for treating psoriasis in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need. This disclosure provides a method for treating or preventing allergies in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need. This disclosure provides a method for treating or preventing graft-versus-host disease in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need. This disclosure provides a method for treating or preventing rejection of a transplanted organ in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need.

[0224] This disclosure provides a method for treating Crohn's disease in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need of treatment. This disclosure also provides a method for treating type 1 diabetes in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need of treatment.

[0225] This disclosure provides a method for treating oophoritis in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need. This disclosure provides a method for treating glomerulonephritis in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need. This disclosure provides a method for treating allergic encephalomyelitis in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need.

[0226] This disclosure provides a method for treating myasthenia gravis in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure to a subject in need thereof. Accordingly, in certain aspects of this disclosure, the disclosure generally includes any T cell-dependent lymphoproliferative disorder or disorder, and any T cell-dependent autoimmune disorder or disorder, more specifically, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentral T cell lymphoma, benign lymphocytic vasculitis, graft-versus-host disease (GVHD), immune disorders associated with graft rejection, psoriasis, inflammation, allergy, oophoritis, glomerulonephritis, encephalomyelitis, Hashimoto's thyroiditis, Graves' disease, Addison's disease, primary thyroiditis The present invention provides CTLA4-Ig molecules produced by cell lines in the production method described herein for the treatment of T-cell-related diseases or disorders, including but not limited to effusion, pernicious anemia, autoimmune atrophic gastritis, rheumatoid arthritis, insulin-dependent diabetes mellitus, Goodpasture syndrome, myasthenia gravis, pemphigus, sympathetic ophthalmitis, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic hepatitis, scleroderma, polymyositis, and mixed connective tissue disease.

[0227] This disclosure provides a method for inhibiting the proliferation (or activation) of T cells, comprising contacting T cells with an effective amount of the CTLA4-Ig composition of this disclosure, with or without combination with another agent, such as methotrexate. This disclosure provides a method for inhibiting an immune response in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure, alone or in combination with methotrexate, to a subject in need thereof. This disclosure provides a method for inducing immune tolerance to a subject in a subject, comprising administering an effective amount of the CTLA4-Ig composition of this disclosure, in combination with methotrexate, to a subject in need thereof.

[0228] Various aspects of this disclosure are described in further detail in the following subsections. This disclosure is further illustrated by the following embodiments, which should not be construed as further limitations. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Examples]

[0229] [Example 1]

[0230] 5L bench scale manufacturing Abatacept is a genetically engineered fusion protein consisting of a human cytotoxic T lymphocyte antigen-4 (CTLA-4) functional binding domain and the Fc domain of a human monoclonal immunoglobulin of the IgG1 class. Abatacept is composed of two homologous glycosylated polypeptide chains, each approximately 46 kDa in size, covalently linked by a single disulfide bond.

[0231] Abatacept is produced as an extracellular protein in large-scale cell cultures using the Chinese hamster ovary (CHO) cell line. The production process begins with thawing working cell bank (WCB) vials. The cultures are grown in a series of baffled shaking flasks, cell bag bioreactors, and seed bioreactors. The final inoculated cultures are transferred to a production bioreactor. Collection from the production bioreactor is performed based on the target molar ratio (MR) of sialic acid (SA) to abatacept protein and the cell culture time. The cell culture collection is clarified by a primary collection process and subsequent sterile-grade filtration. Cell-free collection material is adjusted to achieve a specified pH, and Triton X-100 is added for viral inactivation in preparation for downstream processing.

[0232] Figures 1A, 1B, and 1C show various glycans that may be present at positions T5, T7, and T14. Figure 1D shows the specific locations of N-linked glycosylation sites (T5, T7, and T14) in the primary amino acid sequence of abatacept (also found in SEQ ID NO: 5). Figure 2A shows the mean viable cell density (VCD) profile for each treatment group, and an overview of this bioreactor cell proliferation data by treatment group can be seen in Figure 2B. The treatment groups with a low initial pH (6.9) and a low initial VCD tended to be the lowest among the groups throughout the entire operation, with the two lowest peak VCD values ​​reached between days 11 and 12. Under the early pH shift condition, proliferation was comparable to other conditions until day 3, but gradually deviated from the control group after the pH shift on day 3, reaching a lower peak VCD. The low overall temperature group showed slower proliferation than the control treatment from day 6 onward. The effect of this proliferation can be seen in the flatter shape of the curve and the relatively lower peak VCD. The treatment group with a high initial VCD and the treatment group with the first slower supply tended to be the highest within the group from day 3 onwards. The inoculum was scaled up and passed through two 5L seed bioreactors (N-2 and N-1) before the production bioreactor. The production bioreactor stage was then operated for 14-17 days post-inoculation. Table 1 shows the operating parameters of the centerpoint 5L production bioreactor used in this study. Cell viability profiles can be seen in Figure 3 for each of the test conditions. 0.45 × 10 6 The initial cell density of cells / mL showed the greatest decline in viability by day 14, with a cell viability of approximately 86%. This suggests that the cell density reached a level too high to sustain a higher level of cell viability.

[0233] Glycan profile analysis of a 5L bioreactor Sialic acid content was analyzed via mass spectrometry. The mean values ​​of NANA, NGNA, and N-binding linkages before collection on day 14 are shown in Figure 4A, along with the p-values ​​of Dunnett's test comparing the results of each treatment group to the control. Domain IV+V is defined as the sum of the values ​​of N-binding domains IV and V. Treatments with a p-value of ≤0.05 were considered statistically significant compared to the control. The values ​​of NANA, NGNA, domains I, II, and III on day 14 did not show significant differences compared to the control. Most domain IV+V values ​​did not show significant differences compared to the control. However, treatment groups with lower overall temperatures and lower temperature shifts of 1 showed significantly lower levels of domain IV+V compared to the control. The specification for DS release related to domain IV+V is ≤16%, and therefore this reduction does not pose a risk to product quality. The mean day 14 titer and mean specific productivity (Qp) values, along with the p-values ​​from the controlled Dunnett test comparing the treatment group with the control, can be seen in Figure 4B. A p-value of 0.05 or less was considered statistically significant. Lower initial pH, lower initial VCD, and lower overall temperature were shown to result in significantly lower day 14 titer compared to the control. Conditions of lower initial pH and earlier pH shift timing were found to result in significantly higher specific productivity than the control.

[0234] [Table 2]

[0235] The tests were conducted using the one-of-a-kind (OFAT) method, as shown in Table 2 below, in which a single parameter was varied relative to a centerpoint control under each condition. The glycosylation data can be seen in Figures 4A-4H. Each setting was adjusted as follows.

[0236] [Table 3]

[0237] Experimental results Detailed characterization of N-glycosylation on the abatacept molecule was performed after collection from a bioreactor. Bioreactor samples were purified using a Protein A Waters HPLC system. The purified samples were then concentrated to a final concentration of over 4.0 g / L using a 10 kD cutoff centrifugal filter unit (Millipore). The resulting abatacept protein was reduced, alkylated, digested with trypsin, and then digested with GluC. The trypsin-GluC digests were separated by reverse-phase chromatography and detected by mass spectrometry. The proportion of each glycoform was calculated by dividing the peak area under the extracted ion chromatogram of that glycopeptide by the sum of the peak areas of all target glycopeptides. The glycans were released and labeled using a fluorescent labeling kit, and the samples were then analyzed using a high-performance liquid chromatography system with fluorescence detection (UPLC-FLR). The glycans G0F, G2F, S1G2F, S2G2F, S1G3F, and S2G4F were analyzed by mass spectrometry. The results of the analysis of these six glycans can be seen in Figures 4C to 4H. Figure 4C shows the average liquid chromatography-mass spectrometry (LC-MS) results for the G0F glycoform, reported as relative abundances at the T5 and T7 N-glycosylation sites on the abatacept molecule. Figure 4D shows the average liquid chromatography-mass spectrometry (LC-MS) results for the G2F glycoform, reported as relative abundances at the T5 and T7 N-glycosylation sites on the abatacept molecule. Figure 4E shows the average liquid chromatography-mass spectrometry (LC-MS) results for the S1G2F glycoform, reported as relative abundances at the T5 and T7 N-glycosylation sites on the abatacept molecule. Figure 4F shows the average liquid chromatography-mass spectrometry (LC-MS) results for the S2G2F glycoform, reported as relative abundances at the T5 and T7 N-glycosylation sites on the abatacept molecule.Figure 4G shows the average liquid chromatography-mass spectrometry (LC-MS) results for the S1G3F glycoform, reported as relative abundances at the T5 and T7 N-glycosylation sites on the abatacept molecule. Figure 4H shows the average LC-MS results for the S2G4F glycoform, reported as relative abundances at the T5 and T7 N-glycosylation sites on the abatacept molecule. [Example 2]

[0238] Effect of cell age on pre-collection N-glycoform distribution in the bioreactor step for the CTLA4-Ig process. To understand the impact of the number of inoculum generations on process and quality attributes in the abatacept manufacturing process at lower temperature setpoints, we investigated unsuitable boundaries for the total number of cell generations inoculated into the bioreactor. All conditions were tested as double-repeated tests in a 5L bioreactor. Cell vials were thawed, passaged, and augmented, and finally banked at passages 5, 10, 15, and 21. Before starting this bioreactor test, cell bank vials were thawed and augmented over 6 generations to a maximum of 3L. Each 3L flask was used to inoculate individual n-2 seed bioreactors, which were then combined into an n-1 seed bioreactor. Each n-1 seed bioreactor was used to inoculate two 5L production bioreactors.

[0239] Cell culture performance, including VCD, viability, pH, DO, and nutrient and metabolite levels, was monitored daily. Titer and sialic acid content were measured and analyzed daily from day 10 to day 16. High molecular weight species (HMW), residual DNA, host cell proteins (HCP), and N-linked glycosylation were measured on day 16 (Figure 5). Glycoform distribution was measured by LC / MC on day 16 (Figure 5). [Example 3]

[0240] Temperature range of production bioreactors To evaluate the acceptable temperature range in the production bioreactor step, the effects of bioreactor inoculation live cell density (VCD), pH shift setpoint, PCO2 shift setpoint, first temperature setpoint, second temperature setpoint, and third temperature setpoint on process performance and process quality were analyzed. The process parameters and characterization ranges are shown in Figure 6A. Characterization of the production bioreactor was performed using 5L Sartorius and Finesse vessels and controllers. The operating conditions for the production bioreactor step followed Table X, except for the six experimental ranges shown in Figure 6A. Setpoints or midpoints for the evaluated process parameters were varied among 22 treatments, including two controls, as shown in Table Y. Repeated tests were run only under control and centerpoint conditions.

[0241] [Table 4]

[0242] [Table 5]

[0243] Figure 7 shows a summary of the significant primary effects of process parameters. Process parameter interactions and secondary effects were found to be statistically significant with respect to process performance and product quality. All secondary effects included in the study design influenced either process attributes or quality attributes. The following parameter interactions were determined to influence either process attributes or quality attributes: inoculated VCD × first temperature setpoint, inoculated VCD × third temperature setpoint, inoculated VCD × second temperature setpoint, second temperature setpoint × pCO2 setpoint, second temperature setpoint × pH shift setpoint, and third temperature shift setpoint × pH shift setpoint. [Example 4]

[0244] Industrial-scale culture of suspension mammalian cells expressing CTLA4-Ig This embodiment describes the production of the CTLA4-Ig molecule. The method described in this embodiment can be adapted and extended for the production of secretory proteins such as cytokines and other hormones, secretory proteins that are members of the Ig superfamily or contain a portion of the Ig superfamily proteins, and any protein commonly expressed in CHO cells, including but not limited to these.

[0245] To obtain a sufficient number of viable cells to inoculate a 25,000 L bioreactor, culture flasks (e.g., shaking flasks and Erlenmeyer flasks, rotary bottles, and cell bags) were used for the inoculum augmentation step of the CTLA4-Ig culture process to continuously grow cells from frozen vials.

[0246] Industrial-scale production of CTLA4-Ig: The production phase of this disclosure, carried out in a 25,000 L production bioreactor with a culture operation involving a two-stage temperature shift, produces CTLA4-Ig protein in both high volume and high quality. The bioreactor is replenished with feed medium approximately 76 hours after induction, and this replenishment is performed daily for the production reactor. The 25,000 L culture is incubated in CD-CHO medium at 36°C until approximately 144 hours after induction, and then subjected to a temperature shift (T-shift) from 36°C to 33°C at approximately 144 hours (end of the logarithmic growth phase). The temperature of 33°C is maintained for approximately 144 hours to approximately 240 hours. The 25,000 L culture is then collected approximately 240 hours after induction, subjected to a second and final temperature shift (T-shift) from 33°C to 31°C until approximately 240 hours.

[0247] Samples were collected daily from the production bioreactor for analysis. For example, samples used for cell counting were stained with trypan blue (Sigma, St. Louis, Mo.). Cell number and viability were determined using a hemocytometer to count stained viable cells under a microscope. For metabolite analysis, additional sample aliquots were centrifuged at 2000 rpm (4C) for 20 minutes to pellet the cells. Using conventional methods and protocols in the art, the supernatant was analyzed for protein titer, sialic acid, glucose, lactate, glutamine, glutamic acid, pH, pO2, pCO2, ammonia, and LDH. [Example 5]

[0248] Glycosylation analysis of CTLA4-Ig produced in a 25,000L bioreactor. Glycan content was analyzed by imaging capillary isoelectric focusing (iCIEF). This method is used to separate proteins by their isoelectric point (pI). In this method, the abatacept sample was prepared to a final concentration of approximately 1 mg / mL with water, methylcellulose, amphoteric electrolytes, and a pI marker, and then injected into the imaging capillary isoelectric focusing system (iCIEF) by an autosampler. In electrophoresis, the sample is separated through a pH gradient in a fluorocarbon (FC) coated capillary based on the charge variation of the isoforms. After high-voltage focusing, the movement of the sample is captured by a whole-column CCD camera, and quantitative analysis of the peaks is performed using the appropriate software. The results are reported as the percentage of total glycoforms present in the sample and can be seen in Figures 8A-8B and 9A-9B.

[0249] Analysis of glycosylated domains of CTLA4-Ig produced in a 25,000L bioreactor. The N-linked oligosaccharide profile (glycosylation pattern) of abatacept is determined. Oligosaccharides on abatacept are released by enzymatic hydrolysis using PNGase F. The profile of the released oligosaccharides is determined by high-speed anion exchange chromatography (HPAEC) using electrochemical detection. The oligosaccharide profile of the active pharmaceutical ingredient is evaluated against a sample of a reference material run simultaneously. The results are reported either as the absolute area percentage of the selected domain or as the deviation percentage of the selected domain from the same domain in the reference standard. The results for domains I, II, III, and VI+V can be seen in Figures 10A-10D. The results of sialic acid analysis (NANA and NGNA) can be seen in Figures 10E and 10F. [Example 6]

[0250] In vivo efficacy of abatacept A clinical trial was conducted to compare the pharmacokinetics of abatacept prepared by the reference process (e.g., step F as described in PCT / US2006 / 049074) and the process described herein (e.g., step J). This clinical trial was an open-label, randomized, parallel-group, single-dose study conducted to compare the pharmacokinetics (PK) of abatacept prepared by the process described herein and the reference process after a single dose (750 mg administered as a 30-minute IV infusion) in healthy participants.

[0251] Results from clinical PK trials demonstrated the need to manage the N-glycan composition of abatacept. The N-glycan composition was determined by the HILIC N-linked glycan profiling method, designed to quantify the sum of major branched glycoforms on the CTLA4 region that affect PK.

[0252] Clinical trials demonstrated that CTLA4 G2F, S1G2F, and S2G2F glycoforms had the greatest impact on overall control strategies and PK; therefore, two-sided limit values ​​were proposed for these glycans (Table 3). CTLA4 G2F is a critical quality attribute of abatacept due to its strong correlation with PK removal. S1G2F had minimal impact on PK, and a two-sided limit value was applied for manufacturing consistency. S2G2F was strongly correlated with G2F in both the manufacturing process and removal, and a two-sided limit value was applied for it. G0F, G1F, and S1G1F had negligible impacts on overall removal, and therefore one-sided limit values ​​were proposed. Further rationale is shown in Table 3. The proposed limit values ​​for all glycans in Table 3 ensured glycan control within acceptable PK parameters. Failure of one attribute resulted in batch rejection to maintain consistency with clinical and manufacturing experience.

[0253] [Table 6]

Claims

1. A method for controlling the cell proliferation rate, cell viability, viable cell density and / or titer of CHO cells expressing the protein abatacept in order to produce the protein abatacept, comprising culturing the cells in a bioreactor under suitable conditions, wherein the suitable conditions include (i) culturing the cells for the protein induction phase at a first temperature setting of 36°C, and culturing the cells at a second temperature setting of 33°C and a final temperature setting of 31°C, and (ii) a pH shift over 96 hours, wherein the suitable conditions include, (a) an initial pH setting of 7.0 and a second pH setting of 6.9, or an initial pH setting of 6.9; (b) 0.30 × 10 ６ The initial viable cell density (VCD) setting is cells / mL; (c) The first CO between 15% and 25% ２ Setting value; (d) First supply time of 76 hours; or (e) Any combination of (a) to (d) Methods that further include the following.

2. Suitable conditions, (i) a first temperature setting of 36°C, a second temperature setting of 33°C, and a third temperature setting of 31°C; (ii) A first pH setting of 7.0 and a second pH setting of 6.9; (iii) 0.30×10 ６ The initial viable cell density (VCD) setting is cells / mL; and (iv) The first CO between 15% and 25% ２ Set value The method according to claim 1, including the method described in claim 1.

3. (i) The last temperature setting occurs between 228 and 252 hours; (ii) The second temperature setting occurs between 120 hours and 168 hours; or (iii) Both (i) and (ii) The method according to claim 1 or 2.

4. (i) Reduce the rate of cell proliferation; (ii) Controlling cell viability; (iii) Controlling the titer; (iv) Control the glycosylation profile of abatacept protein; or (v)(i) to (iv) is one of the combinations, The method according to any one of claims 1 to 3.

5. (i) Cell proliferation exhibits an average doubling time of 0–5 days, which is between 30.0 and 40.0 hours; (ii) Cell viability is (a) 10.0 × 10 ６ From 15.0 × 10 cells / mL ６ It shows an average peak live cell density (VCD) of cells / mL; or (b) 0.05 × 10 ９ From 0.11 × 10¹ / mL ９ This shows the mean live cell density integral (IVCD) from 0 to 14 days, which is cells / mL; (iii) Potency is, (a) exhibiting a 14-day average titer of 1.50 g / L to 3.5 g / L; or (b) Shows the average specific productivity from 20.0 pg / cell / day to 40.0 pg / cell / day; (iv) The glycosylation profile contains one or more N-linked glycans; or (v)(i) to (iv) is one of the combinations, The method according to claim 4.

6. the process further comprises modifying bioreactor parameters, wherein the bioreactor parameters are (i) feeding time, (ii) initial pH, (iii) pH shift, (iv) CO ２ concentration, (v) initial cell density, or (vi) any combination thereof. The method according to any one of claims 1 to 5.

7. The method according to claim 5, wherein the N-linked glycan comprises G0F, G1F, G2F, S1G1F, S1G2F, and / or S2G2F.

8. (i) G0F accounts for a relative abundance of 7.0% or less, or 6.5% or less; (ii) G1F accounts for a relative abundance of 7.5% or less or 7%; (iii) G2F accounts for a relative abundance of 25% or less or between 1.5% and 23%; (iv) S1G1F accounts for a relative abundance of 13.5% or less or 12.5%; (v) S1G2F accounts for a relative abundance of 33% or more, or between 32% and 49%; (vi) S2G2F accounts for a relative abundance of 12% or more, or between 14% and 48.5%; or (vii) is one of the combinations of (i) to (vi), The method according to claim 7.

9. (i) G2F accounts for a relative abundance of 1.5% to 23%, S1G2F accounts for a relative abundance of 32% to 49%, and / or S2G2F accounts for a relative abundance of 14% to 48.5%; (ii) G2F accounts for a relative abundance of 25% or less, S1G2F accounts for a relative abundance of 33% or more, and / or S2G2F accounts for a relative abundance of 12% or more; (iii) G0F accounts for a relative abundance of 6.5% or less, G1F accounts for a relative abundance of 7% or less, G2F accounts for a relative abundance of 1.5% to 23%, S1G1F accounts for a relative abundance of 12.5% ​​or less, S1G2F accounts for a relative abundance of 32% to 49%, and / or S2G2F accounts for a relative abundance of 14% to 48.5%; or (iv) G0F accounts for a relative abundance of 7.0% or less, G1F accounts for a relative abundance of 7.5% or less, G2F accounts for a relative abundance of 25% or less, S1G1F accounts for a relative abundance of 13.5% or less, S1G2F accounts for a relative abundance of 33% or more, and / or S2G2F accounts for a relative abundance of 12% or more. The method according to claim 7 or 8.

10. The method according to any one of claims 5 to 9, further comprising measuring the glycosylation profile after 14 days.

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