Cell culture methods and compositions for antibody production

Abstract

The present invention provides cell culture methods for producing anti-α4β7 antibodies, such as vedolizumab, and compositions thereof.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 859,563, filed June 10, 2019, and U.S. Provisional Application No. 62 / 859,596, filed June 10, 2019. The entire contents of the foregoing priority applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on June 10, 2020, is entitled "T103022_1110WO_SL.TXT" and is 14.0 kilobytes in size.

[0003] The present invention relates to methods and compositions for producing anti-α4β7 antibodies in mammalian host cells. [Background technology]

[0004] Mammalian cell culture techniques are commonly used for the production of therapeutic biologics, including therapeutic monoclonal antibodies. Because proteins produced in mammalian cells generally have post-translational modifications more similar to those produced in humans, mammalian cells are typically preferred in the pharmaceutical industry over other forms of eukaryotic cells (e.g., yeast) or prokaryotic cells (e.g., bacteria) for protein production. However, culturing mammalian cells can be challenging, particularly in the context of therapeutic antibodies produced on a commercial scale for human use, as these cells present numerous challenges. Production methods must maximize antibody yield from cells while maintaining the safety, efficiency, and cost-effectiveness of the protein product. Therefore, production requirements are significant, as desired product quality attributes, such as glycosylation profile, aggregate levels, charge heterogeneity, and amino acid sequence integrity, must be maintained (Li et al., 2010, mAbs, 2(5):466-477).

[0005] Given the complexity of the cell culture process, it can be difficult to identify cell culture parameters that can address the challenges associated with therapeutic antibody production, including producing enough protein product to meet manufacturing demands and therapeutic requirements while maintaining high-quality pharmaceutical products. Summary of the Invention

[0006] Although mammalian cell culture processes have been the subject of research over the past several decades, there remains a need for improvements in the large-scale commercial production of recombinant antibodies. Increasing cell viability, lifespan, and specific productivity of mammalian host cell cultures, as well as improving the titer of the recombinant protein produced, can have a real impact on the price of the recombinant protein produced and, in the case of therapeutic proteins, the price and availability of the drug. Furthermore, such increases can be particularly challenging given the need to maintain consistency in the quality of the therapeutic antibodies produced.

[0007] The inventions provided herein disclose, inter alia, cell culture methods and compositions for producing anti-α4β7 antibodies, such as vedolizumab, in mammalian host cells. Also provided herein are compositions comprising anti-α4β7 antibodies, such as vedolizumab, obtained using the methods.

[0008] In one aspect, the invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising culturing mammalian host cells in a production medium and adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition comprising the humanized anti-α4β7 antibody, wherein the mammalian host cells have been genetically engineered to express a humanized anti-α4β7 antibody that is an IgG1 antibody and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable domain comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0009] In some embodiments of the foregoing aspects, the method is a method for producing a composition having a reduced amount of a basic isoform of a humanized anti-α4β7 antibody (as determined by cation exchange chromatography (CEX)), the method comprising culturing mammalian host cells in a production medium and adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having a reduced amount of the basic isoform of a humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplements.

[0010] In some embodiments of the above aspects, the invention features a method for producing a composition having about 16% or less basic isoforms of a humanized anti-α4β7 antibody (as determined by CEX), the method including culturing mammalian host cells in a production medium and adding supplements to the production medium including uridine, manganese, and galactose, thereby producing a composition having about 16% or less basic isoforms of a humanized anti-α4β7 antibody.

[0011] In one embodiment, the composition comprises about 14% or less of the basic isoform of the humanized anti-α4β7 antibody.

[0012] In another embodiment, the composition comprises about 13% or less of the basic isoform of the humanized anti-α4β7 antibody.

[0013] In one embodiment, supplements are added to the production medium or to a feed medium which is then added to the production medium.

[0014] In one embodiment, the cumulative concentration of uridine added to the production medium between supplementation and harvest is about 1 to about 7 mM, the cumulative concentration of manganese added to the production medium between supplementation and harvest is about 0.002 to about 0.015 mM, and / or the cumulative concentration of galactose added to the production medium between supplementation and harvest is about 3 to about 20 mM. In a specific embodiment, the feed medium further comprises zinc. In one embodiment, the cumulative concentration of zinc added to the production medium between supplementation and harvest is about 0.05 mM to about 0.045 mM.

[0015] In one embodiment, manganese is added multiple times to the production medium as a supplement, with each addition being about 0.1-10 μM, about 0.2-1.5 μM, about 0.2-5 μM, about 0.25-2 μM, about 0.3-1.2 μM, or about 0.3-0.8 μM. In a specific embodiment, manganese is added multiple times to the production medium as a supplement, with each addition being about 0.2-1.5 μM.

[0016] In one embodiment, uridine is added to the production medium as a supplement multiple times, with each addition being about 25 to 1000 μM, about 75 to 750 μM, about 55 to 620 μM, about 100 to 600 μM, about 150 to 450 μM, about 100 to 700 μM, about 100 to 600 μM, or about 170 to 630 μM. In a specific embodiment, uridine is added to the production medium as a supplement multiple times, with each addition being about 100 to 700 μM.

[0017] In one embodiment, galactose is added multiple times to the production medium as a supplement, with each addition being about 0.1-10 mM, 0.2-7.5 mM, 0.5-5 mM, 0.4-2.8 mM, 0.5-3.5 mM, 0.7-2.9 mM, 0.75-2.5 mM, or about 1.2 mM or 1.4 mM. In a specific embodiment, galactose is added multiple times to the production medium as a supplement, with each addition being about 0.5-3.5 mM.

[0018] In one embodiment, the supplemental components are added every day or every two days. In a specific embodiment, the supplemental components are added beginning on day 4 of the production stage culture.

[0019] In one embodiment, uridine is added to the feed medium to a final concentration of about 15-120 mM. In one embodiment, uridine is added to the feed medium to a final concentration of about 20-70 mM uridine. In one embodiment, uridine is added to the feed medium to a final concentration of about 1-40 mM uridine.

[0020] In one embodiment, manganese is added to the feed medium to a final concentration of about 0.02-0.3 mM. In one embodiment, manganese is added to the feed medium to a final concentration of about 0.04-0.15 mM. In one embodiment, manganese is added to the feed medium to a final concentration of about 0.0001-0.1 mM.

[0021] In further embodiments, galactose is added to the feed medium to a final concentration of about 85 mM to 600 mM. In one embodiment, galactose is added to the feed medium to a final concentration of about 160 to 340 mM. In one embodiment, galactose is added to the feed medium to a final concentration of about 50 to 150 mM.

[0022] In another embodiment, the feed medium further comprises zinc. In one embodiment, the concentration of zinc in the feed medium is about 90 μM to 120 μM. In one embodiment, the concentration of zinc in the feed medium is about 50 μM to 150 μM.

[0023] In one embodiment, the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0024] In one embodiment, the method further increases the percentage of the major isoform species of the humanized anti-α4β7 antibody compared to the percentage of the major isoform species produced in the absence of a feed medium comprising uridine, manganese, and galactose added to the production medium.

[0025] In one embodiment, the process is a fed-batch process.

[0026] In one embodiment, the feed medium is added to the production medium starting from about day 4 of the production phase.

[0027] In another aspect, the present invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising culturing mammalian host cells in a production medium comprising zinc, thereby producing a composition comprising a humanized anti-α4β7 antibody, wherein the mammalian host cells have been genetically engineered to express a humanized anti-α4β7 antibody that is an IgG1 antibody and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0028] In some embodiments of the foregoing aspects, the method is a method for producing a composition having a reduced amount of a basic isoform of a humanized anti-α4β7 antibody (as determined by cation exchange chromatography (CEX)), the method comprising culturing mammalian host cells in a production medium containing zinc, thereby producing a composition having a reduced amount of the basic isoform of a humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of zinc.

[0029] In some embodiments of the above aspects, the method is a method for producing a composition having about 16% or less basic isoforms of a humanized anti-α4β7 antibody (as determined by CEX), the method comprising culturing mammalian host cells in a production medium containing zinc, thereby producing a composition having about 16% or less basic isoforms of a humanized anti-α4β7 antibody.

[0030] In one embodiment, the composition comprises about 14% or less of the basic isoform of the humanized anti-α4β7 antibody.

[0031] In one embodiment, the composition comprises about 13% or less of the basic isoform of the humanized anti-α4β7 antibody.

[0032] In one embodiment, the concentration of zinc in the production medium is between 2 μM and 60 μM.

[0033] In a further embodiment, the method includes supplementing the production medium with zinc by adding a feed medium containing zinc to the production medium, hi one embodiment, the feed medium is added to the production medium starting at about day 4 of the production phase.

[0034] In one embodiment, the concentration of zinc in the feed medium is between about 90 μM and 120 μM.

[0035] In one embodiment, the production medium contains 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine. In one embodiment, the production medium contains 4.5 to 5.5 g / L of lysine. In one embodiment, the production medium contains 5.5 to 8.8 g / L of lysine. In one embodiment, the production medium contains 5.4 to 7.4 g / L of arginine. In one embodiment, the production medium contains 7.4 to 12 g / L of arginine.

[0036] In a further aspect, the invention features a method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising culturing mammalian host cells in a production medium during a production stage, such that a composition comprising the humanized anti-α4β7 antibody is produced, wherein the production medium has an average temperature of about 37 degrees Celsius, and the host cells are genetically engineered to express a humanized IgG1 anti-α4β7 antibody, wherein the humanized anti-α4β7 comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0037] In some embodiments of the foregoing aspects, the method is a method for producing a composition comprising 2.5% or less HMW species of a humanized anti-α4β7 antibody (as determined by SEC), the method comprising culturing mammalian host cells in a production medium such that during the production step a composition comprising 2.5% or less HMW species of a humanized anti-α4β7 antibody (as determined by SEC) is produced.

[0038] In yet another aspect, the present invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising culturing mammalian host cells in a growth medium during an expansion phase, wherein the mammalian host cells are genetically engineered to express a humanized anti-α4β7 antibody; and culturing the mammalian host cells in a production medium during a production phase, such that a composition comprising the humanized anti-α4β7 antibody is produced, wherein the mammalian host cells are cultured at approximately the same temperature during both the expansion phase and the production phase, and the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0039] In some embodiments of the foregoing aspects, the method is a method for producing a composition comprising a high level of humanized anti-α4β7 antibody monomer (as determined by SEC), the method comprising culturing mammalian host cells in a growth medium during an expansion phase, wherein the mammalian host cells are genetically engineered to express the humanized anti-α4β7 antibody, and culturing the mammalian host cells in a production medium during a production phase, such that a composition comprising a high level of humanized anti-α4β7 antibody monomer is produced.

[0040] In one embodiment, the temperature is between 36 and 38 degrees Celsius. In another embodiment, the average temperature is between 36.5 and 37.5 degrees Celsius. In yet another embodiment, the temperature is an average temperature of about 37 degrees Celsius.

[0041] In one embodiment, the production medium of the methods disclosed herein has a temperature in the range of 36-38 degrees Celsius. In one embodiment, the temperature is in the range of 36.5-37.5 degrees Celsius. In one embodiment, the temperature is an average temperature of about 37 degrees Celsius. In one embodiment, the production medium has a pH in the range of 6.5-7.

[0042] In one embodiment, the production medium of the methods disclosed herein has a pH in the range of 6.8 to 7.0.

[0043] In one embodiment, the production medium of the methods disclosed herein has a glucose level maintained at or below about 7 g / L during the production stage.

[0044] In one embodiment, the production phase is 14 days or less, hi another embodiment, the production phase is in the range of 10 to 17 days.

[0045] In some embodiments of the above aspects, the method is performed in a large-scale bioreactor. In particular embodiments, the large-scale bioreactor is selected from the group consisting of a 200 liter (L) bioreactor, a 2000 L bioreactor, a 3000 L bioreactor, and a 6000 L bioreactor.

[0046] In some embodiments, the production step results in a humanized anti-α4β7 antibody titer of greater than 3 g / L. In certain embodiments, the humanized anti-α4β7 antibody titer is about 3 to about 8 g / L. In other embodiments, the humanized anti-α4β7 antibody titer is about 5 to about 7 g / L.

[0047] In some embodiments of the above aspects, the mammalian host cell is a Chinese hamster ovary (CHO) cell. In particular embodiments, the CHO cell is a GS-CHO cell.

[0048] In some embodiments of the above aspects, the humanized anti-α4β7 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5.

[0049] In some embodiments of the above aspects, the humanized anti-α4β7 antibody is vedolizumab.

[0050] In some embodiments of the foregoing aspects, the method includes recovering and purifying the antibody. In some such embodiments, the purification includes (i) purification steps to remove cellular debris, undesired proteins, salts, minerals, or other undesired elements, and (ii) purification of the antibody from contaminating soluble proteins and polypeptides. In certain embodiments, the method further includes preparing a formulation of the purified antibody suitable for human therapeutic use.

[0051] In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some such embodiments, the liquid pharmaceutical formulation is prepared by ultrafiltration / diafiltration.

[0052] In other embodiments, the pharmaceutical formulation is a lyophilized dry antibody formulation. In some such embodiments, the pharmaceutical antibody formulation is a dry antibody formulation that has been lyophilized from a liquid pharmaceutical antibody formulation that has been prepared by purification followed by ultrafiltration / diafiltration.

[0053] In some embodiments, the invention provides methods for producing a composition having a reduced amount of the G0F glycoform of a humanized anti-α4β7 antibody (as determined by hydrophilic interaction chromatography (HILIC)), the method comprising culturing mammalian host cells in a production medium and adding supplements comprising uridine, manganese, and galactose to the production medium, thereby producing a composition having a reduced amount of the G0F glycoform of a humanized anti-α4β7 antibody compared to control mammalian host cells expressing a humanized anti-α4β7 antibody cultured in the absence of the supplements.

[0054] In one embodiment, the composition comprises at least about a 15% reduced level of the G0F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of supplemental components.

[0055] In one embodiment, the composition comprises at least about a 20% reduction in the G0F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of supplementary components.

[0056] In some embodiments, provided herein are methods for producing a composition having about 65% or less of the G0F glycoform of a humanized anti-α4β7 antibody (as determined by HILIC), the method comprising culturing mammalian host cells in a production medium and adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having about 65% or less of the G0F glycoform of a humanized anti-α4β7 antibody.

[0057] In one embodiment, the composition comprises about 60% or less of the G0F glycoform of the humanized anti-α4β7 antibody.

[0058] In one embodiment, the composition comprises about 55% or less of the G0F glycoform of the humanized anti-α4β7 antibody.

[0059] In some embodiments, provided herein are methods for producing a composition having an increased amount of the G1F glycoform of a humanized anti-α4β7 antibody (as determined by hydrophilic interaction chromatography (HILIC)), the method comprising culturing mammalian host cells in a production medium and adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having an increased amount of the G1F glycoform of a humanized anti-α4β7 antibody compared to control mammalian host cells expressing a humanized anti-α4β7 antibody cultured in the absence of the supplements.

[0060] In one embodiment, the composition comprises at least about a two-fold increase in the G1F form of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of supplementary components.

[0061] In one embodiment, the composition comprises at least about a three-fold increase in the G1F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of supplementary components.

[0062] Further, in some embodiments, provided herein are methods for producing a composition having about 25% or more of the G1F glycoform of a humanized anti-α4β7 antibody (as determined by HILIC), the method comprising culturing mammalian host cells in a production medium and adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having about 25% or more of the G1F glycoform of a humanized anti-α4β7 antibody.

[0063] In one embodiment, the composition comprises about 30% or more of the G1F glycoform of the humanized anti-α4β7 antibody.

[0064] In some embodiments, provided herein are methods for producing a composition having an increased amount of the G2F glycoform of a humanized anti-α4β7 antibody (as determined by hydrophilic interaction chromatography (HILIC)), the method comprising culturing mammalian host cells in a production medium and adding supplements comprising uridine, manganese, and galactose to the production medium, thereby producing a composition having an increased amount of the G2F glycoform of a humanized anti-α4β7 antibody compared to control mammalian host cells expressing a humanized anti-α4β7 antibody cultured in the absence of the supplements.

[0065] In one embodiment, the composition comprises at least about a three-fold increase in the G2F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0066] In one embodiment, the composition comprises at least about a four-fold increase in the G2F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0067] In some embodiments, provided herein are methods for producing a composition having about 3% or more of the G2F glycoform of a humanized anti-α4β7 antibody (as determined by HILIC), the method comprising culturing mammalian host cells in a production medium and adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having about 3% or more of the G2F glycoform of a humanized anti-α4β7 antibody 6.

[0068] In one embodiment, the composition comprises about 4% or more of the G2F glycoform of the humanized anti-α4β7 antibody.

[0069] In one embodiment, supplements are added to the production medium or to a feed medium which is then added to the production medium.

[0070] In one embodiment, the feed medium comprises about 15-100 mM uridine, in one embodiment, about 20-50 mM uridine, in one embodiment, about 1-40 mM uridine.

[0071] In one embodiment, the feed medium comprises about 0.02-0.3 mM manganese. In one embodiment, the feed medium comprises about 0.02-0.1 mM manganese. In one embodiment, the feed medium comprises about 0.001-0.1 mM manganese.

[0072] In one embodiment, the feed medium comprises 85 mM to 600 mM galactose, hi one embodiment, the feed medium comprises about 85 to 100 mM galactose, hi one embodiment, the feed medium comprises about 50 to 150 mM galactose.

[0073] In yet another embodiment, the production medium further comprises zinc. In one embodiment, the concentration of zinc in the feed medium is between about 50 μM and 150 μM.

[0074] In one embodiment, the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0075] In one embodiment, the method further increases the percentage of the major isoform species of the humanized anti-α4β7 antibody compared to the percentage of the major isoform species produced in the absence of a feed medium comprising uridine, manganese, and galactose added to the production medium.

[0076] In one embodiment, the process is a fed-batch process.

[0077] In one embodiment, the feed medium is added to the production medium starting from about day 4 of the production phase. In one embodiment, the feed medium is added to the production medium daily starting from about day 4 of the production phase.

[0078] In one embodiment, the methods disclosed herein are carried out in a large scale bioreactor, hi one embodiment, the large scale bioreactor is selected from the group consisting of a 200 liter (L) bioreactor, a 2000 L bioreactor, a 3000 L, and a 6000 L bioreactor.

[0079] In one embodiment, the production step results in a humanized anti-α4β7 antibody titer of greater than 3 g / L. In one embodiment, the humanized anti-α4β7 antibody titer is about 3 to about 8 g / L. In one embodiment, the humanized anti-α4β7 antibody titer is about 5 to about 7 g / L.

[0080] In one embodiment, the mammalian host cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the CHO cell is a GS-CHO cell.

[0081] In one embodiment, the production medium has a pH of about 6.8 to about 7.1.

[0082] In one embodiment, the humanized anti-α4β7 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5.

[0083] In one embodiment, the anti-α4β7 antibody is vedolizumab.

[0084] In some embodiments, the method includes recovering and purifying the antibody. In some such embodiments, the purification includes (i) purification steps to remove cellular debris, undesired proteins, salts, minerals, or other undesired elements, and (ii) purification of the antibody from contaminating soluble proteins and polypeptides. In certain embodiments, the method further includes preparing a formulation of the purified antibody suitable for human therapeutic use.

[0085] In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some such embodiments, the liquid pharmaceutical formulation is prepared by ultrafiltration / diafiltration.

[0086] In other embodiments, the pharmaceutical formulation is a lyophilized dry antibody formulation. In some such embodiments, the pharmaceutical antibody formulation is a dry antibody formulation that has been lyophilized from a liquid pharmaceutical antibody formulation that has been prepared by purification followed by ultrafiltration / diafiltration.

[0087] In another aspect, provided herein is a cell culture comprising host cells genetically engineered to express a humanized anti-α4β7 antibody and a production medium supplemented with uridine, manganese, and galactose (UMG), wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5.

[0088] In some embodiments of the above aspects, the production medium comprises uridine at a concentration of about 15-100 mM, manganese at a concentration of about 20-200 nM, and galactose at a concentration of about 85-500 mM.

[0089] In some embodiments of the above aspects, the production medium comprises, on the day of harvest, supplemented uridine at a concentration of about 1-7 mM, supplemented manganese at a concentration of about 2-15 μM, and supplemented galactose at a concentration of about 3-20 mM. In some embodiments, the production medium further comprises, on the day of harvest, supplemented zinc at a concentration of about 5-45 μM.

[0090] In some embodiments of the above aspects, on the day of harvest, the production medium comprises uridine at a concentration of about 1-7 mM, manganese at a concentration of about 2-15 μM, and galactose at a concentration of about 3-20 mM. In some embodiments, the production medium further comprises zinc at a concentration of about 5-45 μM on the day of harvest.

[0091] In certain embodiments, the expressed humanized anti-α4β7 antibody has an isoform distribution comprising (a) no more than 16%, no more than 15%, no more than 14%, no more than 13%, or no more than 12% basic isoform, and / or (b) at least 65%, at least 68%, at least 70%, at least 72%, or at least 75% major isoform.

[0092] In other embodiments, the expressed humanized anti-α4β7 antibody has a fucosylated N-glycan content comprising: (a) 65% or less, 60% or less, or 55% or less G0F; and / or (b) 25% or more, 27% or more, or 30% or more G1F; and / or (c) 2.5% or more, 3% or more, 3.5% or more, 4% or more, or 4.5% or more G2F.

[0093] In some embodiments of the above aspects, the expressed humanized anti-α4β7 antibody has a total fucosylated N-glycan (G0F+G1F+G2F) content of at least 92%, at least 93%, at least 94%, or at least 95%.

[0094] In other embodiments, the expressed humanized anti-α4β7 antibody has a total fucosylated N-glycan (G0F+G1F+G2F) content of 92-95%.

[0095] In alternative embodiments, the expressed humanized anti-α4β7 antibody has a total fucosylated N-glycan (G0F+G1F+G2F) content of 91-92%, 91-92.5%, or 91-93%.

[0096] In some embodiments of the above aspects, the cell culture further comprises zinc. In other embodiments, the cell culture further comprises arginine and / or lysine.

[0097] In some embodiments of the above aspects, the host cell is a CHO cell. In particular embodiments, the CHO cell is defective in the gene encoding glutamine synthetase (GS).

[0098] In another aspect, the present disclosure provides a humanized anti-α4β7 antibody produced by cell culture, as described herein above.

[0099] In yet another aspect, the present disclosure provides a composition comprising a humanized anti-α4β7 antibody, the method comprising culturing mammalian host cells genetically engineered to express the humanized anti-α4β7 antibody in a first production medium having a first pH and culturing the mammalian host cells in a second production medium having a second pH, the second pH being lower than the first pH, wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0100] In some embodiments of the foregoing aspects, the second pH is 0.1 to 0.5 pH units lower than the first pH. In particular embodiments, the first pH is in the range of pH 6.8 to 7.2 and the second pH is in the range of pH 6.7 to 6.95.

[0101] In some embodiments, the mammalian host cells are cultured at the first pH for 120 hours or less. In certain embodiments, the mammalian host cells are cultured at the first pH for 85-110 hours. In other embodiments, the mammalian host cells are cultured at the first pH for 90-100 hours.

[0102] In some embodiments, the method further comprises recovering the anti-α4β7 antibody from the second production medium. In certain embodiments, the anti-α4β7 antibody is recovered following culturing the mammalian host cells in the first production medium and the second production medium for a period of 13 to 15 days.

[0103] In some embodiments, the composition has an increased level of a major isoform of an anti-α4β7 antibody compared to a control composition in which mammalian host cells are cultured at a first pH without a pH shift.

[0104] In another aspect, the invention includes a composition comprising a humanized anti-α4β7 antibody produced using any one of the methods disclosed herein. In one embodiment, the methods disclosed herein provide a population of humanized anti-α4β7 antibodies having 92% or more total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F) glycosylation variants.

[0105] Furthermore, the present invention also includes the following embodiments: 1. A method for producing a composition having a reduced amount of the basic isoform of a humanized anti-α4β7 antibody (as determined by cation exchange chromatography (CEX)), said method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having a reduced amount of the basic isoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplements; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0106] 2. A method for producing a composition having about 16% or less of a basic isoform of a humanized anti-α4β7 antibody (as determined by CEX), said method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having about 16% or less of the basic isoform of the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0107] 3. The method of clause 2, wherein said composition comprises about 14% or less of the basic isoform of said humanized anti-α4β7 antibody.

[0108] 4. The method of clause 2, wherein said composition comprises about 13% or less of the basic isoform of said humanized anti-α4β7 antibody.

[0109] 5. The method of any one of clauses 1-4, wherein the supplemental components are added to the production medium or to a feed medium and then the feed medium is added to the production medium.

[0110] 6. The method of clause 5, wherein uridine is added to said feed medium at a final concentration of about 15 to 120 mM.

[0111] 7. The method of clause 6, wherein the uridine is added to the feed medium to a final concentration of about 20-70 mM uridine.

[0112] 8. The method of clause 5, wherein manganese is added to said feed medium at a final concentration of about 0.02 to 0.3 mM.

[0113] 9. The method of clause 8, wherein manganese is added to said feed medium to a final concentration of about 0.04 to 0.15 mM.

[0114] 10. The method of clause 5, wherein galactose is added to said feed medium to a final concentration of about 85 mM to 600 mM.

[0115] 11. The method of clause 10, wherein galactose is added to said feed medium to a final concentration of about 160 to 340 mM.

[0116] 12. The method of any one of clauses 1-11, wherein said feed medium further comprises zinc.

[0117] 13. The method of clause 12, wherein the concentration of zinc in the feed medium is between about 90 μM and 120 μM.

[0118] 14. The method of any one of clauses 1-13, wherein the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0119] 15. The method of any one of clauses 1-14, wherein the method further increases the percentage of the major isoform species of the humanized anti-α4β7 antibody compared to the percentage of the major isoform species produced in the absence of a feed medium comprising uridine, manganese, and galactose added to the production medium.

[0120] 16. The method of any one of clauses 1 to 15, wherein said method is a fed-batch method.

[0121] 17. The method of clause 16, wherein said feed medium is added to said production medium from about day 4 of the production stage.

[0122] 18. A method for producing a composition having a reduced amount of a basic isoform of a humanized anti-α4β7 antibody (as determined by cation exchange chromatography (CEX)), the method comprising culturing mammalian host cells in a production medium containing zinc, thereby producing a composition having a reduced amount of the basic isoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of zinc; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0123] 19. A method for producing a composition having about 16% or less of a basic isoform of a humanized anti-α4β7 antibody (as determined by CEX), the method comprising culturing mammalian host cells in a production medium containing zinc, thereby producing a composition having about 16% or less of a basic isoform of the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0124] 20. The method of clause 19, wherein said composition comprises about 14% or less of the basic isoform of said humanized anti-α4β7 antibody.

[0125] 21. The method of clause 19, wherein said composition comprises about 13% or less of the basic isoform of said humanized anti-α4β7 antibody.

[0126] 22. The method of any one of clauses 18 to 21, wherein the concentration of zinc in the production medium is between 2 μM and 60 μM.

[0127] 23. The method of any one of clauses 18 to 22, wherein the method comprises supplementing the production medium with zinc by adding a feed medium containing zinc to the production medium.

[0128] 24. The method of clause 23, wherein said feed medium is added to said production medium from about day 4 of the production stage.

[0129] 25. The method of clause 24, wherein the concentration of zinc in the feed medium is between about 90 μM and 120 μM.

[0130] 26. The method of any one of clauses 1 to 25, wherein the production medium comprises 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine.

[0131] 27. The method of clause 26, wherein the production medium contains 4.5 to 5.5 g / L of lysine.

[0132] 28. The method of clause 26, wherein the production medium contains 5.5 to 8.8 g / L of lysine.

[0133] 29. The method of clause 26, wherein the production medium contains 5.4 to 7.4 g / L of arginine.

[0134] 30. The method of clause 26, wherein the production medium contains 7.4 to 12 g / L of arginine.

[0135] 31. A method for producing a composition comprising 2.5% or less HMW species of a humanized anti-α4β7 antibody (as determined by SEC), said method comprising: culturing mammalian host cells in a production medium such that a composition containing 2.5% or less HMW species of said humanized anti-α4β7 antibody (as determined by SEC) is produced during the production stage; the production medium has an average temperature of about 37 degrees Celsius, the host cells are genetically engineered to express a human IgG1 anti-α4β7 antibody; The method, wherein the humanized anti-α4β7 antibody comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and comprises a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[0136] 32. A method for producing a composition containing a high level of humanized anti-α4β7 antibody monomer (as determined by SEC), said method comprising: culturing mammalian host cells in a growth medium during an expansion stage, wherein the mammalian host cells are genetically engineered to express a humanized anti-α4β7 antibody; and culturing the mammalian host cells in a production medium during a production phase such that a composition containing a high level of monomeric form of the humanized anti-α4β7 antibody (as determined by SEC) is produced; the mammalian host cells are cultured at about the same temperature during both the expansion step and the production step; The method, wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and comprises a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[0137] 33. The method of clause 31 or 32, wherein the temperature is 36 to 38 degrees Celsius.

[0138] 34. The method of clause 31 or 32, wherein the average temperature is between 36.5 and 37.5 degrees Celsius.

[0139] 35. The method of clause 31 or 32, wherein the temperature is an average temperature of about 37 degrees Celsius.

[0140] 36. The method of any one of clauses 1 to 35, wherein the production medium has a temperature in the range of 36 to 38 degrees Celsius.

[0141] 37. The method of clause 36, wherein the temperature is in the range of 36.5 to 37.5 degrees Celsius.

[0142] 38. The method of clause 36, wherein the temperature is an average temperature of about 37 degrees Celsius.

[0143] 39. The method of any one of clauses 1 to 38, wherein the production medium has a pH in the range of 6.5 to 7.

[0144] 40. The method of clause 39, wherein the production medium has a pH in the range of 6.8 to 7.0.

[0145] 41. The method of any one of clauses 1 to 40, wherein the production medium has a glucose level that is maintained at or below about 7 g / L during the production stage.

[0146] 42. The method of any one of clauses 1 to 41, wherein the production stage is 14 days or less.

[0147] 43. The method of any one of clauses 1 to 42, wherein the production stage is in the range of 10 to 17 days.

[0148] 44. The method of any one of clauses 1 to 43, carried out in a large-scale bioreactor.

[0149] 45. The method of clause 44, wherein the large scale bioreactor is selected from the group consisting of a 200 liter (L) bioreactor, a 2000 L bioreactor, a 3000 L, and a 6000 L bioreactor.

[0150] 46. ​​The method of any one of clauses 1-45, wherein said production step results in a titer of said humanized anti-α4β7 antibody of greater than 3 g / L.

[0151] 47. The method of clause 46, wherein the humanized anti-α4β7 antibody has a titer of about 3 to about 8 g / L.

[0152] 48. The method of clause 46, wherein the humanized anti-α4β7 antibody has a titer of about 5 to about 7 g / L.

[0153] 49. The method of any one of clauses 1-48, wherein said mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0154] 50. The method of clause 49, wherein said CHO cells are GS-CHO cells.

[0155] 51. The method of any one of clauses 1-50, wherein said humanized anti-α4β7 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5.

[0156] 52. The method of any one of clauses 1-50, wherein said humanized anti-α4β7 antibody is vedolizumab.

[0157] 53. A composition comprising a humanized anti-α4β7 antibody produced using the method of any one of clauses 1 to 52.

[0158] 54. The composition of clause 53, comprising a population of humanized anti-α4β7 antibodies having glycosylation variants of 54.92% or more total asialo, agalacto, core-fucosylated biantennary glycans (G0F), asialo, monogalacto, core-fucosylated biantennary glycans (G1F), and / or asialo, digalacto, core-fucosylated biantennary glycans (G2F).

[0159] 55. A method for producing a composition having a reduced amount of the G0F glycoform of a humanized anti-α4β7 antibody (as determined by hydrophilic interaction chromatography (HILIC)), the method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having a reduced amount of the G0F glycoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplements; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0160] 56. The method of clause 55, wherein the composition comprises a level of the G0F glycoform of the humanized anti-α4β7 antibody that is reduced by at least about 15% compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0161] 57. The method of clause 55, wherein the composition comprises at least about a 20% reduction in the G0F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0162] 58. A method for producing a composition having about 65% or less G0F glycoforms of a humanized anti-α4β7 antibody (as determined by HILIC), said method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having about 65% or less of the G0F glycoform of the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0163] 59. The method of clause 58, wherein said composition comprises about 60% or less of the G0F glycoform of said humanized anti-α4β7 antibody.

[0164] 60. The method of clause 58, wherein said composition comprises about 55% or less of the G0F glycoform of said humanized anti-α4β7 antibody.

[0165] 61. A method for producing a composition having an increased amount of the G1F glycoform of a humanized anti-α4β7 antibody (as determined by HILIC), said method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having an increased amount of the G1F glycoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplements; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0166] 62. The method of clause 61, wherein said composition comprises at least about a two-fold increase in the G1F glycoform of said humanized anti-α4β7 antibody compared to control mammalian host cells expressing said humanized anti-α4β7 antibody cultured in the absence of said supplement.

[0167] 63. The method of clause 61, wherein said composition comprises at least about a three-fold increase in the G1F glycoform of said humanized anti-α4β7 antibody compared to control mammalian host cells expressing said humanized anti-α4β7 antibody cultured in the absence of said supplement.

[0168] 64. A method for producing a composition having about 25% or more of the G1F glycoform of a humanized anti-α4β7 antibody (as determined by HILIC), said method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium including uridine, manganese, and galactose, thereby producing a composition having about 25% or more of the G1F glycoform of the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0169] 65. The method of clause 64, wherein said composition comprises about 30% or more of the G1F glycoform of said humanized anti-α4β7 antibody.

[0170] 66. A method for producing a composition having an increased amount of the G2F glycoform of a humanized anti-α4β7 antibody (as determined by HILIC), the method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium comprising uridine, manganese, and galactose, thereby producing a composition having an increased amount of the G2F glycoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplements; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0171] 67. The method of clause 66, wherein said composition comprises at least about a three-fold increase in the G2F glycoform of said humanized anti-α4β7 antibody compared to control mammalian host cells expressing said humanized anti-α4β7 antibody cultured in the absence of said supplement.

[0172] 68. The method of clause 66, wherein said composition comprises at least about a four-fold increase in the G2F glycoform of said humanized anti-α4β7 antibody compared to control mammalian host cells expressing said humanized anti-α4β7 antibody cultured in the absence of said supplement.

[0173] 69. A method for producing a composition having about 3% or more G2F glycoforms of a humanized anti-α4β7 antibody (as determined by HILIC), the method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium including uridine, manganese, and galactose, thereby producing a composition having about 3% or more of the G2F glycoform of the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[0174] 70. The method of clause 69, wherein said composition comprises about 4% or more of the G2F glycoform of said humanized anti-α4β7 antibody.

[0175] 71. The method of any one of clauses 55 to 70, wherein the supplemental component is added to the production medium or to a feed medium and then the feed medium is added to the production medium.

[0176] 72. The method of clause 71, wherein the feed medium comprises about 15-100 mM uridine.

[0177] 73. The method of clause 72, wherein the feed medium comprises about 20-50 mM uridine.

[0178] 74. The method of clause 71, wherein the feed medium comprises about 0.02 to 0.3 mM manganese.

[0179] 75. The method of clause 74, wherein the feed medium comprises about 0.02 to 0.1 mM manganese.

[0180] 76. The method of clause 71, wherein the feed medium comprises between 85 mM and 600 mM galactose.

[0181] 77. The method of clause 76, wherein the feed medium comprises 85 to 100 mM galactose.

[0182] 78. The method of any one of clauses 55 to 77, wherein the production medium further comprises zinc.

[0183] 79. The method of clause 78, wherein the concentration of zinc in the production medium is between about 50 μM and 150 μM.

[0184] 80. The method of any one of clauses 55-79, wherein the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement.

[0185] 81. The method of any one of clauses 55 to 80, wherein the method further increases the percentage of the major isoform species of the humanized anti-α4β7 antibody compared to the percentage of the major isoform species produced in the absence of a feed medium comprising uridine, manganese, and galactose added to the production medium.

[0186] 82. The method of any one of clauses 55 to 81, wherein said method is a fed-batch method.

[0187] 83. The method of clause 82, wherein said feed medium is added to said production medium from about day 4 of the production stage.

[0188] 84. The method of any one of clauses 1 to 83, wherein the production medium has a pH of about 6.8 to about 7.1. [Brief explanation of the drawings]

[0189] [Figure 1] We provide results from a predictive profiler based on experiments testing a variety of culture conditions, including pH, temperature, galactose Gal+ addition, UMG addition, and feeding strategy conditions in production cell cultures. [Figure 2A] The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2B] The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2C] The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2D]The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2E] The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2F] The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2G] The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 2H]The graphs show the effects of uridine, galactose, and manganese (UMG) supplementation (33xUMG, 50xUMG, 66xUMG) and pH (pH 7.05 vs. pH 6.85) on antibody titers (A), acidic species (B), basic species (C), major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H). Results without UMG supplementation (indicated by a "+" symbol) are shown for comparison. [Figure 3] The graphs show the comparative effects of various arginine and lysine concentrations on the percentage of basic species (A) and antibody titers (B). The x-axis labels correspond to high (H), medium (M), or low (L) concentrations of lysine and arginine, as outlined in Table 5. [Figure 4A] Predicted maximum desirability results from JMP analysis of culture conditions containing different levels of lysine and arginine (low lysine and low arginine (LL) - A; low lysine and medium arginine (LM) - B; low lysine and high arginine (LH) - C). Concentrations corresponding to high (H), medium (M), or low (L) concentrations of lysine and arginine are outlined in Table 5. [Figure 4B] Predicted maximum desirability results from JMP analysis of culture conditions containing different levels of lysine and arginine (low lysine and low arginine (LL) - A; low lysine and medium arginine (LM) - B; low lysine and high arginine (LH) - C). Concentrations corresponding to high (H), medium (M), or low (L) concentrations of lysine and arginine are outlined in Table 5. [Figure 4C] Predicted maximum desirability results from JMP analysis of culture conditions containing different levels of lysine and arginine (low lysine and low arginine (LL) - A; low lysine and medium arginine (LM) - B; low lysine and high arginine (LH) - C). Concentrations corresponding to high (H), medium (M), or low (L) concentrations of lysine and arginine are outlined in Table 5. [Figure 5A]Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5B] Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5C] Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5D] Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5E]Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5F] Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5G] Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5H] Time course data comparing the effect of zinc on antibody titers (A), percentage of basic species (B), percentage of acidic species (C), percentage of major species (D), percentage of G0F species (E), percentage of G1F species (F), percentage of G2F species (G), and total glycan species (H) after 14, 15, 16, 17, and 18 days of culture are shown. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 6A]Time course data comparing the effect of zinc, days of incubation, and temperature (33°C, 35°C, and 37°C) on the percentage of basic species (A), total glycan species (B), aggregate (high molecular weight (HMW)) formation (C), titer (D), and acidic isoforms (E) are shown in graphs. The solid black lines in A, C, and E represent the top process criterion for each attribute, while the solid black line in B represents the bottom acceptance criterion. The numbers on the x-axis correspond to the zinc concentrations outlined in Table 6. [Figure 6B] Time course data comparing the effect of zinc, days of incubation, and temperature (33°C, 35°C, and 37°C) on the percentage of basic species (A), total glycan species (B), aggregate (high molecular weight (HMW)) formation (C), titer (D), and acidic isoforms (E) are shown in graphs. The solid black lines in A, C, and E represent the top process criterion for each attribute, while the solid black line in B represents the bottom acceptance criterion. The numbers on the x-axis correspond to the zinc concentrations outlined in Table 6. [Figure 6C] Time course data comparing the effect of zinc, days of incubation, and temperature (33°C, 35°C, and 37°C) on the percentage of basic species (A), total glycan species (B), aggregate (high molecular weight (HMW)) formation (C), titer (D), and acidic isoforms (E) are shown in graphs. The solid black lines in A, C, and E represent the top process criterion for each attribute, while the solid black line in B represents the bottom acceptance criterion. The numbers on the x-axis correspond to the zinc concentrations outlined in Table 6. [Figure 6D] Time course data comparing the effect of zinc, days of incubation, and temperature (33°C, 35°C, and 37°C) on the percentage of basic species (A), total glycan species (B), aggregate (high molecular weight (HMW)) formation (C), titer (D), and acidic isoforms (E) are shown in graphs. The solid black lines in A, C, and E represent the top process criterion for each attribute, while the solid black line in B represents the bottom acceptance criterion. The numbers on the x-axis correspond to the zinc concentrations outlined in Table 6. [Figure 6E]Time course data comparing the effect of zinc, days of incubation, and temperature (33°C, 35°C, and 37°C) on the percentage of basic species (A), total glycan species (B), aggregate (high molecular weight (HMW)) formation (C), titer (D), and acidic isoforms (E) are shown in graphs. The solid black lines in A, C, and E represent the top process criterion for each attribute, while the solid black line in B represents the bottom acceptance criterion. The numbers on the x-axis correspond to the zinc concentrations outlined in Table 6. [Figure 7A] Graphical representations of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), the percentage of basic species (B), the percentage of major species (C), and antibody titers (D) from two sets of experiments. In A–D, run 2 is represented by open circles, and data points for run 1 are represented by closed circles. [Figure 7B] Graphical representations of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), the percentage of basic species (B), the percentage of major species (C), and antibody titers (D) from two sets of experiments. In A–D, run 2 is represented by open circles, and data points for run 1 are represented by closed circles. [Figure 7C] Graphical representations of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), the percentage of basic species (B), the percentage of major species (C), and antibody titers (D) from two sets of experiments. In A–D, run 2 is represented by open circles, and data points for run 1 are represented by closed circles. [Figure 7D] Graphical representations of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), the percentage of basic species (B), the percentage of major species (C), and antibody titers (D) from two sets of experiments. In A–D, run 2 is represented by open circles, and data points for run 1 are represented by closed circles. [Figure 8A] The correlation between isoform distribution and pH shift parameters is shown graphically. The correlation between final cell culture pH (after pH shift) and % acidic isoform species (left panel) or % major isoform (right panel) is shown. [Figure 8B] The correlation between isoform distribution and pH shift parameters is shown graphically. The correlation between pH shift duration and % acidic isoform species (left panel) or % major isoform (right panel) is shown. [Figure 9] Figure 1 shows N-glycan structures that may be present in a population of anti-α4β7 antibodies, such as vedolizumab. Glycan legends are provided in the figure. DETAILED DESCRIPTION OF THE INVENTION

[0190] I. Definition In order that the present invention may be more readily understood, certain terms are first defined.

[0191] The cell surface molecule "α4β7 integrin" or "α4β7" (used interchangeably throughout) is a heterodimer of the α4 chain (CD49D, ITGA4) and the β7 chain (ITGB7). The human α4 integrin and β7 integrin genes, GenBank (National Center for Biotechnology Information, Bethesda, Md.) RefSeq accession numbers NM_000885 and NM_000889, respectively, are expressed by B and T lymphocytes, particularly memory CD4+ lymphocytes. Typical of many integrins, α4β7 can exist in either a resting or activated state. Ligands for α4β7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM (e.g., MAdCAM-1)). Antibodies that bind to α4β7 integrin are referred to herein as "anti-α4β7 antibodies."

[0192] As used herein, an antibody, or antigen-binding fragment thereof, that has "binding specificity for the α4β7 complex" binds to α4β7 but not to α4β1 or α E Vedolizumab is an example of an antibody that has binding specificity for the α4β7 complex.

[0193] The term "about" means that the value that follows is not the exact value, but rather the center of a range that is + / - 5% of the value. When the value is a relative value expressed as a percentage, the term "about" means that the value that follows is not the exact value, but rather the center of a range that is + / - 5% of the value, whereby the upper limit of the range cannot exceed 100% of the value.

[0194] As used herein, the term "aggregate" or "aggregates" refers to an association of two or more antibodies or antibody fragments. For example, an aggregate may be a dimer, trimer, tetramer, or multimer larger than a tetramer of antibodies and / or antibody fragments. Antibody aggregates may be soluble or insoluble. The association between the aggregated molecules may be either covalent or noncovalent, regardless of the mechanism by which they are associated. The association may be direct between the aggregated molecules or indirect through other molecules linking them together. Examples of the latter include, but are not limited to, disulfide bonds with other proteins, hydrophobic association with lipids, charge association with DNA, affinity association with leached protein A, or mixed-mode association with multiple components. Aggregates may form irreversibly during protein expression in cell culture, protein purification in downstream processes, or during storage of pharmaceutical products. The presence of aggregates in solution can be determined, for example, using size exclusion chromatography (SEC) (e.g., SEC with UV detection, SEC with light scattering detection (SEC-LSD)), field-flow fractionation, analytical ultracentrifugation sedimentation velocity, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced).

[0195] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions are further divided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody has a fragment crystallizable (Fc) region. In certain embodiments, the antibody is of the IgG1 isotype and has a kappa light chain.

[0196] As used herein, the terms "charged species," "charged isoform," or "charged isoform species" refer to a variant of an antibody or antigen-binding portion thereof (e.g., vedolizumab, or an antigen-binding portion thereof) characterized by an overall charge different from the predominant species of the antibody or antigen-binding portion thereof. Charged isoform species of an antibody or antigen-binding portion thereof can be detected by various methods known in the art, such as cation exchange chromatography (CEX), e.g., cation exchange-high performance liquid chromatography (CEX-HPLC), CEX-mass spectrometry, or isoelectric focusing. For example, generally, when an antibody preparation is separated using CEX, CEX-HPLC, or CEX-mass spectrometry, the majority of the antibody elutes from the CEX resin at a retention time characteristic of the predominant (major) isoform of that antibody. This can be visualized by plotting the amount of antibody eluted from the resin as a function of the retention time of the CEX resin. When visualized in this way, the predominant isoform of the antibody or antigen-binding portion thereof is the fraction of the antibody or antigen-binding portion thereof eluting from the CEX resin within the largest peak. Using this method, charged isoform species can be identified by having a different retention time from the major isoform. For example, when the charged isoform species are detected by CEX, CEX-HPLC, or CEX mass spectrometry, acidic isoform species can elute from the resin at a shorter retention time than the major isoform of an antibody, or antigen-binding portion thereof, and basic isoform species can elute from the resin at a longer retention time than the major isoform of an antibody, or antigen-binding portion thereof.

[0197] As used herein, the term "acidic species" or "acidic isoform species" refers to a variant of an antibody or antigen-binding portion thereof (e.g., vedolizumab, or an antigen-binding portion thereof) characterized by an overall acidic charge. Acidic species of an antibody or antigen-binding portion thereof can be detected by various methods known in the art, such as cation exchange chromatography (CEX), e.g., cation exchange high-performance liquid chromatography (CEX-HPLC), CEX-mass spectrometry, or isoelectric focusing. Generally, acidic species of an antibody or antigen-binding portion thereof elutes from a CEX resin with a shorter retention time than the major isoform of the antibody or antigen-binding portion thereof. Acidic species of an antibody may include, but are not limited to, charge variants, structural variants, and / or fractionation variants. In some embodiments, a composition comprising an antibody or antigen-binding portion thereof may contain more than one type of acidic isoform species. In some embodiments, multiple acidic isoform species can be identified based on differences in retention time during CEX-HPLC separation. For example, when a composition comprising an antibody, such as vedolizumab, is analyzed using CEX, one or more acidic isoform peaks can be identified, each representing one or more acidic isoform species of the antibody.

[0198] As used herein, the term "basic species" or "basic isoform species" refers to a variant of an antibody or antigen-binding portion thereof (e.g., vedolizumab) characterized by an overall basic charge. Basic species of an antibody or antigen-binding portion thereof can be detected by various methods known in the art, such as cation exchange chromatography (CEX), e.g., cation exchange high-performance liquid chromatography (CEX-HPLC), CEX-mass spectrometry, or isoelectric focusing. Generally, basic species of an antibody or antigen-binding portion thereof elutes from a CEX resin with a longer retention time than the major isoform of the antibody or antigen-binding portion thereof. Basic species of an antibody may include, but are not limited to, charge variants, structural variants, and / or fractionation variants. In some embodiments, a composition comprising an antibody or antigen-binding portion thereof may contain more than one type of basic isoform species. In some embodiments, multiple basic isoform species can be identified based on differences in retention time during CEX-HPLC separation. For example, when a composition containing an antibody, such as vedolizumab, is analyzed using CEX, one or more basic isoform peaks can be identified, each representing one or more basic isoform species of the antibody. In one embodiment, the basic isoform of vedolizumab is vedolizumab with a carboxyl-terminal lysine (C-Lys). Host cell impurities or other impurities not associated with the antibody or its antigen-binding portion by primary sequence are not considered "basic species" or "basic isoform species" of the antibody or its antigen-binding portion.

[0199] "CDRs" or "complementarity determining regions" are regions of hypervariability interspersed within more conserved regions called "framework regions" (FR).

[0200] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to Fab, Fab', F(ab'), and Fv fragments, single-chain antibodies, functional heavy-chain antibodies (nanobodies), and any portion of an antibody having specificity for at least one desired epitope (e.g., an isolated portion of the complementarity-determining regions having sufficient framework sequence to specifically bind to the epitope) that competes with the intact antibody for specific binding. Antigen-binding fragments can be produced by recombinant techniques or by enzymatic or chemical cleavage of antibodies.

[0201] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human antibody (e.g., murine) that retains or substantially retains the antigen-binding properties of the parent antibody, but is less immunogenic in humans.

[0202] Polypeptides, such as antibodies, produced by recombinant mammalian host cell lines using cell culture methods are referred to as "recombinant polypeptides," "proteins," or, in the case of antibodies, "recombinant antibodies." The expressed protein can be produced intracellularly or secreted into the medium from which it can be recovered or collected. In one embodiment, the recombinant antibody is an antibody with binding specificity to the α4β7 complex, such as a recombinant anti-α4β7 antibody, e.g., vedolizumab. Because the methods and compositions described herein relate to compositions and cell culture methods for producing recombinant antibodies, the term "antibody" is used interchangeably herein with the term "recombinant antibody," unless otherwise specified.

[0203] The term "recombinant host cell" or "host cell" refers to a cell that has been genetically engineered to express a recombinant polypeptide, e.g., an antibody. In one embodiment, a recombinant host cell contains an expression vector that includes nucleic acid encoding an antibody heavy chain, light chain, or both. It should also be understood that the term "host cell" is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations, due either to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Furthermore, unless otherwise specified, when the term "cell," e.g., host cell, mammalian cell, or mammalian host cell, is used, it should be understood that the term is intended to include a population of cells.

[0204] As used herein, the term "cell culture process" collectively refers to cell culture steps involved in the production of a recombinant polypeptide, e.g., an antibody. The term "cell culture process" generally refers to a process of growing or maintaining cells under controlled conditions. A cell culture process can be performed in vitro or ex vivo. In some embodiments, a cell culture process has both an expansion phase and a production phase. In some embodiments, the expansion phase and the production phase are separated by a transition or shift phase. "Culturing" cells refers to contacting the cells with a cell culture medium under conditions suitable for growing or maintaining the cells. In certain embodiments, cell culture refers to a method for generating or maintaining a population of host cells capable of producing a recombinant polypeptide of interest, e.g., an anti-α4β7 antibody. For example, once an expression vector is incorporated into a suitable mammalian host cell, e.g., a Chinese hamster ovary (CHO) host cell, the host can be cultured under conditions suitable for expression of the relevant nucleotide coding sequence. A "cell culture" can also refer to a solution containing cells.

[0205] The terms "culture medium" and "cell culture medium" (plural, "media") refer to a nutrient source used to grow or maintain cells. As will be understood by one of skill in the art, a nutrient source may contain components necessary for cells to grow and / or survive, or may contain components that aid in the growth and / or survival of cells. Vitamins, essential or non-essential amino acids (e.g., cysteine ​​and cystine), and trace elements (e.g., copper) are examples of medium components. Examples of cell culture media include growth media and production media.

[0206] Cell culture media can also be supplemented with, e.g., "media supplements" or "supplements," which include one or more components that aid in the cell culture process, e.g., by increasing recombinant polypeptide production or improving cell viability. In one embodiment, the supplements are not formulated with the cell culture media, e.g., are not formulated with the production medium or feed medium. The supplements can be prepared in concentrated forms where combination with a feed solution or medium results in a low final concentration of the supplements. The supplements can include one or more components already present in the starting, e.g., stock or base, medium, and / or the supplements can include one or more components that are new in the medium. In one embodiment, the supplements are added to a feed solution.

[0207] Supplementary components can affect specific aspects of the cell culture, for example, improving cell growth or increasing recombinant polypeptide production, depending on the cell type, growth format, and product (protein of interest) characteristics. Examples of substances that can be added by supplementary components include, but are not limited to, one or more trace elements, one or more hormones, one or more amino acids, one or more vitamins, one or more fatty acids, one or more non-ionic detergents, one or more nucleotides, and / or one or more sugars. In some embodiments, supplementary components include insulin, plant hydrolysates, and / or animal hydrolysates. One or more supplementary components can be added at one or more stages of the cell culture process.

[0208] Cell culture media and / or supplements may be "defined" or "undefined" to a certain degree, based on the nature of the component(s), e.g., whether they are supplied as a known chemical composition such as one or more elements, inorganic salts or organic ions or sugars, or as a mixture, e.g., complex components such as hydrolysates, with likely known or unknown sources of variability. The presence of complex components such as proteins or hydrolysates in the medium reduces the degree of definition.

[0209] The terms "growth phase," "growth phase," "expansion phase," and "expansion phase," used interchangeably herein, refer to a period during which cultured host cells are rapidly dividing and increasing in number. During the expansion phase, cells may generally be cultured in a growth medium (or expansion medium) under conditions designed to maximize cell growth. The growth phase may precede the production phase in time, for example, in a batch culture, whereby the two phases may (or may not) be separated by a transition phase.

[0210] As used herein, the term "production phase" or "production stage" refers to the period during which a host cell is producing maximum amounts of a recombinant polypeptide, such as a recombinant antibody. The production phase is typically characterized by fewer cell divisions than during the expansion phase, and may also include the use of media and culture conditions designed to maximize polypeptide production.

[0211] The term "growth medium" refers to a cell culture medium that promotes the growth, i.e., increase in number, of cultured cells and is used during the growth or expansion phase of the cell culture process.

[0212] A "production medium" is a cell culture medium that promotes the production of a recombinant polypeptide, eg, an antibody of interest, eg, an anti-α4β7 antibody.

[0213] As used herein, "feed solution" or "feed medium" refers to a cell culture medium that is added to a cell culture in a growth medium or production medium to improve or maintain the profile of the protein produced by the cells in the growth medium or production medium. For example, a feed solution can be added to maintain a particular protein titer level produced by the cells. Feed solutions are known in the art. In one embodiment, the feed solution is supplemented with additional nutrients identified as beneficial to the production of proteins from mammalian cells.

[0214] It will be understood that growth can also occur in a production medium, and production can occur in a growth medium, such that the growth medium and production medium can be the same, however, in one embodiment, a production medium is selected that promotes production of the polypeptide of interest more than if a growth medium were used.

[0215] As used herein, the term "batch culture" refers to a culture in which all components for cell culture (including cells and all culture nutrients) are supplied to the culture vessel at the beginning of the culture process.

[0216] As used herein, the term "fed-batch cell culture" refers to a batch culture in which cells and medium are initially fed to a culture vessel and additional supplemental components, e.g., nutrients, are supplied continuously (via a feeding solution) or in discrete increments, with or without periodic cell and / or product harvesting prior to the end of the culture.

[0217] As used herein, the term "perfusion culture" refers to a culture in which cells and supplementary components are supplied to the culture vessel at the beginning of the culture process, additional supplementary component(s) are continuously supplied to the culture, and product is continuously harvested from the medium during the culture process.

[0218] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of driving the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0219] "Nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, any substrate that can be incorporated into a polymer by DNA polymerase or RNA polymerase or by a synthetic reaction. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer.

[0220] "Isolated nucleic acid" means and encompasses a non-naturally occurring, recombinant, or naturally occurring sequence that is outside or separated from its normal context. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules are therefore distinguished from the nucleic acid molecule as it exists in natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express proteins, for example, where the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0221] As used herein, "purified" (or "isolated") refers to a nucleic acid molecule (e.g., a polynucleotide) or an amino acid molecule (e.g., a polypeptide or protein) that is substantially free of other components. In some embodiments, a purified polynucleotide or purified polypeptide is recovered or separated from other components present in the environment in which it is produced. For example, an isolated polypeptide is one that is separated from other components of the cell in which it is produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA). An isolated polynucleotide is one that is separated from other nuclear components (e.g., histones) and / or upstream or downstream nucleic acid sequences.

[0222] The term "culture vessel" refers to a vessel used to culture cells. The culture vessel can be of any size as long as it is useful for culturing cells.

[0223] As used herein, the term "inoculation" or "seeding" refers to the addition of cells to a medium to initiate a culture, or the process of providing a cell culture to a bioreactor or another vessel for cultivation. The cells may have previously been grown in another bioreactor or vessel. Alternatively, the cells may be frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term refers to any number of cells, including a single cell.

[0224] As used herein, the term "titer" refers to the total amount of recombinantly expressed polypeptide, e.g., antibody, produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of antibody per milliliter or grams per liter of medium. Titer can be expressed or assessed in terms of a relative measure, such as the percentage increase in titer compared to obtaining protein product under different culture conditions.

[0225] As used herein, the term "recovery" refers to the separation of the cell culture medium (containing the expressed protein of interest) from the cells and cell debris of the cell culture, e.g., for expressed proteins secreted from the host cells. (When non-secreted proteins are recovered, the cells are harvested and the medium is discarded.) The medium containing the protein of interest is called the "culture supernatant." Recovery is carried out using any of several techniques, including, but not limited to, centrifugation, microfiltration, depth filtration, and filtration through absolute pore size membranes. Subsequent steps to isolate the desired protein after recovery, e.g., from the supernatant or cells, including clarification, are generally considered to be purification steps.

[0226] The term "clarified harvest" refers to a liquid material derived from a culture supernatant containing a recombinant polypeptide of interest, e.g., an anti-α4β7 antibody. A clarified harvest is obtained from a cell culture medium that has undergone one or more process steps to separate the polypeptide of interest from the cells and cellular debris of the cell culture and / or to remove finer solid particles and particulate impurities from the liquid. Examples of such separation techniques include, but are not limited to, sedimentation, flocculation, centrifugation, and / or filtration.

[0227] As used herein, the term "upstream processing" in the context of recombinant polypeptide, e.g., antibody, preparations refers to activities that involve the production and collection of a polypeptide (e.g., an antibody) from cells (e.g., during cell culture of a protein, e.g., an antibody of interest).

[0228] As used herein, the term "downstream processing" refers to one or more techniques used after an upstream process to purify a protein, e.g., an antibody of interest. For example, downstream processing includes the generation of a protein product using, for example, affinity chromatography, including protein A affinity chromatography, size exclusion chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, hydrophobic interaction chromatography (HIC), or displacement chromatography.

[0229] As used herein, the term "glycosylation profile" refers to a complex of post-translationally modified species, including oligosaccharides. In the context of an anti-α4β7 antibody, the glycosylation profile describes the N-linked glycosylation in the Fc region of the antibody. In the context of vedolizumab, the glycosylation profile refers to the glycosylated species attached to asparagine 301 of the heavy chain of SEQ ID NO: 13.

[0230] II. Methods and Compositions of the Invention Provided herein are methods and compositions for producing anti-α4β7 antibodies, such as vedolizumab, in mammalian, e.g., non-human, cell culture. The invention is based, at least in part, on cell culture parameters that can be used to achieve high anti-α4β7 antibody titer levels, i.e., greater than 1 g / L, e.g., 3-10 g / L, 4-8 g / L, or 5-7 g / L, in mammalian cell culture. Also provided herein are methods and compositions for achieving reduced levels of basic isoforms of anti-α4β7 antibodies, such as vedolizumab; methods and compositions for achieving low aggregation levels of anti-α4β7 antibodies, such as vedolizumab; and methods and compositions for achieving specific glycan types of anti-α4β7 antibodies, such as vedolizumab. Also provided herein are compositions comprising anti-α4β7 antibodies, such as vedolizumab, having reduced levels of basic isoform species; low levels of high molecular weight aggregates; and / or specific glycan types.

[0231] In particular, the methods and compositions disclosed herein can be used to produce the anti-α4β7 antibody vedolizumab, or antibodies having the antigen-binding region of vedolizumab. Vedolizumab is also known by its trademark ENTYVIO® (Takeda Pharmaceuticals, Inc.). Vedolizumab is a humanized antibody containing mutated human IgG1 framework regions and antigen-binding CDRs from the murine antibody Act-1 (described in U.S. Patent No. 7,147,851, incorporated herein by reference).

[0232] Vedolizumab specifically binds to the α4β7 integrin, blocks its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and fibronectin, and inhibits the migration of memory T lymphocytes across the endothelium into inflamed gastrointestinal parenchyma. Vedolizumab does not bind to or inhibit the function of α4β1 and αEβ7 integrins, and does not antagonize the interaction of α4 integrin with vascular cell adhesion molecule-1 (VCAM-1).

[0233] The α4β7 integrin is expressed on the surface of a distinct subset of memory T lymphocytes that preferentially migrate to the gastrointestinal tract. MAdCAM-1 is expressed on intestinal endothelial cells and plays an important role in the homing of T lymphocytes to intestinal lymphoid tissues. The interaction between α4β7 integrin and MAdCAM-1 has been implicated as an important cause of mucosal inflammation, such as the chronic inflammation characteristic of ulcerative colitis and Crohn's disease. Vedolizumab can be used to treat inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, HIV, pouchitis, including chronic pouchitis, fistulizing Crohn's disease, graft-versus-host disease, and celiac disease.

[0234] The heavy chain variable region of vedolizumab is provided in SEQ ID NO: 1, and the light chain variable region of vedolizumab is provided in SEQ ID NO: 5. Vedolizumab comprises a heavy chain variable region comprising a CDR1 set forth in SEQ ID NO: 2, a CDR2 set forth in SEQ ID NO: 3, and a CDR3 set forth in SEQ ID NO: 4. Vedolizumab comprises a light chain variable region comprising a CDR1 set forth in SEQ ID NO: 6, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 8. The nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 9. The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 10. The full-length nucleic acid sequence encoding the light chain of vedolizumab is set forth as SEQ ID NO: 11. The full-length nucleic acid sequence encoding the heavy chain of vedolizumab is set forth as SEQ ID NO: 12. The nucleic acid sequence encoding vedolizumab is also set forth in U.S. Patent Publication 2010 / 0297699, the entire contents of which are incorporated herein. Vedolizumab and the sequence of vedolizumab are further described in U.S. Patent Publication No. 2014 / 0341885 and U.S. Patent Publication No. 2014 / 0377251, the entire contents of each of which are expressly incorporated herein by reference.

[0235] The methods and compositions provided herein are useful for producing anti-α4β7 antibodies, particularly vedolizumab or antibodies having the binding regions, i.e., CDRs or variable regions, of vedolizumab, or antigen-binding fragments of anti-α4β7 antibodies in mammalian cells.

[0236] The methods and compositions disclosed herein relate to mammalian cell culture processes. Mammalian cells have become the primary system for the production of mammalian proteins for clinical applications, such as human therapeutics, primarily due to their ability to produce properly folded and assembled heterologous proteins and their ability to undergo post-translational modifications, including modifications similar to those made by human cells. Chinese hamster ovary (CHO) cells, as well as cell lines derived from various other mammalian sources, such as mouse myeloma (NS0), baby hamster kidney (BHK), human embryonic kidney (HEK-293), and human retinal cells, have been approved by regulatory authorities for the production of biopharmaceuticals, including therapeutic antibodies. Of these, CHO cells are one of the most commonly used industrial hosts, widely used for the production of heterologous proteins. Thus, methods for large-scale production of antibodies in CHO cells, including dihydrofolate reductase-negative (DHFR-) or glutamine synthase-negative (GS-) CHO cells, are well known in the art (see, e.g., Trill et al., Curr. Opin. Biotechnol. 6(5):553-60 (1995); Birch and Racher, Adv. Drug Delivery Reviews 58:671-685 (2006); and U.S. Pat. No. 6,610,516). Examples of CHO cell lines suitable for use in the compositions and methods provided herein include, but are not limited to, GS-CHO, CHO-K1 DUX B11, and DP-12 CHO cells. CHO cells suitable for use in the compositions and methods provided herein are described in the following references: U.S. Pat. Nos. 4,766,075; 4,853,330; 5,185,259; 5,122,464; 5,591,639; 5,879,936; and Lubiniecki et al., in Advances in Animal Cell Biology and Technology for Bioprocesses, Spier et al., eds. (1989), pp. 442-451.Known CHO derivatives suitable for use herein include, for example, CHO / -DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)), CHO-K1 DUX B11 (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA, 80:2495-2499 (1983); Urlaub and Chasin, supra), and DP-12 CHO cells (EP 307,247 published March 15, 1989, or U.S. Pat. No. 5,721,121).

[0237] Other examples of suitable mammalian cell lines include the SV40-transformed monkey kidney CVI line (COS-7, ATCC (商標) CRL1651); human embryonic kidney line 293S (Graham et al., J. Gen. Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC (商標) CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243 (1980)); monkey kidney cells (CVI-76, ATCC (商標) CCL 70); African green monkey kidney cells (VERO-76, ATCC (商標) CRL-1587); human cervical cancer cells (HELA, ATCC (商標) CCL2); canine kidney cells (MDCK, ATCC (商標) CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC™ CRL 1442); human lung cells (W138, ATCC (商標)CCL 75); human hepatocytes (HepG2.HB 8065); mouse mammary tumor cells (MMT 060562, ATCCV CCL 51); rat hepatoma cells (HTC, MI.54, Baumann et al., J. Cell Biol., 85:1 (1980)), 3T3 cells; 293T cells (Pear, WS, et al. al., Proc. Natl. Acad. Sci. USA, 90:8392-8396 (1993)); NS0 cells (Sato et al. Tissue Culture Association, 24: 1223 (1988)); SP2 / 0 (Sato et al. J. Exp. Med., 165: 1761 (1987)); and TR-1 cells (Mather et al., Annals NY Acad. Sci., 383:44 (1982)), and hybridoma cell lines.

[0238] Many host cell types can produce the encoded recombinant polypeptide, but the product encoded by a particular nucleic acid produced in one host cell may differ from the product encoded by that nucleic acid in another host cell. The differences may reside in one or more biochemical properties. Examples of biochemical properties include basic protein structure, such as primary, secondary, or tertiary structure, or post-translational modifications, such as signal peptide processing, glycosylation, N-terminal acetylation, lipidation, or phosphorylation. Specific differences may depend on the cellular enzymatic machinery and / or medium or growth conditions. In the case of recombinant therapeutic antibodies, changes in biochemical properties may affect one or more antibody characteristics, such as binding capacity, antibody effector function, immunogenicity, clearance, solubility, or storage stability.

[0239] In some embodiments, products that differ from the reference product can be reduced or eliminated by purification, e.g., downstream processing techniques. In other embodiments, products that differ from the reference product can be reduced or eliminated by controlling the cellular enzymatic machinery, e.g., upstream processing techniques. In some embodiments, controlling the cellular enzymatic machinery includes mutating the cell to recombinantly modify its genetic background, e.g., mutating an enzyme or modifying its expression. In some embodiments, controlling the cellular enzymatic machinery includes controlling components in the culture medium, such as providing a specific culture medium or adding one or more supplemental components. In some embodiments, controlling the cellular enzymatic machinery includes maintaining or adjusting growth conditions, such as temperature, pH, or atmospheric gases.

[0240] Methods for producing anti-α4β7 antibodies, such as vedolizumab, have been described (see, e.g., U.S. Pat. No. 7,402,410 and U.S. Patent Application Publication No. 20070122404). These publications described specific properties of antibodies, such as binding affinity, effector functions, and biochemical properties, such as charge profile, molecular weight, and glycosylation pattern. Antibodies exhibited certain properties when cultured in NS0 cells, which changed when cultured in CHO cells. The properties of recombinant proteins, e.g., antibodies, can also change when switching to a different variant of CHO cells, e.g., switching from DHFR-cells to GS-cells. Described herein are methods and media compositions that control these variations in, e.g., recombinant protein, e.g., antibody, production and limit or minimize changes in properties when expressing an antibody, e.g., vedolizumab, in GS-CHO cells (also referred to herein simply as "GS-CHO" cells). In certain embodiments, described herein are methods and compositions for producing anti-α4β7 antibodies, such as vedolizumab, in GS-CHO cells.

[0241] Examples of properties that may change when producing anti-α4β7 antibodies such as vedolizumab in cell culture include their charge profile, glycosylation profile, and high molecular weight (HMW) impurity species. Culture conditions such as temperature, pH, shear stress, dissolved oxygen, and medium composition may contribute to these changes. The charge of the enzyme may be altered by variations in the presence or absence of C-terminal lysine, N-terminal pyroglutamic acid, or sialic acid, and / or variations in deamidation or oxidation. The glycosylation profile may be altered by variations in the presence or absence of sialic acid or terminal galactose, processing of high-mannose species, etc. (See Hossler et al. (2009) Glycobiology 19:936-949). Medium supplements may control such changes.

[0242] In some embodiments, properties of anti-α4β7 antibodies produced in cell culture, including but not limited to, charge variation, glycan variation, and aggregate content, can be controlled by adjusting the amount of sugars (e.g., galactose), metal cofactors (e.g., manganese), and / or nucleosides (e.g., uridine) in the medium, e.g., production-stage medium. In some embodiments, properties of anti-α4β7 antibodies produced in cell culture, including but not limited to, charge variation, glycan variation, and aggregate content, can be controlled by adjusting the amount of lysine and arginine in the medium, e.g., production-stage medium. In some embodiments, properties of anti-α4β7 antibodies produced in cell culture, including but not limited to, charge variation, glycan variation, and aggregate content, can be controlled by adjusting the amount of zinc used in the medium, e.g., production-stage medium. Additionally, temperature shifts can be used during production. While it is known in the art that temperature shifts can prove beneficial for antibody production (Moore et al. (1997) Cytotechnology 23:47-54), provided herein are methods based on maintaining cell culture temperature, i.e., methods in which culture conditions do not involve substantial shifts, e.g., not more than 1 degree above or below 37 degrees Celsius.

[0243] A. Zinc supplementation In some embodiments, provided herein are methods and compositions for producing a humanized anti-α4β7 antibody, e.g., vedolizumab, or an antigen-binding portion thereof, in a CHO cell culture supplemented with zinc during the production phase. In some embodiments, zinc is used as a media supplement to control charge variation of the anti-α4β7 antibody in the CHO cell culture. In other embodiments, zinc is used as a media supplement to control the level of high molecular weight (HMW) aggregates in the preparation of an anti-α4β7 antibody produced in the CHO cell culture. The metal ion can be in the form of a hydrochloride, sulfate, nitrate, bromide, acetate, stearate, citrate, or phosphate salt. The media supplement can be provided to the culture in concentrated form along with the feed at the start of the batch, during the expansion phase, or during the production phase. The media supplement can be provided in concentrated form in a feed solution that is also a concentrated supplement. In such embodiments, the supplement can be diluted more than once for each stage of supplement preparation.

[0244] In some embodiments, metal ions, such as zinc, can be added to a production-stage culture. The presence of zinc for the production of an anti-α4β7 antibody, such as vedolizumab, can reduce the level of the antibody's basic isoform (compared to a control process, which is the same process except for the addition of zinc). In some embodiments, zinc can be added to a production-stage culture more than once. In one embodiment, zinc is added as a supplement to the initial production medium of the production-stage culture. In one embodiment, zinc is added to the production-stage culture, for example, directly to the production culture after the start date or in a feed solution. In one embodiment, zinc is added as a supplement to the initial production medium and as a supplement to the production medium after the start date, for example, zinc is added in a feed solution added to the production-stage culture. In some embodiments, zinc is added multiple times in a supplement after the start date of the production-stage culture. For example, zinc is added as a supplement daily, every 2 days, every 3 days, every 4 days, every 1-3 days, every 2-4 days, or every week. In some embodiments, zinc added multiple times after the initiation date of the production-stage culture is not added on days 1, 2, 3, 4, 5, or 6 of the production-stage culture, but is added daily or every two days thereafter. In one embodiment, zinc is added as a supplement to the starting medium and as a daily supplement to the production-stage medium. In another embodiment, zinc is added as a supplement to the starting medium and as a daily supplement to the production-stage medium from day 4 of the production-stage culture. Zinc can be supplemented up to one day prior to harvest, two days prior to harvest, or three days prior to harvest. In one embodiment, zinc is added as a supplement to the starting medium and as a daily supplement to the production-stage medium from day 4 of the production-stage culture until one day prior to harvest.

[0245] In certain embodiments, zinc is included in a method for producing a composition having about 16% or less basic isoform of a humanized anti-α4β7 antibody (as determined by CEX), wherein the humanized antibody, e.g., vedolizumab, is produced in mammalian host cells, e.g., GS-CHO cells, in a production medium containing zinc. In certain embodiments, adding a supplement containing zinc to the production medium provides a composition having about 14% or less basic isoform of a humanized anti-α4β7 antibody. In certain embodiments, including zinc in a supplement for the production medium provides a composition having about 13% or less basic isoform of a humanized anti-α4β7 antibody. In certain embodiments, including zinc in a supplement for the production medium provides a composition having about 12% or less basic isoform of a humanized anti-α4β7 antibody. In certain embodiments, including zinc in a supplement for the production medium provides a composition having about 11% or less basic isoform of a humanized anti-α4β7 antibody. In some embodiments, the level of a basic isoform can be measured on day 14 of cell culture, i.e., 14 days after inoculation of the cell culture. In other embodiments, the level of a basic isoform can be measured on day 15 of cell culture.

[0246] In certain embodiments, zinc is included in a method for producing a composition having about 70% or more of a major isoform of a humanized anti-α4β7 antibody (as determined by CEX), wherein the humanized antibody, e.g., vedolizumab, is produced in mammalian host cells, e.g., GS-CHO cells, in a production medium containing zinc. In certain embodiments, adding a supplement containing zinc to the production medium provides a composition having about 71% or more of a major isoform of a humanized anti-α4β7 antibody. In certain embodiments, adding a supplement containing zinc to the production medium provides a composition having about 72% or more of a major isoform of a humanized anti-α4β7 antibody. In certain embodiments, adding a supplement containing zinc to the production medium provides a composition having about 73% or more of a major isoform of a humanized anti-α4β7 antibody. In certain embodiments, adding a supplement containing zinc to the production medium provides a composition having about 74% or more of a major isoform of a humanized anti-α4β7 antibody. In some embodiments, the level of the major isoform can be measured on day 14 of cell culture. In other embodiments, the level of the major isoform can be measured on day 15 of cell culture.

[0247] In other embodiments, zinc supplementation can be used to limit the level of HMW contamination in preparations containing anti-α4β7 antibodies. In some embodiments, adding zinc to the culture medium at concentrations of about 10-200 μM, about 50-150 μM, or about 100-130 μM can reduce the level of HMW aggregates (as determined by SEC) to <5%, <4%, <3%, <2.5%, <2%, <1.5%, or <1%.

[0248] Zinc can be added directly to the production medium or in a feed supplement to the production medium.

[0249] Zinc ions can be supplemented to a medium, e.g., a production-stage medium, at a final concentration of 10-200 μM, 10-100 μM, 15-90 μM, 20-80 μM, 10-80 μM, 10-70 μM, about 14-55 μM, about 10-60 μM, about 10-30 μM, about 10-20 μM, about 14 μM, about 50 μM, about 55 μM, about 57 μM, or about 15 μM. As described above, zinc ions can be added multiple times. In one embodiment, zinc is added to the production medium of a production-stage culture, resulting in a production medium having a zinc concentration of about 2-60 μM, 5-57 μM, 5-50 μM, 5-40 μM, 8-30 μM, 10-20 μM, 12-15 μM, or about 14 μM. In one embodiment, the cumulative concentration of zinc in the production medium, accounted for by supplementation up to the time of harvest, is about 15.5 μM, with each supplement adding about 1 to about 4 μM of zinc to the medium. In one embodiment, zinc is added to the production medium of the production-stage culture so that the production medium has a zinc concentration of about 50-150 μM, 75-150 μM, 100-150 μM, 80-130 μM, or 100-130 μM. In some embodiments, zinc is added to the production medium of the production stage culture so that the production medium has a zinc concentration of about 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM. In some cases, too much zinc in the starting culture can reduce cell viability and / or reduce antibody titer.

[0250] In some embodiments, zinc is added to the culture medium (e.g., production-phase culture medium) over a period of 10-16 days, e.g., 10-17 days, 10-15 days, or 12-14 days. In some embodiments, zinc supplementation can be added incrementally to the cell culture, e.g., as part of a feed solution. For example, zinc can be added to the medium on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, zinc can be added daily or every other day. In some embodiments, the daily or every other day addition of zinc can begin when the cells reach the production phase. Thus, in some embodiments, zinc can be added to the production medium daily or every other day starting on day 4, 5, or 6 of cell culture. In one embodiment, zinc can be added daily from about day 4 to about day 10. In one embodiment, zinc can be added daily from about day 4 to about day 14. In one embodiment, zinc is added as a supplement after the start of the production phase to replenish the production medium from a feed at a concentration of about 10-80 μM, or at a concentration of about 50-150 μM. In one embodiment, zinc is added to supplement the starter medium of a production-stage culture so that the production medium has a zinc concentration of about 50-150 μM, 2-60 μM, 5-57 μM, 5-50 μM, 5-40 μM, 8-30 μM, 10-20 μM, 12-15 μM, or about 14 μM, and is also added multiple times to supplement the production medium after the start date (e.g., from days 2-10, 2-8, 2-6, 3-6, or 4 of culture) with 0.1-10 μM, 0.5-5 μM, 0.75-4 μM, 0.9-3 μM, 1.0-2.7 μM, or about 1.4 μM or 1.9 μM increments for each addition to the production-stage culture.In another embodiment, zinc is added to supplement the starter medium of the production culture so that the production medium has a zinc concentration of about 2-50 μM, 5-40 μM, 8-30 μM, 10-20 μM, 12-15 μM, or about 14 μM, and is added daily to the production-stage medium beginning on day 4 of the production-stage culture, supplementing with 0.1-10 μM, 0.5-5 μM, 0.75-4 μM, 0.9-3 μM, 1.0-2.7 μM, or about 1.4 μM, or 1.9 μM for each addition to the production-stage culture. In some embodiments, a feed solution is added to the production medium so that zinc is added to the production medium so that the production medium has a total zinc concentration of about 10-20 μM (e.g., 15-17 μM). In some embodiments, the zinc supplement components described herein are added to CHO cell culture media, such as the culture media provided in International Patent Publication WO 98 / 08934 A1, the entire contents of which are incorporated herein by reference. In some embodiments, the zinc supplement components described herein are added to CD-CHO media. In some embodiments, the zinc supplement components described herein are added to CD-CHO AGT (Catalog No. 12490-001 (Invitrogen, Carlsbad, CA, USA)).

[0251] In one embodiment, zinc is added to supplement the feed solution so that the feed solution has a concentration of about 90-120 μM, about 95-120 μM, about 100-120 μM, about 105-120 μM, about 110-120 μM, or about 117 μM. Such a feed supplement can then be added to the production medium.

[0252] In some embodiments, provided herein is a cell culture comprising a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, and a production medium containing or supplemented with zinc. In other embodiments, provided herein is a cell culture obtained by culturing a host cell expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with zinc.

[0253] The aforementioned cell cultures can incorporate any of the embodiments described herein. For example, in some embodiments, the host cells are CHO cells, e.g., GS-CHO cells, or DHFR cells. - The host cell is a CHO cell. In some embodiments, the host cell expresses an antibody or antigen-binding portion thereof comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5. In some embodiments, the host cell expresses an antibody or antigen-binding portion thereof comprising a heavy chain variable region comprising a CDR1 domain set forth in SEQ ID NO: 2, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR3 domain set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 domain set forth in SEQ ID NO: 6, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR3 domain set forth in SEQ ID NO: 8. In some embodiments, the host cell expresses vedolizumab or an antigen-binding portion thereof. In some embodiments, the host cell comprises a nucleic acid set forth in SEQ ID NO: 9 (encoding the light chain variable region of the anti-α4β7 antibody) and a nucleic acid set forth in SEQ ID NO: 10 (encoding the heavy chain variable region of the anti-α4β7 antibody). In some embodiments, the host cell comprises a nucleic acid set forth in SEQ ID NO: 11 (encoding the light chain of vedolizumab) and a nucleic acid set forth in SEQ ID NO: 12 (encoding the heavy chain of vedolizumab).

[0254] In some embodiments, the cell culture contains zinc at a concentration of about 10-100 μM, 10-100 μM, about 15-90 μM, about 20-80 μM, about 10-80 μM, about 10-70 μM, about 14-55 μM, about 10-60 μM, about 10-30 μM, about 10-20 μM, about 14 μM, about 50 μM, about 55 μM, about 57 μM, or about 15 μM. In some embodiments, the cell culture contains zinc at a concentration of about 2-60 μM, 5-57 μM, 5-50 μM, 5-40 μM, 8-30 μM, 10-20 μM, 12-15 μM, or about 14 μM. In some embodiments, the cell culture comprises zinc at a concentration of about 5-45 μM, 50-150 μM, 75-150 μM, 100-150 μM, 80-130 μM, or 100-120 μM zinc. In some embodiments, the cell culture comprises zinc at a concentration of about 1-10 μM, 10-30 μM, 30-50 μM, 50-70 μM, or 70-90 μM zinc. In some embodiments, the cell culture comprises zinc at a concentration of about 1-30 μM, 10-40 μM, 20-50 μM, 30-60 μM, 40-70 μM, or 60-90 μM zinc. In some embodiments, the cell culture comprises zinc at a concentration of about 1-50 μM, 20-60 μM, 30-70 μM, 40-80 μM, or 50-100 μM zinc. In some embodiments, the cell culture contains zinc at a concentration of about 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM.

[0255] In some embodiments, provided herein is a cell culture obtainable by culturing GS-CHO host cells expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with zinc at a concentration of 50-150 μM, 100-120 μM, or 100-120 μM. In some embodiments, the antibody is vedolizumab, or an antigen-binding portion thereof.

[0256] In some embodiments, the cells of the cell culture express an anti-α4β7 antibody, or antigen-binding portion thereof, with reduced levels of basic isoforms (as determined by CEX) compared to equivalent cell cultures containing medium lacking zinc or medium not supplemented with zinc. In some embodiments, the expressed antibody contains about 16% or less basic isoforms. In some embodiments, the expressed antibody contains about 15% or less basic isoforms. In some embodiments, the expressed antibody contains about 14% or less basic isoforms. In some embodiments, the expressed antibody contains about 13% or less basic isoforms. In some embodiments, the expressed antibody contains about 12% or less basic isoforms. In some embodiments, the expressed antibody contains about 11% or less basic isoforms.

[0257] In some embodiments, provided herein are methods for producing a monoclonal antibody, the method comprising: (i) culturing a cell culture provided herein comprising host cells expressing an anti-α4β7 antibody, or antigen-binding portion thereof, and a production medium containing or supplemented with zinc for a period of time sufficient for the host cells to express the anti-α4β7 antibody, or antigen-binding portion thereof; and (ii) recovering the anti-α4β7 antibody, or antigen-binding portion thereof, from the cell culture. In some embodiments, the population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from the cell culture comprises decreased levels of the basic isoform and / or increased levels of the major isoform (as determined by CEX) compared to a population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from an equivalent cell culture comprising medium lacking or not supplemented with zinc. In some embodiments, the population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from the cell culture contains decreased levels of aggregates and / or increased levels of monomers (as determined by SEC) compared to a population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from an equivalent cell culture containing medium lacking zinc or medium not supplemented with zinc. In some embodiments, the cell culture is cultured for 5 to 20 days. In some embodiments, the cell culture is cultured for 10 to 16 days. In some embodiments, the cell culture is cultured for 13 to 15 days. In some embodiments, the cell culture is cultured for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. Further provided are anti-α4β7 antibodies obtained or obtainable by the aforementioned methods provided herein.

[0258] In each embodiment described herein, the cell culture medium can, in some embodiments, be further supplemented with sugars, nucleosides, and / or metal cofactors. For example, the cell culture medium can be further supplemented with uridine, manganese, and zinc. Additionally or alternatively, the cell culture medium can be further supplemented with lysine and / or arginine.

[0259] B. Supplementation with sugars, nucleosides, and / or metal cofactors In some embodiments, provided herein are methods and compositions for producing a humanized anti-α4β7 antibody, e.g., vedolizumab, or an antigen-binding portion thereof, in a CHO cell culture supplemented with sugars, nucleosides, and / or metal cofactors during the production stage.

[0260] In some embodiments, the medium supplement for controlling the glycosylation profile of an anti-α4β7 antibody in CHO cell culture comprises a sugar. For example, the sugar in the supplement can be glucose, fucose, or galactose.

[0261] In some embodiments, the media supplement for controlling the glycosylation profile of an anti-α4β7 antibody in CHO cell culture comprises a nucleoside. For example, the nucleoside in the supplement can be adenosine, uridine, cytidine, guanosine, thymidine, and / or inosine.

[0262] In some embodiments, the medium supplement for controlling the glycosylation profile of an anti-α4β7 antibody in CHO cell culture comprises a metal cofactor. For example, the metal cofactor in the supplement can be magnesium, manganese, iron, or copper.

[0263] In some embodiments, a media supplement for controlling the glycosylation profile of an anti-α4β7 antibody in CHO cell culture comprises a sugar and a nucleoside. In some embodiments, a media supplement for controlling the glycosylation profile of an anti-α4β7 antibody in CHO cell culture comprises a sugar and a metal cofactor. In some embodiments, a media supplement for controlling the glycosylation profile of an anti-α4β7 antibody in CHO cell culture comprises a sugar, a nucleoside, and a metal cofactor.

[0264] In some embodiments, provided herein are methods and compositions for producing a humanized anti-α4β7 antibody, e.g., vedolizumab, or an antigen-binding portion thereof, in a CHO cell culture supplemented with galactose, uridine, and manganese during the production phase. In some embodiments, the supplemented components are present in the same media supplement. In some embodiments, the supplemented components are present in different media supplements. In some embodiments, the different media supplements are combined before adding to the cell culture. In some embodiments, the supplemented components for controlling the glycosylation profile of an anti-α4β7 antibody are added multiple times. For example, they may be added after the start of the production-phase culture, or they may not be added on days 1, 2, 3, 4, 5, or 6 of the production-phase culture, but are added every day or every two days thereafter. In one embodiment, the components for controlling the glycosylation profile are added to the production-phase medium in daily supplements. In another embodiment, the components for controlling the glycosylation profile are added to the production-phase medium in daily supplements starting on day 4 of the production-phase culture.

[0265] In some embodiments, a media supplement for controlling glycosylation is provided to a CHO cell culture for producing an anti-α4β7 antibody during the expansion phase. In other embodiments, it is added during the production phase. In some embodiments, the media supplement for controlling glycosylation is provided at a concentration of 20-400x, 25-300x, 30-250x, 40-120x, about 50x, about 60x, about 100x, or about 200x the final concentration in the medium. In some embodiments, the amount of media supplementation ignores consumption by the cells, which may metabolize some of the supplemented components to other chemical forms.

[0266] In one embodiment, a metal cofactor component, such as manganese, is provided to the cell culture in a media supplement containing a metal ion, such as zinc. In some embodiments, the concentration of the metal cofactor, e.g., manganese, can be 10,000-50,000 times its final concentration in the media, 20,000-40,000 times its final concentration in the media, or about 30,000 times its final concentration in the media.

[0267] In some embodiments, manganese may be present in a medium, e.g., a production-phase medium, or may be added to replenish the medium at a concentration of 0.1-100 μM, 0.5-50 μM, 1.0-25 μM, 2.0-15 μM, 3-10 μM, 1-50 μM, 1-100 μM, 20-50 μM, 30-60 μM, 40-70 μM, 50-80 μM, 70-100 μM, 20-70 μM, 30-80 μM, 40-90 μM, or 50-100 μM. In one embodiment, the concentration of manganese in the production-phase medium is approximately 5.15 μM. Thus, the production-phase medium can be replenished according to a schedule to achieve an average manganese concentration of approximately 5.15 μM. In some embodiments, manganese may be present in or added to supplement the medium, e.g., production stage medium, at a concentration of about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM.

[0268] In one embodiment, manganese is added multiple times after the start date as a supplement to replenish the production phase medium at 0.1-10 μM, 0.2-1.5 μM, 0.2-5 μM, 0.25-2 μM, 0.3-1.2 μM, 0.3 μM-0.8 μM, or about 0.5 μM, or 0.56 μM increments for each addition. In one embodiment, manganese is added multiple times after the start date as a supplement to replenish the production phase medium at about 0.2-1.5 μM increments for each addition. In one embodiment, manganese is added multiple times after the start date as a supplement to replenish the production phase medium at about 0.31-1.2 μM increments for each addition. In some embodiments, the manganese supplement is added daily or every two days starting on day 4 of the production phase culture. In some embodiments, the supplement is not added on the harvest day.

[0269] In one embodiment, manganese is added as a supplement to the feed medium to achieve a final concentration of manganese in the feed medium of 0.02 mM to 0.2 mM, 0.03 mM to 0.15 mM, 0.03 mM to 0.10 mM, 0.03 mM to 0.05 mM, 0.03 mM to 0.04 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.1 mM, or about 0.14 mM. In one embodiment, manganese is added as a supplement to the feed medium to achieve a final concentration of manganese in the feed medium of 0.1 to 100 μM. In one embodiment, manganese is added as a supplement to the feed medium to achieve a final concentration of manganese in the feed medium of 39 μM. In one embodiment, the manganese-supplemented feed medium is added (e.g., multiple times, e.g., daily or every two days) to the production medium after the initiation date of the production-stage culture (e.g., from days 2-10, 2-8, 2-6, 3-6, or 4 of the production-stage culture). In one embodiment, the manganese-supplemented feed medium is added to the production medium starting on day 4 of the production-stage culture.

[0270] Uridine may be added for more than one reason. It can be added to a nutritional supplement along with other nucleosides to support cell growth. Uridine can also be added as a supplement to control the glycosylation profile of anti-α4β7 antibodies. In some embodiments, uridine may be present in a medium, e.g., a production phase medium, or added to supplement the medium, at a concentration of about 0.1 to 20 mM, about 0.9 to 3.0 mM, about 1 to 20 mM, about 0.5 to 12 mM, about 1 to 8 mM, about 1.5 to 4 mM, about 0.1 to 1.5 mM, about 1 to 5 mM, about 1 to 7 mM, about 1 to 6 mM, about 1 to 5 mM, about 1 to 4 mM, about 2 to 4 mM, about 2 to 5 mM, about 2 to 3 mM, about 1 mM to 10 mM, about 10 mM to 15 mM, about 10 mM to 20 mM, about 10 mM to 30 mM, about 1 mM to 40 mM, about 1 mM to 50 mM, or about 10 mM to 30 mM. In some embodiments, uridine may be present in a medium, e.g., a production medium, or added to supplement the medium, at a concentration of about 0.9 mM, 1.0 mM, about 2 mM, about 1.5 mM, about 2.0 mM, about 2.7 mM, about 2.5 mM, about 2.7 mM, about 2.8 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In some embodiments, the amounts listed for production medium describe the amount provided in the basal medium or supplement and do not take into account the amount consumed, metabolized, or produced by the cells. In some embodiments, the amounts listed for production medium are cumulative amounts as the sum of all additions up to the harvest date. In one embodiment, uridine can be supplemented to the production stage medium at 0.1 to 20 mM, 0.5 to 12 mM, 1 to 8 mM, 1.5 to 5 mM, 1.6 to 4.8 mM, or about 2.4 mM.

[0271] In one embodiment, uridine is added multiple times after the start date as a supplement to replenish the production-phase medium, with each addition being 25-1000 μM, 75-750 μM, 55-620 μM, 100-600 μM, 150-450 μM, 100-600 μM, 170-630 μM, or about 250 μM, or about 300 μM. In some embodiments, these uridine supplements are added daily or every two days starting from day 4 of the production-phase culture; furthermore, uridine supplements may not be added on the harvest day. In one embodiment, the supplement containing a nucleoside, e.g., uridine, is present at a concentration of 10-500 times the final concentration in the medium, 20-400 times, 25-300 times, 40-250 times, about 50 times, about 60 times, about 100 times, or about 200 times the final concentration in the medium.

[0272] In one embodiment, uridine is added as a supplement to the feed medium to achieve a final concentration of uridine in the feed medium of about 1-40 mM, 15-25 mM, 15-100 mM, 20-90 mM, 15-70 mM, 15-50 mM, 15-30 mM, about 18 mM, about 19 mM, about 19.3 mM, about 20 mM, about 33 mM, about 50 mM, or about 66 mM. In one embodiment, the uridine-supplemented feed medium is added to the production medium (e.g., multiple times, e.g., daily or every two days) after the initiation date of the production-stage culture (e.g., from days 2-10, 2-8, 2-6, 3-6, or 4 of the production-stage culture). In a specific embodiment, the uridine-supplemented feed medium is added to the production medium starting on day 4 of the production-stage culture.

[0273] In one embodiment, the sugar, e.g., galactose-containing supplement is present at a concentration of 10 to 500 times, 20 to 400 times, 25 to 300 times, 30 to 250 times, 40 to 120 times, about 50 times, about 60 times, about 100 times, or about 200 times the final concentration in the medium to control the glycosylation profile of the anti-α4β7 antibody. In some embodiments, galactose is present in an amount of 0.1 to 100 mM, 1 to 75 mM, 2.5 to 50 mM, 3 to 20 mM, 5 to 35 mM, about 8 to 25 mM, 0.1 to 10 mM, 0.1 to 20 mM, 0.1 to 30 mM, 1 to 10 mM, 1 to 20 mM, 1 to 30 mM, 1 to 40 mM, 1 to 50 mM, 1 to 60 mM, 1 to 70 mM, 1 to 80 mM, 1 to 90 mM, 1 to 100 mM, It may be present in the medium, e.g., production stage medium, or added to supplement the medium, at a concentration of 20-40 mM, 40-60 mM, 60-80 mM, 80-100 mM, 20-50 mM, 30-60 mM, 40-70 mM, 50-80 mM, 70-100 mM, 20-70 mM, 30-80 mM, 40-90 mM, 50-100 mM, or 50-150 mM. In some embodiments, galactose can be present in or added to supplement media, e.g., production stage media, at a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 μM, about 12.5 mM, about 12 mM, about 12.8 mM, about 13 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. In one embodiment, galactose is added multiple times after the start date as a supplement to replenish the production-phase medium at about 0.1-10 mM, 0.2-7.5 mM, 0.5-5 mM, 0.4-2.8 mM, 0.5-3.5 mM, 0.7-2.9 mM, 0.75-2.5 mM, or about 1.2 mM or 1.4 mM per addition. In some embodiments, these galactose supplements are added daily or every two days starting on day 4 of the production-phase culture, and may not be added on the harvest day.

[0274] In one embodiment, galactose is added as a supplement to the feed medium to achieve a final concentration of galactose in the feed medium of 50-150 mM, 85-500 mM, 90-400 mM, 90-300 mM, 90-200 mM, 90-100 mM, about 95 mM, about 96 mM, about 97 mM, about 100 mM, about 165 mM, about 250 mM, or about 330 mM. In one embodiment, the galactose-supplemented feed medium is added to the production medium (e.g., multiple times, e.g., daily or every two days) after the initiation date of the production-stage culture (e.g., from days 2-10, 2-8, 2-6, 3-6, or 4 of the production-stage culture). In a specific embodiment, the galactose-supplemented feed medium is added to the production medium starting on day 4 of the production-stage culture.

[0275] In some embodiments, the production medium is supplemented with uridine, manganese, and galactose (UMG) to control the glycosylation profile of the anti-α4β7 antibody. In some embodiments, the UMG supplement can be added incrementally to the cell culture, e.g., as part of a feed solution. For example, a feed solution containing the UMG supplement can be added every day or every two days.

[0276] In some embodiments, the supplement provides 0.1-0.7 mM uridine, 0.2-1.5 μM manganese, and 0.5-3.5 mM galactose to the production-phase medium. In some embodiments, UMG can be added to the medium on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, UMG can be added daily or every other day. In some embodiments, the daily or every other day addition of UMG can begin when the cells reach the production phase. Thus, in some embodiments, UMG can be added to the production medium daily or every other day starting on day 4, 5, or 6 of cell culture. In one embodiment, UMG can be added daily from about day 4 to about day 10. In one embodiment, UMG can be added daily from about day 4 to about day 14. In some embodiments, the production-stage medium is supplemented with manganese at an average daily addition of 1.0-25 μM, 2.0-15 μM, 3-10 μM, or 5 μM, e.g., about 0.2-1.5 μM or 0.3-1.2 μM; uridine at an average daily addition of 1-8 mM, 1.5-5 mM, 1.6-4.8 mM, or about 2.4 mM, or 2.7 mM, e.g., about 100-700 μM; and galactose at an average daily addition of 0.5-3.5 mM, 0.7-2.9 mM, 2.5-50 mM, 5-35 mM, about 8-25 mM, or about 12 mM or 12.6 mM, e.g., about 0.5-3.5 mM. In certain embodiments, the average daily addition begins on day 4 of the production-stage culture. In some embodiments, the UMG supplement described herein is added to a CHO cell culture medium, such as the culture medium provided in International Patent Publication WO 98 / 08934 A1, the entire contents of which are incorporated herein by reference. In some embodiments, the UMG supplement described herein is added to a CD-CHO medium. In some embodiments, the UMG supplement described herein is added to a CD-CHO AGT (Catalog No. 12490-001 (Invitrogen, Carlsbad, CA, USA)).

[0277] In one embodiment, UMG is added to the production medium such that the cumulative concentration of UMG added from supplementation to harvest is about 1-7 mM uridine, about 2-15 μM manganese, and about 3-20 mM galactose. In some embodiments, the cumulative concentration of zinc added to the production medium from supplementation to harvest is about 5-45 μM.

[0278] In one embodiment, the uridine, manganese, and galactose (UMG) are present in a feed medium having a final concentration of uridine of 1 to 40 mM, 15 to 100 mM, 15 to 90 mM, 15 to 70 mM, 15 to 50 mM, 15 to 30 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 33 mM, about 50 mM, or about 66 mM; and a manganese concentration in the feed medium of about 0.0001 to 0.1 mM, 0.02 mM to 0.2 mM, 0.03 mM to 0.15 mM, 0.03 mM to 0.10 mM, 0.03 mM to 0.05 mM, 0.03 mM to 0.04 mM. 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.1 mM, or about 0.14 mM manganese; and galactose in the feed medium is added as a supplement to a final concentration of galactose of 85 mM to 500 mM, 90 mM to 400 mM, 90 mM to 300 mM, 90 mM to 200 mM, 50 mM to 150 mM, 90 mM to 100 mM, about 95 mM, about 96 mM, about 97 mM, about 100 mM, about 165 mM, about 250 mM, or about 330 mM. In one embodiment, the feed medium supplemented with uridine, manganese, and galactose is added to the production medium (e.g., multiple times, e.g., daily or every two days) after the initiation date of the production-stage culture (e.g., from days 2-10, 2-8, 2-6, 3-6, or 4 of the production-stage culture). In a particular embodiment, the feed medium supplemented with uridine, manganese, and galactose is added to the production medium, e.g., daily, starting on day 4 of the production-stage culture.

[0279] In certain embodiments, a complex supplement containing uridine, manganese, and galactose (UMG) is added to the production medium (or a feed solution subsequently added to the production medium) to produce a composition containing an anti-α4β7 antibody, such as vedolizumab, having reduced levels of basic antibody isoforms. In one embodiment, the level of basic isoforms is about 16% or less (as determined by CEX). In one embodiment, the level of basic isoforms is about 15% or less (as determined by CEX). In one embodiment, the level of basic isoforms is about 14% or less (as determined by CEX). In one embodiment, the level of basic isoforms is about 13% or less (as determined by CEX). In one embodiment, the level of basic isoforms is about 12% or less (as determined by CEX).

[0280] The addition of UMG to the production medium can also affect the levels of acidic and / or major species in compositions of anti-α4β7 antibodies, such as vedolizumab, produced by mammalian cells.

[0281] The addition of UMG to the production medium can also affect the levels of G0F, G1F, and / or G2F glycoforms in compositions of anti-α4β7 antibodies, such as vedolizumab, produced by mammalian cells. A depiction of N-glycan structures that may be present in a population of anti-α4β7 antibodies, such as vedolizumab, is provided in Figure 9.

[0282] In certain embodiments, a complex supplement containing uridine, manganese, and galactose (UMG) is added to the production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having reduced levels of GOF glycoforms. In one embodiment, the level of GOF glycoforms is about 70% or less (as determined by hydrophilic interaction chromatography (HILIC)). In one embodiment, the level of GOF glycoforms is about 69% or less (as determined by HILIC). In one embodiment, the level of GOF glycoforms is about 68% or less (as determined by HILIC). In one embodiment, the level of GOF glycoforms is about 67% or less (as determined by HILIC). In one embodiment, the level of GOF glycoforms is about 66% or less (as determined by HILIC). In one embodiment, the level of GOF glycoforms is about 65% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 64% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 63% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 62% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 61% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 60% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 59% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 58% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 57% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 56% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 55% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 40-75%. In one embodiment, the level of G0F glycoforms is about 45-65% (as determined by HILIC).In one embodiment, the level of G0F glycoforms is about 50-60% (as determined by HILIC).

[0283] In certain embodiments, a complex supplement comprising uridine, manganese, and galactose (UMG) is added to a production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having a reduced level of GOF glycoforms compared to control mammalian host cells expressing the anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 20-40% (e.g., 20-40%, 20-30%, 20-25%) reduction in GOF glycoforms of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 20% reduction in GOF glycoforms of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 25% reduction in the G0F glycoform of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of supplemental components, the control being performed under substantially similar conditions except for the parameters designated as different, e.g., in the absence of supplemental components.

[0284] In certain embodiments, a complex supplement containing uridine, manganese, and galactose (UMG) is added to the production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having an increased level of G1F glycoforms. In one embodiment, the level of G1F glycoforms is about 20% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 21% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 22% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 23% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 24% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 25% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 26% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 27% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 28% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 29% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 30% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 31% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 32% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 33% or greater (as determined by HILIC). In one embodiment, the level of G1F glycoforms is about 20-45%. In one embodiment, the level of G1F glycoforms is about 25-45% (as determined by HILIC). In one embodiment, the level of the G1F glycoform is about 30-40% (as determined by HILIC).

[0285] In certain embodiments, a complex supplement comprising uridine, manganese, and galactose (UMG) is added to a production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having an increased amount of the G1F glycoform compared to a control cell culture comprising mammalian host cells expressing the anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 2-fold to 3.5-fold (e.g., 2-3.5-fold, 2-3.3-fold, 2-3-fold) increase in the G1F glycoform of the humanized anti-α4β7 antibody compared to a control cell culture comprising mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 2-fold increase in the G1F glycoform of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a three-fold increase in the G1F glycoform of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement, the control cell culture being cultured under substantially the same conditions except for certain parameters, e.g., the supplement.

[0286] In certain embodiments, a complex supplement containing uridine, manganese, and galactose (UMG) is added to the production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having increased levels of G2F glycoforms. In one embodiment, the level of G2F glycoforms is about 2% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 2.5% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 3% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 3.5% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 4% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 4.5% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 5% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 5.5% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 6% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 6.5% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 7% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is 10% or less. In one embodiment, the level of G2F glycoforms is about 2-4% (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 3-5% (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 2-7% (as determined by HILIC).

[0287] In certain embodiments, a complex supplement containing uridine, manganese, and galactose (UMG) is added to the production medium (or a feed solution subsequently added to the production medium) to produce a composition containing an anti-α4β7 antibody, e.g., vedolizumab, having increased levels of G2F glycoforms. In one embodiment, the level of G2F glycoforms is about 2% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 3% or greater (as determined by HILIC). In one embodiment, the level of G2F glycoforms is about 4% or greater (as determined by HILIC).

[0288] In certain embodiments, a complex supplement comprising uridine, manganese, and galactose (UMG) is added to a production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having an increased level of G2F glycoforms compared to control mammalian host cells expressing the anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 2- to 5-fold (e.g., 2- to 5-fold, 2- to 4-fold, 3- to 4-fold) increase in G2F glycoforms of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 3-fold increase in G2F glycoforms of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a four-fold increase in the G2F glycoform of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement, the control cell culture being cultured under substantially the same conditions except for certain parameters, e.g., the supplement.

[0289] In certain embodiments, a complex supplement comprising uridine, manganese, and galactose (UMG) is added to a production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, having increased amounts of G1F glycoforms and G2F glycoforms compared to a control cell culture comprising mammalian host cells expressing anti-α4β7 cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a 2- to 5-fold (e.g., 2- to 5-fold, 2- to 4-fold, 3- to 4-fold) increase in G1F glycoforms and at least about a 2- to 5-fold (e.g., 2- to 5-fold, 2- to 4-fold, 3- to 4-fold) increase in G2F glycoforms of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement. In one embodiment, the composition comprises at least about a three-fold increase in the G1F glycoform and the G2F glycoform of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of supplementation. In one embodiment, the composition comprises at least about a two-fold increase in the G1F glycoform and at least about a four-fold increase in the G2F glycoform of the humanized anti-α4β7 antibody compared to a cell culture comprising control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of supplementation. The control cell culture is cultured under substantially the same conditions, except for certain parameters, e.g., supplementation.

[0290] In certain embodiments, a complex supplement comprising uridine, manganese, and galactose (UMG) is added to the production medium (or a feed solution subsequently added to the production medium) to produce a composition comprising an anti-α4β7 antibody, wherein the composition has glycosylation variants of 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F). In one embodiment, the compositions and methods described herein can produce a population of humanized anti-α4β7 antibodies having 91-96%, 92-95%, 91-92%, 91-92.5%, 91-93%, or 91-95% total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F) glycosylation variants. In one embodiment, the compositions and methods described herein can produce a population of humanized anti-α4β7 antibodies having 92-98%, 92-97%, 92-96%, or 92-95% total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F) glycosylation variants.

[0291] Media supplements consist of water, basal medium, or a mixture of ascorbate, citrate, carbonate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), histidine, glutamate, acetate, succinate, gluconate, histidine, phosphate, maleate, cacodylate, 2-[N-morpholino]ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris), N-[2-acetate], thiazolinone ... The medium supplement may be provided in a buffer solution such as 2-iminodiacetic acid (ADA), glycylglycine, and other organic acids or zwitterionic buffers. In some embodiments, the medium supplement has a pH of 5.5-7.0, 6.0-7.5, or 5.9-6.1. In other embodiments, the medium supplement has a pH of 1.5-5.5, 1.8-3.0, 3.2-4.5, or 1.9-2.1. In other embodiments, the medium supplement has a pH of 7.5-9.0.

[0292] In some embodiments, zinc and UMG are used to supplement the feed solution that is added daily starting on day 4 of the production stage culture.

[0293] In certain embodiments, the metal-containing supplement component, e.g., zinc or manganese, is a low pH buffer, such as a citrate or acetate buffer. Citrate may also function to chelate the metal ion, limiting toxicity of the metal ion supplement component. In one embodiment, the metal-containing supplement component comprises zinc and manganese, e.g., zinc and manganese in a 100-140 mM or 115-125 mM citrate buffer. In one embodiment, the buffer for the metal-containing supplement component comprises 118-122 mM citric acid, pH 1.9-2.1. Thus, in some embodiments, provided herein is a cell culture obtained by culturing GS-CHO host cells expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium supplemented with a solution of 50-150 μM zinc and 10-50 mM manganese in 115-125 mM citrate buffer, pH 1.9-2.1. In some embodiments, provided herein is a cell culture obtained by culturing GS-CHO host cells expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium supplemented with zinc and manganese in a 115-125 mM citrate buffer feed supplement, pH 1.9-2.1, to provide concentrations of 10-100 μM zinc and 0.1-100 μM manganese. In some embodiments, the citrate buffer metal supplement is added to the feed supplement daily, for example, starting on day 4 of the production culture.

[0294] In some embodiments, provided herein is a cell culture comprising a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, and a production medium containing or supplemented with metal ions, nucleosides, sugars, and / or metal cofactors. In other embodiments, provided herein is a cell culture obtained by culturing a host cell expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with metal ions, nucleosides, sugars, and / or metal cofactors.

[0295] In some embodiments, provided herein is a cell culture comprising a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, and a production medium containing or supplemented with sugars, nucleosides, and / or metal cofactors. In other embodiments, provided herein is a cell culture obtained by culturing a host cell expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with sugars, nucleosides, and / or metal cofactors.

[0296] In some embodiments, provided herein is a cell culture comprising a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, and a production medium containing or supplemented with uridine, manganese, and galactose (UMG). In other embodiments, provided herein is a cell culture obtained by culturing a host cell expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with uridine, manganese, and galactose (UMG).

[0297] The aforementioned cell cultures can incorporate any of the embodiments described herein. For example, in some embodiments, the host cells are CHO cells, e.g., GS-CHO cells, or DHFR cells. -The host cell is a CHO cell. In some embodiments, the host cell expresses an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5. In some embodiments, the host cell expresses an antibody or antigen-binding portion thereof comprising a heavy chain variable region comprising a CDR1 domain set forth in SEQ ID NO: 2, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR3 domain set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 domain set forth in SEQ ID NO: 6, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR3 domain set forth in SEQ ID NO: 8. In some embodiments, the host cell expresses vedolizumab or an antigen-binding portion thereof. In some embodiments, the host cell comprises a nucleic acid set forth in SEQ ID NO: 9 (encoding the light chain variable region of the anti-α4β7 antibody) and a nucleic acid set forth in SEQ ID NO: 10 (encoding the light chain variable region of the anti-α4β7 antibody). In some embodiments, the host cell comprises a nucleic acid set forth in SEQ ID NO: 11 (encoding the light chain of vedolizumab) and a nucleic acid set forth in SEQ ID NO: 12 (encoding the heavy chain of vedolizumab).

[0298] In some embodiments, the cell culture contains uridine at a concentration of 0.1 to 20 mM. For example, in some embodiments, the cell culture contains uridine at a concentration of 0.1 to 20 mM, 1 to 20 mM, 0.5 to 12 mM, 1 to 8 mM, 1.5 to 4 mM, 0.1 to 1.5 mM, 0.1 to 5 mM, 5 to 10 mM, 10 to 15 mM, 15 to 20 mM, 0.1 to 10 mM, 10 to 20 mM, 1 to 7 mM, 7 to 14 mM, or 14 to 20 mM. In other embodiments, the cell culture contains uridine at a concentration of 10 to 50 mM, 20 to 60 mM, 30 to 70 mM, 40 to 80 mM, 50 to 90 mM, 60 to 100 mM, or 0.1 to 100 mM. In some embodiments, the cell culture comprises uridine at a concentration of about 10 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 25 mM, about 27 mM, about 30 mM, about 33 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 66 mM, or about 70 mM.

[0299] In some embodiments, the cell culture comprises manganese at a concentration of 0.1 to 100 μM. For example, in some embodiments, the cell culture comprises manganese at a concentration of 0.1 to 100 μM, 0.5 to 50 μM, 1.0 to 25 μM, 2.0 to 15 μM, 3 to 10 μM, 0.1 to 10 μM, 0.1 to 20 μM, 0.1 to 30 μM, 1 to 10 μM, 1 to 20 μM, 1 to 30 μM, 1 to 40 μM, 1 to 50 μM, 1 to 60 μM, 1 to 70 μM , 1-80 μM, 1-90 μM, 1-100 μM, 20-40 μM, 40-60 μM, 60-80 μM, 80-100 μM, 20-50 μM, 30-60 μM, 40-70 μM, 50-80 μM, 70-100 μM, 20-70 μM, 30-80 μM, 40-90 μM, or 50-100 μM concentrations of manganese. In other embodiments, the cell culture comprises manganese at a concentration of about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 2 μM, about 3 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM.

[0300] In some embodiments, the cell culture contains galactose at a concentration of about 0.1 to 100 mM. For example, in some embodiments, the cell culture contains galactose at a concentration of 1 to 75 mM, 2.5 to 50 mM, 5 to 35 mM, about 8 to 25 mM, 0.1 to 10 mM, 0.1 to 20 mM, 0.1 to 30 mM, 1 to 10 mM, 1 to 20 mM, 1 to 30 mM, 1 to 40 mM, 1 to 50 mM, 1 to 60 mM, 1 to 70 mM, 1 to 80 mM, Include galactose at concentrations of 1-90 mM, 1-100 mM, 20-40 mM, 40-60 mM, 60-80 mM, 80-100 mM, 20-50 mM, 30-60 mM, 40-70 mM, 50-80 mM, 70-100 mM, 20-70 mM, 30-80 mM, 40-90 mM, or 50-100 mM. In some embodiments, the cell culture comprises galactose at a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 μM, about 12.5 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0301] In some embodiments, provided herein is a cell culture obtainable by culturing a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with uridine, manganese, and galactose (UMG). For example, in some embodiments, the cell culture can contain 0.1-20 mM uridine (and ranges therein), 0.1-100 μM manganese (and ranges therein), and 0.1-100 mM galactose (and ranges therein). In some embodiments, the cell culture can further contain zinc, as described herein. In some embodiments, the cell culture can further contain lysine and / or arginine, as described herein. In some embodiments, the cell culture can further contain zinc, lysine, and arginine. In some embodiments, provided herein is a cell culture comprising a host cell (or population of host cells) expressing an anti-α4β7 antibody or antigen-binding portion thereof (e.g., vedolizumab), and a production medium, wherein the production medium is supplemented with a cumulative concentration of about 1 to about 7 mM uridine, about 2 to about 15 μM manganese, about 3 to about 20 mM galactose, and / or about 0.005 to 0.045 μM zinc, e.g., during the production phase, e.g., from day 4 until harvest.

[0302] In some embodiments, provided herein is a cell culture obtainable by culturing a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with uridine, manganese, galactose, and zinc. For example, in some embodiments, the cell culture can contain 0.1-20 mM uridine (and ranges therein), 0.1-100 μM manganese (and ranges therein), 0.1-100 mM galactose (and ranges therein), and 10-100 μM zinc (and ranges therein).

[0303] In some embodiments, provided herein is a cell culture obtainable by culturing a host cell (or population of host cells) expressing an anti-α4β7 antibody, or antigen-binding portion thereof, in a production medium containing or supplemented with uridine, manganese, galactose, zinc, lysine, and / or arginine. For example, in some embodiments, the cell culture can contain 0.1-20 mM uridine (and ranges therein), 0.1-100 μM manganese (and ranges therein), 0.1-100 mM galactose (and ranges therein), 10-100 μM zinc (and ranges therein), 5.0-8.8 g / L lysine (and ranges therein), and / or 3.0-12.0 g / L arginine (and ranges therein).

[0304] In some embodiments, the cells of the cell culture express an anti-α4β7 antibody, or antigen-binding portion thereof, with reduced levels of basic isoforms (as determined by CEX) compared to equivalent cell cultures comprising medium lacking uridine, manganese, and galactose, or medium not supplemented with uridine, manganese, and galactose. In some embodiments, the expressed antibody comprises about 16% or less basic isoforms (as determined by CEX). In some embodiments, the expressed antibody comprises about 15% or less basic isoforms (as determined by CEX). In some embodiments, the expressed antibody comprises about 14% or less basic isoforms (as determined by CEX). In some embodiments, the expressed antibody comprises about 13% or less basic isoforms (as determined by CEX). In some embodiments, the expressed antibody comprises about 12% or less basic isoforms (as determined by CEX). In some embodiments, the expressed antibody comprises about 11% or less basic isoforms (as determined by CEX).

[0305] In some embodiments, the cells of the cell culture express an anti-α4β7 antibody, or antigen-binding portion thereof, with reduced levels of GOF glycoforms (as determined by HILIC) compared to a comparable cell culture comprising medium lacking uridine, manganese, and galactose, or medium not supplemented with uridine, manganese, and galactose, hi some embodiments, the cells of the cell culture express an anti-α4β7 antibody with GOF content of 70% or less, 65% or less, 60% or less, or 55% or less (as determined by HILIC). In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a GOF content of 85% or less, 80% or less, 75% or less, 70% or less, 69% or less, 68% or less, 67% or less, 66% or less, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, 60% or less, 59% or less, 58% or less, 57% or less, 56% or less, or 55% or less (as determined by HILIC). In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a GOF content of 45-65%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a GOF content of 50-60%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a GOF content of 45-85%. In some embodiments, the cells of the cell culture express anti-α4β7 antibodies with a GOF content of 45-82%.In some embodiments, the GOF content of an anti-α4β7 antibody produced by a cell culture comprising medium containing or supplemented with uridine, manganese, and / or galactose as described herein is reduced by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40% compared to the GOF content of an anti-α4β7 antibody produced by a comparable cell culture comprising medium lacking uridine, manganese, and / or galactose or medium not supplemented with uridine, manganese, and / or galactose.

[0306] In some embodiments, the cells of the cell culture express an anti-α4β7 antibody, or antigen-binding portion thereof, with increased levels of the G1F glycoform (as determined by HILIC) compared to a comparable cell culture containing medium lacking uridine, manganese, and galactose, or medium not supplemented with uridine, manganese, and galactose. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G1F content of 10% or more, 15% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, or 33% or more (as determined by HILIC). In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G1F content of 25-45%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody having a G1F content of 30-40%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody having a G1F content of 10-45%. In some embodiments, the G1F content of an anti-α4β7 antibody produced by a cell culture containing or supplemented with uridine, manganese, and / or galactose described herein is increased by at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25-fold, or at least 3.5-fold compared to the G1F content of an anti-α4β7 antibody produced by a comparable cell culture containing medium lacking uridine, manganese, and / or galactose or medium not supplemented with uridine, manganese, and / or galactose.

[0307] In some embodiments, the cells of the cell culture express an anti-α4β7 antibody, or antigen-binding portion thereof, with increased levels of G2F glycoforms (as determined by HILIC) compared to a comparable cell culture containing medium lacking uridine, manganese, and galactose, or medium not supplemented with uridine, manganese, and galactose. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G2F content of 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more, or 8% or more (as determined by HILIC). In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G2F content of 2-4%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G2F content of 3-5%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G2F content of 2-7%. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody with a G2F content of 0.5-7.5%. In some embodiments, the G2F content of an anti-α4β7 antibody produced by a cell culture containing or supplemented with uridine, manganese, and / or galactose described herein is increased by at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25-fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25-fold, at least 4.5-fold, at least 4.75-fold, or at least 5-fold compared to the G1F content of an anti-α4β7 antibody produced by a comparable cell culture containing medium lacking uridine, manganese, and / or galactose or medium not supplemented with uridine, manganese, and / or galactose.

[0308] The cell cultures provided herein, in some embodiments, are capable of producing a population of humanized anti-α4β7 antibodies, wherein the population has glycosylation variants of 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more total asialo, agalacto, core-fucosylated biantennary glycans (G0F), asialo, monogalacto, core-fucosylated biantennary glycans (G1F), and / or asialo, digalacto, core-fucosylated biantennary glycans (G2F) (as determined by HILIC). In one embodiment, the cell culture is capable of producing a population of humanized anti-α4β7 antibodies having 91-96%, 92-95%, 91-92%, 91-92.5%, 91-93%, or 91-95% total asialo, agalactic, core-fucosylated biantennary glycans (G0F), asialo, monogalactic, core-fucosylated biantennary glycans (G1F), and / or asialo, digalactic, core-fucosylated biantennary glycans (G2F) glycosylation variants (as determined by HILIC). In one embodiment, the cell culture is capable of producing a population of humanized anti-α4β7 antibodies having 92-98%, 92-97%, 92-96%, or 92-95% total asialo, agalactic, core-fucosylated biantennary glycans (G0F), asialo, monogalactic, core-fucosylated biantennary glycans (G1F), and / or asialo, digalactic, core-fucosylated biantennary glycans (G2F) glycosylation variants.

[0309] In some embodiments, provided herein are methods for producing a monoclonal antibody, the method comprising: (i) culturing a cell culture comprising host cells expressing an anti-α4β7 antibody, or antigen-binding portion thereof, and a production medium containing or supplemented with uridine, manganese, and / or galactose for a period of time sufficient for the host cells to express the anti-α4β7 antibody, or antigen-binding portion thereof; and (ii) recovering the anti-α4β7 antibody, or antigen-binding portion thereof, from the cell culture. In some embodiments, the population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from the cell culture comprises a reduced level of the basic isoform (as determined by CEX) compared to a population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from an equivalent cell culture comprising medium lacking uridine, manganese, and / or galactose or medium not supplemented with uridine, manganese, and / or galactose. In some embodiments, the population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from the cell culture contains a reduced level of GOF compared to a population of anti-α4β7 antibodies, or antigen-binding portions thereof, recovered from an equivalent cell culture containing medium lacking uridine, manganese, and / or galactose or medium not supplemented with uridine, manganese, and / or galactose. In some embodiments, the production medium further comprises or is further supplemented with zinc. In some embodiments, the production medium further comprises or is further supplemented with lysine and / or arginine. In some embodiments, the cell culture is cultured for 5 to 20 days. In some embodiments, the cell culture is cultured for 10 to 16 days. In some embodiments, the cell culture is cultured for 13 to 15 days. In some embodiments, the cell culture is cultured for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. Further provided are anti-α4β7 antibodies obtained or obtainable by the aforementioned methods provided herein.

[0310] In one embodiment, provided herein is a composition comprising vedolizumab having 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F) glycosylation variants. In one embodiment, provided herein are compositions comprising vedolizumab having 91-96%, 92-95%, 91-92%, 91-92.5%, 91-93%, or 91-95% total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F) glycosylation variants. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, and galactose. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, and zinc. In some embodiments, the aforementioned compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, zinc, arginine, and / or lysine.

[0311] In one embodiment, provided herein is a composition comprising vedolizumab having 85% or less, 80% or less, 75% or less, 70% or less, 69% or less, 68% or less, 67% or less, 66%, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, 60% or less, 59% or less, 58% or less, 57% or less, 56% or less, or 55% or less asialo-, agalacto-, core-fucosylated biantennary glycans (G0F) (as determined by HILIC). In one embodiment, provided herein is a composition comprising vedolizumab having 45-65%, or 50-60% asialo-, agalacto-, core-fucosylated biantennary glycans (G0F) (as determined by HILIC). In some embodiments, the aforementioned composition can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, and galactose. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, and zinc. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, zinc, arginine, and / or lysine.

[0312] In one embodiment, provided herein is a composition comprising vedolizumab having 10% or more, 15% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, or 33% or more asialo-, monogalactoc-, core-fucosylated biantennary glycans (G1F) (as determined by HILIC). In one embodiment, provided herein is a composition comprising vedolizumab having 25-45%, or 30-40% asialo-, monogalactoc-, core-fucosylated biantennary glycans (G1F) (as determined by HILIC). In some embodiments, the aforementioned composition can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, and galactose. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, and zinc. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, zinc, arginine, and / or lysine.

[0313] In one embodiment, provided herein is a composition comprising vedolizumab having 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more, or 8% or more asialo-, digalactoc-, core-fucosylated biantennary glycans (G2F) (as determined by HILIC). In one embodiment, provided herein is a composition comprising vedolizumab having 2-4%, 3-5%, or 2-7% asialo-, digalactoc-, core-fucosylated biantennary glycans (G2F) (as determined by HILIC). In some embodiments, the aforementioned composition can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, and galactose. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, and zinc. In some embodiments, the compositions can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in a production medium supplemented with uridine, manganese, galactose, zinc, arginine, and / or lysine.

[0314] In some embodiments, methods for producing anti-α4β7 antibodies in CHO cell culture include providing the culture with a media supplement comprising a metal ion and a metal cofactor, and another media supplement comprising a nucleoside and a sugar. In some embodiments, methods for producing anti-α4β7 antibodies in CHO cell culture include providing the culture with a media supplement comprising a metal ion, and another media supplement comprising a nucleoside, a sugar, and a metal cofactor. In some embodiments, methods for producing anti-α4β7 antibodies in CHO cell culture include providing the culture with a media supplement comprising a metal ion, a nucleoside, a sugar, and a metal cofactor. In some embodiments, methods for producing anti-α4β7 antibodies in CHO cell culture include providing the culture with a media supplement comprising a metal ion, a nucleoside, a sugar, and a metal cofactor.

[0315] The exemplary media supplement components and their methods of use provided in the Examples are considered embodiments of the present invention.

[0316] C. Lysine and / or arginine supplementation In some embodiments of the foregoing aspects, the cell culture medium, e.g., production stage medium, can be further supplemented with lysine and / or arginine. Thus, in some aspects, the methods and compositions provided herein can employ cell culture medium, e.g., production stage medium, supplemented with zinc, lysine, and / or arginine. In other aspects, the methods and compositions provided herein can employ cell culture medium, e.g., production stage medium, supplemented with uridine, manganese, galactose, lysine, and / or arginine. In other aspects, the methods and compositions provided herein can employ cell culture medium, e.g., production stage medium, supplemented with uridine, manganese, galactose, zinc, lysine, and / or arginine.

[0317] In one embodiment, the production medium contains 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine. In one embodiment, the production medium contains 4.5 to 5.5 g / L of lysine. In one embodiment, the production medium contains 5.5 to 8.8 g / L of lysine. In one embodiment, the production medium contains 5.4 to 7.4 g / L of arginine. In one embodiment, the production medium contains 7.4 to 12 g / L of arginine.

[0318] The medium can be supplemented with uridine, manganese, galactose, and zinc, as described above. For example, in some embodiments, the cell culture medium, e.g., production-phase medium, is supplemented with 0.1-20 mM uridine, 0.1-100 μM manganese, 0.1-100 mM galactose, and 1-100 μM zinc, and is further supplemented with 5.0-8.8 g / L lysine and / or 3.0-12.0 g / L arginine.

[0319] III. Upstream production methods The present invention relates to large-scale recombinant production of antibodies, such as anti-α4β7 antibodies, in mammalian host cells using conditions and / or supplemental components identified herein that result in anti-α4β7 antibodies, such as vedolizumab, at titers greater than 3 g / L. High-level recombinant antibody expression in mammalian cell culture systems is a known challenge in the art.

[0320] The overall process includes inoculating mammalian cells engineered to express an anti-α4β7 antibody into a cell culture medium, followed by a growth phase, a production phase, and finally a harvest phase in which the recombinant antibody is harvested. Between the various phases, there may be transitional phases in certain embodiments.

[0321] Thus, as a first step, a nucleic acid (e.g., a cDNA) encoding the desired recombinant anti-α4β7 antibody can be inserted into a replicable vector for expression. A variety of vectors are publicly available and known to those skilled in the art. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is described below. Optional signal sequences, origins of replication, marker genes, enhancer elements, and transcription terminator sequences that can be used are known in the art and are described in further detail in PCT Publication WO 97 / 25428 or U.S. Patent No. 7,053,202.

[0322] Expression vectors typically contain a promoter that is recognized by a host organism and operably linked to a nucleic acid sequence encoding a protein. Promoters are untranslated sequences (generally within about 100 to 1000 bp) located upstream (5') of the start codon of a structural gene and control the transcription and translation of the specific nucleic acid sequence to which they are operably linked. Such promoters are typically divided into two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Numerous promoters recognized by a variety of potential host cells are now known. These promoters are operably linked to DNA encoding the desired protein by recovering the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.

[0323] Expression vectors that provide transient expression in mammalian cells can be used. Generally, transient expression involves the use of an expression vector that can replicate efficiently in a host cell, resulting in the host cell accumulating many copies of the expression vector and, consequently, synthesizing high levels of the desired polypeptide encoded by the expression vector (Sambrook et al., supra). A transient expression system comprising an appropriate expression vector and host cells allows for convenient positive identification of polypeptides encoded by cloned DNA and rapid screening of such polypeptides for desired biological or physiological properties. Mammalian host cells are transfected, preferably transformed, with the above-described expression vector and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Such cells are then grown and, ultimately, transferred, after several rounds of replication, to larger vessels for subsequent growth and, ultimately, production of the desired polypeptide.

[0324] Mammalian cells, such as CHO cells, may be cultured in small-scale cultures, e.g., up to 5 L, e.g., 5 ml, 25 ml, 50 ml, 100 ml, 250 ml, 1 L, 3 L, or 5 L vessels. Alternatively, the cultures may be medium-sized vessels, e.g., 10 L, 20 L, 100 L, or 200 L vessels. Alternatively, the cultures may be large-scale cultures in vessels of more than 200 L, such as 500 L, 1000 L, 2000 L, 3000 L, 5000 L, 10,000 L, and 15,000 L vessels. Large-scale cell cultures, such as for the production of therapeutic antibodies, are typically maintained for days or even weeks during which the cells produce the protein(s) of interest.

[0325] For purposes of this invention, a cell culture medium is a medium suitable for growing animal cells, such as mammalian cells, in in vitro cell culture. Examples of types of cell culture medium include expansion cell culture medium and production cell culture medium.

[0326] Cell culture medium formulations are well known in the art. Typically, cell culture media are composed of buffers, salts, carbohydrates, amino acids, vitamins, and trace amounts of essential elements. Cell culture media may or may not contain serum, peptone, protein hydrolysates, and / or proteins. Various tissue culture media, including serum-free and defined media, are commercially available. For example, any one or combination of the following cell culture media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as EX-CELL™ 300 series (JRH Biosciences, Lenexa, Kans.), among others. Cell culture media can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements, depending on the requirements of the cells being cultured and / or the desired cell culture parameters. CHO cell media are known in the art, such as CD-CHO (Invitrogen), CD-CHO-AGT™ medium (ThermoFisher Scientific), HYCELL™ CHO medium (GE Healthcare Life Sciences), or CHOMACS CD medium (Militenyi Biotech). In some embodiments, commercially available media, as described above, can be used as starting media for production-stage cultures to produce anti-α4β7 antibodies, such as vedolizumab, in GS-CHO cells. In one preferred embodiment, the antibody is produced in GS-CHO cells grown in CD-CHO medium, which is supplemented as described herein.

[0327] Prior to the production phase, mammalian cells are first cultured in a growth phase under environmental conditions that maximize cell growth and viability. Following the growth phase, a production phase is initiated, whereby cell culture conditions that maximize polypeptide production are used. The growth and production phases can be preceded by or separated by one or more transition phases. For example, in one embodiment, the production phase of the cell culture process is preceded by a cell culture transition phase that involves production phase parameters of the cell culture.

[0328] During the growth phase, mammalian cells are grown under conditions and for a period of time that maximizes proliferation. Culture conditions, such as temperature, pH, and dissolved oxygen (DO), will be used for a particular host and will be apparent to those skilled in the art. Generally, pH is adjusted to a level of approximately 6.5 to 7.5 using either an acid (e.g., CO) or a base (e.g., NaCO or NaOH). A suitable temperature range for culturing mammalian cells, such as CHO cells, is between approximately 30 and 40 degrees Celsius, preferably between 36 and 38 degrees Celsius.

[0329] In commercial processes for the production of proteins by mammalian cells, there are generally multiple, e.g., different, e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth stages occurring in successively larger culture vessels prior to the final production stage.

[0330] Once the cells have grown to sufficient numbers, they are transferred to a large-scale production vessel, e.g., a bioreactor, to begin the production phase, whereby the mammalian host cells are cultured under conditions that promote the production of the polypeptide of interest, i.e., the antibody. One skilled in the art can choose to use one or more of the cell culture media described herein that have been developed for recombinant polypeptide production in a particular cultured host cell. Alternatively, the methods and compositions of the present invention can be used in combination with commercially available cell culture media.

[0331] Typically, the growth phase occurs at a higher temperature than the production phase. For example, the growth phase can occur at a first temperature of about 35°C to about 38°C, and the production phase can occur at a second temperature of about 30°C to about 34°C. However, as described in the Examples, one improvement identified herein is maintaining substantially similar temperatures between the growth phase and the production phase of mammalian cells in cell culture for the production of anti-α4β7 antibodies, such as vedolizumab, resulting in increased antibody titers from the cell culture. Indeed, by maintaining similar temperatures between the two phases, the antibody titer of vedolizumab was greater than 1 g / L, e.g., about 5-7 g / L.

[0332] Thus, in one embodiment, the invention features a method of producing a humanized anti-α4β7 antibody in a mammalian host cell, wherein the mammalian host cell is cultured in cell culture medium during an expansion phase, followed by a production phase, and both the expansion phase and the production phase are carried out at about the same average temperature, e.g., an average temperature for both phases of 36-38° C. In one embodiment, the average temperature for both the expansion phase and the production phase is 36.5-37.5° C., e.g., about 37° C.

[0333] Alternatively, the invention features a method for producing a humanized anti-α4β7 antibody in a mammalian host cell, wherein the mammalian host cell is cultured in cell culture medium during an expansion phase and then in cell culture medium during a production phase, and both the expansion phase and the production phase are carried out at about the same average temperature range, e.g., any temperature in the range of 36-38 degrees Celsius, e.g., 36.5-37.5 degrees Celsius.

[0334] The length of the production phase can vary depending on the cells and the antibody being expressed. In certain embodiments, the production phase is about 14 days or less. In certain embodiments, the production phase is about 15 days or less. In certain embodiments, the production phase is about 16 days or less. Alternatively, the production phase is 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 10-16 days, 11-15 days, 13-17 days, or 12-14 days. These figures include partial days, e.g., 13.5 days.

[0335] In one embodiment, the pH of the cell culture medium is in the range of 6.0 to 8.0, 6.5 to 7.5, 6.7 to 7.0, 6.7 to 6.9, 6.95 to 7.05, or 7.1 to 7.2. Intermediate pH values, such as 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0, are also intended to be part of the present invention, as are all other pH values ​​described herein. Ranges of values ​​using any combination of the above values ​​as upper and / or lower limits are intended to be within the scope of the present invention. In some embodiments, the pH of the culture may shift from one pH to another, e.g., to a pH lower than that at the time of inoculation. For example, the pH can be shifted from a pH range of 6.9-7.1, 6.95-7.05, or pH 7.00 ±0.1, ±0.05, or ±0.02 to a pH range of 6.7-7.0, 6.75-6.85, or pH 6.8 ±0.1 or ±0.02. The timing of the shift can be after 2, 3, 4, or 5 days of culture. In some embodiments, the pH shift occurs on day 4 or 5 of the production-stage culture.

[0336] Thus, in one embodiment, provided herein is a method for producing a humanized anti-α4β7 antibody in a mammalian host cell genetically engineered to express the antibody, wherein the mammalian host cell is cultured in a production medium at a first pH and then shifted to a second pH, the second pH being lower than the first pH. For example, in some embodiments, the second pH can be shifted 0.1 to 0.5 pH units lower than the first pH during the production phase of the host cell culture. In one embodiment, the starting pH can be in the range of pH 6.8 to pH 7.2. After the pH shift has occurred, the adjusted pH can be decreased by 0.1 to 0.5 pH units, e.g., 0.1, 0.2, 0.3, 0.4, or 0.5 pH units. Thus, in some embodiments, the second pH can be in the range of about 6.7 to 6.95.

[0337] As described in the Examples below, a pH shift during production can, for example, reduce the amount of a basic isoform of an antibody, reduce the amount of an acidic isoform of an antibody, and / or increase the amount of a major isoform of an antibody.

[0338] In some embodiments, the pH of the culture medium is maintained at a pH in the range of 6.5 to 7.0 during the production phase.

[0339] In some embodiments, the pH of the culture medium is maintained at a pH in the range of 6.7 to 7.0 during the production phase.

[0340] In certain embodiments, the pH of the cell culture medium during the production phase is about 6.85.

[0341] During the production phase, the culture can be supplemented with a concentrated feed medium containing nutrients, amino acids, and other components consumed during the production phase of the cell culture. Concentrated feed media can be based on almost any cell culture medium formulation. Such concentrated feed media can contain most or a subset of the components of a cell culture medium, for example, at approximately 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 25-40x, 30x, 50x, 100x, 40-120x, 200x, 400x, 600x, 800x, or even approximately 1000x the usual amounts. Concentrated feed media are often used in fed-batch processes.

[0342] In one embodiment, the production phase is a fed-batch culture. Fed-batch culture is a culture method widely used for large-scale protein production from mammalian cells. See, for example, Chu and Robinson (2001), Current Opin. Biotechnol. 12:180-87. Antibody production can be demanding on cells; basal or starting media cannot sustain high cell densities and high levels of antibody production. Without fresh nutrients, such as amino acids and an energy source, yields can decline and cells can die. For example, a culture that consumes its supply of amino acids, such as tyrosine, will cease antibody production. A fed-batch culture of mammalian cells is a culture in which the culture is continuously or periodically fed with a concentrated feed medium containing nutrients. Feeding can occur on a predetermined schedule, such as daily, every other day, or every third day. In one embodiment, one or more additional nutrients, e.g., selected from the group consisting of glucose, zinc, manganese, uridine, and galactose, are added to the cell culture medium, e.g., via a media supplement, starting on or around day 4 of the production phase. The feed solution is added on a schedule including daily, every other day, every two days, and combinations thereof. In some embodiments, tyrosine is added twice during the production phase, e.g., in a bolus on days 4 and 11. In other embodiments, tyrosine is added daily to the production phase culture, e.g., in a feed supplement. In some embodiments, glucose is added to the production phase culture. In some embodiments, glucose consumption is monitored, e.g., by measuring glucose or its metabolic product, e.g., lactate. In some embodiments, a feed supplement containing glucose is added to control the glucose level at a level of 1-10 g / L, 2-7 g / L, 2.5-6 g / L, or about 7 g / L.

[0343] In certain embodiments, fed-batch methods are used in the growth phase of mammalian cell culture processes to replenish growing cells.

[0344] In certain embodiments, cell cultures of the present invention are carried out in large-scale bioreactors and use a fed-batch procedure. In one embodiment of fed-batch culture, mammalian host cells and medium are initially fed into a culture vessel, and additional culture nutrients are fed continuously or in discrete increments, with or without periodic cell and / or product harvest before the end of the culture. Fed-batch culture can include, for example, semi-continuous fed-batch culture, in which the entire culture (including cells and medium) is periodically removed and replaced with fresh medium; fed-batch culture is distinguished from simple batch culture, in which all components for cell culture (including cells and all culture nutrients) are fed into the culture vessel at the beginning of the culture process.

[0345] The methods described herein can be used to achieve cell cultures with humanized anti-α4β7 antibody titers of greater than 1 g / L. In one embodiment, the methods described herein are used to achieve humanized anti-α4β7 antibody titers of about 2 to about 6 g / L, about 3 to about 5 g / L, about 5 to about 9 g / L, or about 4.5 to about 7 g / L.

[0346] The methods disclosed herein can be used to achieve antibody compositions with specific glycosylation patterns. In one embodiment, the methods described herein provide a population of humanized anti-α4β7 antibodies, wherein the population has 88% or more, 90% or more, or 91% or more total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F) glycosylation variants.

[0347] The methods disclosed herein can also be used to achieve an antibody composition having a certain amount of a major antibody isoform. In one embodiment, the methods disclosed herein provide a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of a major antibody isoform of 61% or greater, as determined by cation exchange chromatography (CEX). In another embodiment, the methods disclosed herein provide a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of a major antibody isoform of 62% or greater, as determined by CEX. In one embodiment, the methods disclosed herein provide a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of a major antibody isoform of 63% or greater, as determined by CEX. In one embodiment, the methods disclosed herein provide a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of a major antibody isoform of 64% or greater, as determined by CEX. In another embodiment, the methods disclosed herein provide a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of a major antibody isoform of 65% or greater, as determined by CEX.

[0348] IV. Downstream Production Methods Compositions comprising an anti-α4β7 antibody or antigen-binding portion thereof, e.g., vedolizumab of the present invention, can be produced by the upstream cell culture methods and compositions provided herein. These upstream process techniques can optionally be combined with downstream production methods to isolate, purify, and / or formulate the antibody or antigen-binding portion thereof. Following the production stage, the recombinant antibody can be harvested. Typically, mammalian cells are engineered to secrete the protein of interest into the cell culture medium, so the first step in the purification process is to separate the cells from the medium. The harvested medium can be further clarified, for example, by filtration. The medium, e.g., the clarified harvest, can then be subjected to several additional purification steps to remove cellular debris, unwanted proteins, salts, minerals, or other undesirable elements. Recombinant antibodies can be purified from contaminating soluble proteins and polypeptides. The following procedures are exemplary of suitable purification procedures, which can include one or more of the following: affinity chromatography using a resin that binds to the Fc region of the antibody, such as protein A; ion exchange columns or cation exchange chromatography (CEX) using resins, e.g., SP-Sepharose; (商標) or CM-Sepharose (商標) Fractionation on hydroxyapatite; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed-mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; e.g., Sephadex G-75 (商標) gel filtration using HCl; ultrafiltration and / or diafiltration, or a combination of the above. Exemplary purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the recombinant protein is highly pure and suitable for human therapeutic use, for example, in pharmaceutical antibody formulations described below. After purification, the highly pure recombinant protein can be subjected to ultrafiltration / diafiltration (UF / DF) into a pharmaceutical formulation suitable for human administration.

[0349] Following diafiltration and ultrafiltration, the antibody formulation may remain liquid or may be lyophilized to a dry antibody formulation. In one aspect, the dry, lyophilized antibody formulation is provided in a single-dose vial containing 150 mg, 180 mg, 240 mg, 300 mg, 360 mg, 450 mg, or 600 mg of anti-α4β7 antibody and can be reconstituted with a liquid, such as sterile water, for administration. In another aspect, the anti-α4β7 antibody, e.g., vedolizumab, is a stable liquid pharmaceutical composition stored in a container, e.g., a vial, syringe, or cartridge, at about 2-8°C until administration to a subject in need thereof. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody contains about 0%-5.0%, 0%-2%, ≦2%, ≦1%, ≦0.6%, or ≦0.5% aggregates.

[0350] Thus, in some embodiments, provided herein are reconstituted lyophilized antibody formulations or stable liquid pharmaceutical compositions comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody comprises about 11%-16%, 12%-15%, <14%, <13%, <12%, or <11% of the basic isoform species. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody comprises 65%-75%, 66%-74%, 67%-73%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, or at least 70% of the major isoform. In some embodiments, a reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises between 92% and 98%, between 92% and 97%, between 92% and 96%, between 92% and 95%, at least 92%, at least 93%, at least 94%, or at least 95% total asialo-, agalacto-, core-fucosylated biantennary glycan (G0F), asialo-, monogalacto-, core-fucosylated biantennary glycan (G1F), and / or asialo-, digalacto-, core-fucosylated biantennary glycan (G2F) glycosylation variant (G0F+G1F+G2F) content. In some embodiments, a reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises a GOF content of 45%-65%, 50%-65%, 55%-65%, 45%-60%, 50%-60%, 55%-60%, 45%-55%, 47%-61%, 47%-63%, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, 60% or less, 57% or less, 55% or less, 53% or less, 52% or less, or 50% or less.In some embodiments, a reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises a G1F content of 25% to 45%, 26% to 42%, 27% to 40%, 30% to 40%, 30% to 45%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, or at least 43%. In some embodiments, a reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises a G2F content of 2% to 8%, 2.5% to 7.5%, 3% to 7%, 3.5% to 6.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, or at least 5.5%, at least 6%, at least 6.5%, or at least 7%.

[0351] In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises, but is not limited to, an amino acid (e.g., arginine, histidine, and / or histidine monohydrochloride), a sugar (e.g., sucrose), a surfactant (e.g., polysorbate 80), and / or a buffer (e.g., citrate, phosphate, etc.). In one embodiment, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises L-arginine, L-histidine, L-histidine monohydrochloride, sucrose, and / or polysorbate 80. In another embodiment, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises citrate, arginine, histidine, and / or polysorbate 80.

[0352] The syringe or cartridge can be a 1 mL or 2 mL container (e.g., for a 160 mg / mL dose) or can be larger than 2 mL, e.g., for higher doses (at least 320 mg or 400 mg or more). The syringe or cartridge can contain at least about 20 mg, at least about 50 mg, at least about 70 mg, at least about 80 mg, at least about 100 mg, at least about 108 mg, at least about 120 mg, at least about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 400 mg, or at least about 500 mg of anti-α4β7 antibody. In some embodiments, the container, e.g., syringe or cartridge, can be manufactured to deliver about 20-120 mg, about 40-70 mg, about 45-65 mg, about 50-57 mg, or about 54 mg of an anti-α4β7 antibody, e.g., vedolizumab. In other embodiments, the syringe or cartridge can be manufactured to deliver about 90-120 mg, about 95-115 mg, about 100-112 mg, or about 108 mg of an anti-α4β7 antibody, e.g., vedolizumab. In other embodiments, the syringe or cartridge can be manufactured to deliver about 140-250 mg, about 150-200 mg, about 160-170 mg, about 160-250 mg, about 175 mg-210 mg, about 220-260 mg, or about 160 mg, about 165 mg, about 180 mg, or about 200 mg of an anti-α4β7 antibody, e.g., vedolizumab.

[0353] The formulation can be administered parenterally, such as intravenously, subcutaneously, or intramuscularly. Intravenous injection can be by infusion, such as with sterile isotonic saline, further diluted with a buffer, e.g., phosphate-buffered saline, or Ringer's (lactate or dextrose) solution. In some embodiments, the anti-α4β7 antibody is administered by subcutaneous injection at a dose of about 54 mg, 108 mg, or about 165 mg or about 216 mg, for example, about every 2, 3, or 4 weeks after initiation of treatment, or after the third subsequent dose.

[0354] V. Analysis method Various parameters of the antibodies or antigen-binding portions thereof reported herein can be measured using standard analytical methods and techniques, such as those described below.

[0355] In various embodiments described herein, cation exchange chromatography (CEX) can be used to determine the relative amounts of the major isoform, basic isoform(s), and acidic isoform(s) present in a population of antibodies or antigen-binding portions thereof, e.g., vedolizumab. The CEX method fractionates antibody species according to overall surface charge. After dilution to low ionic strength using a mobile phase, the test sample is loaded onto a CEX column, e.g., Dionex Pro-Pac, equilibrated with an appropriate buffer, e.g., 10 mM sodium phosphate, pH 6.6. (商標) The antibody can be injected onto a WCX-10 column (Thermo Fisher Scientific, Waltham, MA, USA). The antibody can be eluted using a sodium chloride gradient in the same buffer. Protein elution can be monitored at 280 nm, and peaks are assigned to acidic, basic, or major isoform classification. Acidic peaks elute from the column at shorter retention times than the major isoform peak, and basic peaks elute from the column at longer retention times than the major isoform peak. The % major isoform, total percent acidic species, and total percent basic species are reported. The major isoform retention time of the sample is compared to that of a reference standard to determine conformation.

[0356] In one embodiment, the CEX assay involves diluting a test sample to low ionic strength and injecting it onto a CEX column equilibrated with 10 mM sodium phosphate, pH 6.6, eluting the column with a gradient of NaCl in this buffer, monitoring peaks at 280 nm, assigning the peaks as acidic, major, or basic, with the acidic peak eluting first with the shortest retention time, the major peak eluting second, and the basic peak eluting with the longest retention time, and quantifying the peak areas and calculating their amounts as a percentage of the total peak area.

[0357] In various embodiments described herein, hydrophilic interaction phase separation (HILIC) can be used to determine the glycoform profile of an antibody or its antigen-binding portion, such as vedolizumab. The HILIC method fractionates free fluorescently labeled carbohydrates. Intact glycans can be released from a sample of the antibody or its antigen-binding portion by digestion with N-glycosidase F. The released glycans can be immediately labeled with a fluorescent tag, such as the InstantAB fluorescent tag, using standard techniques such as those used in the GlykoPrep Rapid Glycoprotein Sample Preparation System from Prozyme (Hayward, CA, USA). The labeled glycans can be fractionated using ultra-high performance liquid chromatography. In some embodiments, the labeled glycans are fractionated using an ACQUITY UPLC BEH amide column (Waters Corporation, Milford, MA, USA) and an acetonitrile / ammonium formate gradient system. The labeled glycans can be detected by fluorescence emission at 344 nm using an excitation wavelength of 278 nm. Thus, as used in the context of the present invention, HILIC is a HILIC method for fractionating free fluorescently labeled glycoforms, preferably in which intact glycoforms are released from a sample of antibody or antigen-binding portion thereof by digestion with N-glycosidase F. The released glycoforms are then immediately labeled with a fluorescent tag, preferably an InstantAB fluorescent tag, using standard labeling techniques, preferably those used in the GlykoPrep Rapid Glycoprotein Sample Preparation System from Prozyme (Hayward, CA, USA), and fractionated using high performance liquid chromatography, preferably an ACQUITY UPLC BEH Amide Column (Waters Corporation, Milford, MA, USA) and an acetonitrile / ammonium formate gradient system, and the labeled glycoforms are detected by fluorescence emission at 344 nm using an excitation wavelength of 278 nm.Assay controls can be performed by confirming adequate resolution of commercially available standards, such as InstantAB-labeled glucose homopolymer ladder (Agilent Technologies, Inc., Santa Clara, CA, USA). Quantitation is based on the relative area percentage of detected sugars. Peak area percentages of G0F (asialo-, agalactosylated biantennary glycan, core fucosylated); G1F (asialo-, monogalactosylated biantennary glycan, core fucosylated); and G2F (asialo-, digalactosylated biantennary glycan, core fucosylated) species are reported.

[0358] In various embodiments described herein, size exclusion chromatography (SEC) can be used to determine the relative levels of monomer, high molecular weight (HMW) aggregates, and low molecular weight (LMW) degradation products present in a population of an antibody or antigen-binding portion thereof, e.g., vedolizumab. The SEC method provides size-based separation of antibody monomer from HMW species and LMW degradation products. Test samples and reference standards can be analyzed using commercially available SEC columns with appropriate buffers. For example, in some preferred embodiments, SEC analysis can be performed using a G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA, USA), or preferably two G3000 SWxl columns connected in tandem, and an isocratic phosphate-sodium chloride buffer system, pH 6.8. Elution of protein species is monitored at 280 nm. Purity is determined by assessing the area of ​​the main peak (monomer) and total peaks. In one embodiment, SEC analysis involves injecting the sample onto two G3000 SWxl columns connected in tandem, monitoring the elution of protein species at 280 nm, and measuring the major peak (monomer) and total peak areas in an isocratic phosphate-sodium chloride buffer system at pH 6.8. Sample purity (calculated as % monomer), % HMW aggregates, and / or % LMW degradation products are reported.

[0359] Residual CHO host cell protein (HCP) impurities present in antibody preparations If necessary, measurement can be performed by enzyme-linked immunosorbent assay (ELISA) using standard techniques. Many ELISA kits designed for this purpose are commercially available, such as the CHO HCP ELISA Kit 3G from Cygnus Technologies (Southport, NC, USA). Host cell proteins in the test sample can be captured using an immobilized polyclonal anti-CHO HCP antibody. The captured protein can then be detected using an appropriate detection agent, such as a horseradish peroxidase-labeled version of the same antibody. In this exemplary embodiment, the amount of captured peroxidase, which is directly proportional to the concentration of CHO HCP, can be measured colorimetrically at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the CHO HCP assay involves capturing HCP using a polyclonal anti-CHO HCP antibody, which is detected after binding to a horseradish peroxidase-labeled version of the polyclonal anti-CHO HCP antibody, which converts the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to a substance that is quantified colorimetrically at 450 nm. HCP concentration can be determined by comparison to a CHO HCP standard curve, such as that included in the test kit, and is reported as a percentage of the total level of protein in the antibody preparation.

[0360] The following examples illustrate improved methods and compositions for producing antibodies in mammalian cell culture. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. [Example]

[0361] Vedolizumab was previously produced in a dihydrofolate reductase-deficient (DHFR) Chinese hamster ovary (CHO) cell line (Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA, 77:4216-4220, U.S. Patent Application Publication No. 20070122404). Selected clones showed stable expression of vedolizumab, but production levels were below 2 g / L. Given the high demand for materials, researchers sought to develop more productive cell lines.

[0362] After testing various selection systems on thousands of clones and evaluating several clones in bioreactors, the glutamine synthase-deficient (GS-) Chinese hamster ovary (GS-CHO) cell line was selected. In one example, the GS-CHO system yielded 6.7 g / L antibody. Additional studies showed that culture conditions and media supplements can affect certain quality attributes. The following example describes experiments to improve the quality of vedolizumab produced in GS-CHO cells.

[0363] Example 1. Impact of cell culture production on product quality attributes To improve product quality attributes, a screening design was created using the Plackett-Burman method to evaluate the impact of five process parameter modifiers in eight bioreactor runs. Cells were thawed, passaged for three days using a standard scale-up strategy, and transferred from shake flasks to a 3 L production bioreactor with a working volume of 1.75 L. A bolus-feed strategy with two feeds (unless otherwise specified) was used for the 15-day bioreactor production run.

[0364] Design: Five different factors were selected for this screening study: 1) temperature shift (to 33°C); 2) change in feeding strategy (2 g / L vs. 6 g / L glucose); 3) change in pH (6.85 vs. 7.05); 4) addition of uridine, manganese chloride, galactose (UMG) to the feed solution; and 5) Sigma Gal+ / ExCell® glycosylation adjustment additive. These five factors were tested in eight different bioreactor runs based on the Plackett-Burman screening design described in Table 1.

[0365] [Table 1]

[0366] [Table 2]

[0367] Feed: 3 x 10 to production bioreactor culture 5 Viable cells / mL were inoculated, and on day 4, feed medium was added to the cultures based on cell growth rate and glucose consumption rate according to the study design. The feed dosage was capped at 7 g / L glucose concentration. Temperature shifts were initiated on day 7 from 37°C to 33°C or 35°C according to the study design. All production bioreactor cultures were harvested on day 18 or when target cell viability fell below 50%, whichever came first.

[0368] Product Quality: The results of the conditions tested in Table 1 were analyzed with JMP software to explore conditions (using predictive profiler) that improve product quality attributes.

[0369] The results of the predicted profiles are shown in Figure 1. In general, conditions that achieve increased antibody titers, decreased basic and acidic species of vedolizumab, and increased G2F isoforms of vedolizumab are generally desirable.

[0370] As shown in Figure 1, the model predicted that operation at a feed delivery based on glucose consumption rate, 37 degrees Celsius, a shift to pH 6.85, and the addition of UMG to the feed solution was optimal. In contrast, the results in Figure 1 suggest that the addition of Gal+ is not essential, as it had little effect on the G2 isoform, acidic or basic species of vedolizumab, or antibody titer. Furthermore, a temperature shift from 37 degrees Celsius to 33 degrees Celsius had a negative effect on titer, suggesting that maintaining cell production at 37 degrees Celsius is advantageous, whereas a pH below 7.05 (e.g., from 6.85 to 7) improved titer while maintaining low levels of the G2 isoform.

[0371] The predictive profiler further demonstrated the benefit of using a combination of UMG to achieve carbohydrate targets, as well as higher antibody titer levels and lower levels of acidic species. A glucose consumption-based feeding strategy and a lower pH (6.85) compared to pH 7 showed advantages in achieving a lower proportion of basic species.

[0372] Example 2: Effect of UMG supplementation and pH on product quality attributes The purpose of this experiment was to test the effect of pH and UMG level of the feed solution on the quality attributes of the product. This experiment is a follow-up check of Example 1.

[0373] Design: GS-CHO cells were used in this experiment. The experiment was designed to accommodate pH factors investigated at 6.85 or 7.05, and UMG as a feed replenisher (in production) investigated at concentrations of 33x, 50x, and 66x. An additional condition involving a pH shift was added on day 4 (V10). On day 1 of the experiment, the titration pump in V01 significantly overpumped the titrant due to a loose connection on the pH probe, necessitating the reactor shutdown. Because V10 had similar media and cells, the run template for V10 was quickly replaced to reflect the run template for V01. As described in Example 1, a temperature shift was not used because no benefit was anticipated.

[0374] Cells were fed using a consumption-based feeding method, in which current growth and consumption rates are estimated to predict glucose requirements.

[0375] The experiment evaluated the effect of UMG supplementation in feed medium (investigated at 33x (33 mM uridine, 0.066 mM manganese, and 165 mM galactose), 50x (50 mM uridine, 0.1 mM manganese, and 250 mM galactose), and 66x (66 mM uridine, 0.132 mM manganese, and 330 mM galactose in the feed medium) and pH (investigated at 7.05 and 6.85) on product quality attributes, including antibody titer quantity, acidic species, basic species, major species, G0F species, G1F species, G2F species, and total glycans. Results were compared to cultures grown in CD-CHO production medium without UMG supplementation. The experimental design is shown in Table 3.

[0376] [Table 3]

[0377] Results: Using the results of the experiment, predictive profiles were generated to further study the effects of various conditions on cell culture and the quality characteristics of the vedolizumab product. The results of the predictive profiles are shown in Figures 2A-2H (antibody titer vs. UMG (Figure 2A), % acidic species (CEX) vs. UMG (Figure 2B), % basic species (CEX) vs. UMG (Figure 2C), percentage of major species (CEX) vs. UMG (Figure 2D), percentage of G0F species vs. UMG (Figure 2E), percentage of G1F species (Figure 2F), percentage of G2F species vs. UMG (Figure 2G), and total glycans vs. UMG (Figure 2H)). In each of Figures 2A-2H, the vessel without UMG supplementation is indicated by a dot at the left edge of the shaded region (the shaded region represents UMG supplementation). Additionally, the two pH values ​​tested (pH 7.05 and pH 6.85) are shown in Figures 2A-2H.

[0378] As illustrated in Figures 2D, 2F, 2G, and 2G, cell cultures with increasing UMG supplementation had higher percentages of major species, G1F species, G2F species, and total glycans, respectively, compared to cultures without UMG supplementation. Furthermore, cultures with UMG supplementation exhibited lower titers (Figure 2A), lower acidic species (Figure 2B), lower basic species (Figure 2C), and lower G0F species (Figure 2E) compared to cultures without UMG supplementation.

[0379] Across the various UMG concentrations tested, UMG concentration appeared to have minimal effect on titer and acidic species, a low effect on the percentage of basic species (i.e., basic species decreased slightly with increasing UMG concentration), and a high effect on the percentage of major species (i.e., major species increased with increasing UMG concentration). There were no significant changes in the carbohydrate profile in response to the various UMG concentrations.

[0380] Finally, with regard to pH, operation at low pH (6.85) appeared to perform better than operation at high pH (pH 7.05), as illustrated in Figures 2A-2H.

[0381] In another experiment, GS-CHO cells recombinantly expressing vedolizumab were cultured at a 3000 L scale in CD-CHO production medium supplemented with a feed medium containing uridine (20.91 mM), manganese (0.039 mM), galactose (96.69 mM), and zinc (0.117 mM). The feed was added to the culture daily starting on day 4. The amounts of UMG added in each daily addition were as follows: 0.17–0.63 mM uridine, 0.31–1.2 μM manganese, and 0.77–2.9 mM galactose. By the harvest date, the average cumulative supplemented concentration of uridine in the production medium after daily supplementation from day 4 to day 13 of culture was approximately 2.76 mM, the average cumulative supplemented concentration of manganese was approximately 0.00515 mM, and the average cumulative supplemented concentration of galactose was approximately 12.8 mM. Zinc was also added daily as a feed supplement at a concentration of about 0.117 mM on days 4 through 13, for an average cumulative supplement concentration of about 0.0154 mM in the production medium by day 14. The averages used in this example refer to the average of the lots tested.

[0382] The antibody was harvested after 14 days of culture and purified and the levels of fucosylated glycans were determined using HILIC. The results are shown in Table 4.

[0383] [Table 4]

[0384] Example 3: Effect of lysine and arginine on product quality attributes The objective of this experiment was to test the effect of lysine and arginine levels in the feed medium on the quality attributes of vedolizumab, specifically the potency and percentage of basic species.

[0385] Design: This experiment was designed to evaluate the effect of lysine and arginine concentrations on antibody titers when produced in GS-CHO cells and levels of basic species (C-terminal lysine levels) as determined by CEX. The study was performed in a similar manner to Examples 1 and 2.

[0386] Results: Results comparing the effect of various arginine and lysine concentrations on the percentage of basic species are shown in Figure 3A, and results showing the effect on antibody titers are shown in Figure 3B. The x-axis labels in both Figures 3A and 3B correspond to high (H), medium (M), or low (L) concentrations of lysine and arginine, as outlined in Table 5. For example, "LM" refers to low levels of lysine (see Table 5) and medium levels of arginine (see Table 5). These results indicated that, compared to the control, low levels of lysine and arginine minimized the reduction in basic species, but low levels of these amino acids had a negative impact on antibody titers (5-4g, approximately 20%).

[0387] [Table 5]

[0388] Predictive analysis was performed based on the arg / lys experiment. JMP analysis predicted that the optimal conditions for reducing basic species would be low lysine and low arginine levels (LL), as shown in Figure 4A. Figures 4B and 4C show the predicted profiles for the combinations "LM" (low lysine and medium arginine) and "LH" (low lysine and high arginine). However, due to the impact on titer production, low lysine (5 g / L) and medium arginine (6.5 g / L) levels were used in Example 4.

[0389] Example 4: Effect of zinc on produce quality attributes The aim of this experiment was to test the effect of zinc levels on the quality attributes of vedolizumab during cell culture, more specifically during the production phase of the culture system.

[0390] Three levels of zinc were tested, as listed in Table 6 below. Cultures ranged from 14 to 18 days of recovery. Zinc supplementation was evaluated in combination with 22xUMG and reduced lysine and arginine ("LM" as described in Example 3). The concentrations of lysine and arginine in the feed were 5 g / L and 6.4 g / L, respectively.

[0391] [Table 6]

[0392] As shown in Figure 5A, the zinc concentrations tested did not substantially affect antibody titers. Also shown in Figure A is the effect on the number of days in the production culture. A benefit in titer production was observed with longer culture days. Figures 5B–5G show the effect of zinc on the percentage of basic species (Figure 5B), the percentage of acidic species (Figure 5C), the percentage of major species (Figure 5D), the percentage of G0F species (Figure 5E), the percentage of G1F species (Figure 5F), the percentage of G2F species (Figure 5G), and the total glycan species (Figure 5H).

[0393] As shown in Figures 5B and 5C, a decreasing trend in basic and acidic profiles was observed with increasing zinc levels. Furthermore, it was observed that similar basic profiles were achieved up to day 16 of culture. As shown in Figure 5D, the highest level of the main (major) species was obtained at 57.2 μM zinc (4Zn) on day 14 of harvest.

[0394] Zinc levels and culture days had minimal impact on carbohydrate profiles. Overall, the data in Figures 5A-5H suggest that culture and harvest should be terminated by day 16.

[0395] Temperature was also tested in combination with various zinc concentrations to determine whether it affected the various product qualities of vedolizumab. Temperatures of 33, 35, and 37 degrees Celsius were tested in combination with zinc levels ranging from 0 to 4 (see Table 6 above). Overall, 37 degrees Celsius was most effective in maintaining desirable vedolizumab product quality. For example, Figure 6A shows the percent basic isoforms of the antibody under various zinc conditions and temperatures. The percent levels of basic species after supplementation with 57.2 μM zinc (4Zn) at 33 and 37°C were below the specified upper limit of basic species (isoforms) (shown by the black line, i.e., 13% basic antibody isoforms (CEX)), thus meeting the specification requirements. In contrast, as shown in Figure 6B, total glycans after supplementation with 57.2 μM zinc (4Zn) at 37°C but not at 33°C exceeded the lower acceptance standard (shown by the black line). Additionally, as shown in Figure 6C, increased protein aggregation (HMW species, with an acceptable standard of approximately 1.4% or less) was observed at lower temperatures. The data in Figure 6D indicate that extending incubation time promoted titer generation, with generally higher vedolizumab titers being generated at 37°C compared to 33°C and 35°C on day 14. The data in Figure 6E suggest that acidic species of the antibody increased with increasing temperature and prolonged incubation. The solid black line in Figure 6E represents the upper acceptable limit for acidic species.

[0396] Overall, the data in Figures 6A-6E suggest that vedolizumab production in GS-CHO cells is best achieved by stopping fermentation by day 16 and maintaining a production temperature of approximately 37°C. Additionally, 4Zn provides additional benefits as a supplement.

[0397] Example 5: Effect of culture days on product quality characteristics The objective of this experiment was to test the effect of incubation days on the quality attributes of vedolizumab.

[0398] The percentage of acidic antibody species, the percentage of basic antibody species, the percentage of major antibody species, and the vedolizumab titer were assessed after 12, 13, 14, 15, 16, or 17 days of culture in GS-CHO cells after two separate runs.

[0399] As shown in Figure 7D, an increase in titer was observed with increasing culture days. However, this increase in titer was accompanied by an increase in acidic species and a decrease in the major species, as shown in Figures 7A and 7B, respectively. While basic species did not appear to be affected during run 1, a trend toward increased basic species was observed during run 2. The data indicate that it is generally best to stop and harvest the fermentation by day 16. The lowest levels of acidic species were obtained when antibody was harvested by day 15.

[0400] Example 6: Effect of pH on product quality attributes The objective of this experiment was to test the effect of pH during production cell culture on the quality attributes of vedolizumab. Specifically, the introduction of a pH shift into the production medium was evaluated for its impact on the quality attributes of vedolizumab.

[0401] The initial pH of the production medium was evaluated in the range of pH 6.8 to pH 7.2. During the pH shift, the pH of the medium was decreased to the final pH, which was evaluated in the range of pH 6.6 to pH 7.0. The onset time of the pH shift was investigated from 86 to 108 hours, and the completion time of the pH shift (i.e., the time to reach the final pH) was investigated from 88 to 144 hours. The intervening 2- to 36-hour intervals account for the pH ramp time.

[0402] The highest % major antibody isoform (determined by CEX) and lowest % acidic antibody isoform (determined by CEX) were observed at a final pH of 6.7 or higher (FIG. 8A) and a pH shift completion time of 122 hours or less (FIG. 8B). This data suggests that pH shifts during production cell culture may reduce the levels of acidic antibody isoform species in vedolizumab preparations.

[0403] Example 7: Determination of product quality characteristics The following analytical assays and methods were used in the preceding examples to determine the product quality attributes of vedolizumab.

[0404] Cation exchange chromatography (CEX) fractionates vedolizumab antibody species (major isoforms, basic species, and acidic species) according to overall surface charge. After dilution to low ionic strength using the mobile phase, test samples were eluted onto a Dionex Pro-Pac buffer equilibrated with 10 mM sodium phosphate, pH 6.6. (商標) The sample is injected onto a WCX-10 column (Thermo Fisher Scientific, Waltham, MA, USA) and eluted using sodium chloride in the same buffer. Protein elution is monitored at 280 nm, and peaks are assigned to acidic, basic, or major isoform classifications. The % major isoform, total percent acidic species, and total percent basic species are reported. The retention time of the major isoform in the sample is compared to that of a reference standard to determine conformation.

[0405] The carbohydrate profile of vedolizumab was generated by fractionation of free fluorescently labeled carbohydrates by hydrophilic interaction phase separation (HILIC). Intact glycans were released from the protein sample by digestion with N-glycosidase F and then immediately labeled with InstantAB fluorescent tags (Agilent Technologies, Inc., Santa Clara, CA, USA) using the GlykoPrep Rapid Glycoprotein Sample Preparation System from Prozyme (Hayward, CA, USA). The labeled glycans were fractionated using an ACQUITY UPLC BEH amide column (Waters Corporation, Milford, MA, USA) and an acetonitrile / ammonium formate gradient system. Detection was achieved by fluorescence emission at 344 nm using an excitation wavelength of 278 nm. Assay controls were performed to determine the appropriate resolution of a commercially available InstantAB-labeled glucose homopolymer ladder (Agilent Technologies, Inc., Santa Clara, CA, USA). Quantitation is based on the relative area percentages of the detected sugars. Peak area percentages are reported for G0F (asialo-, agalactosylated biantennary glycan, core fucosylated); G1F (asialo-, monogalactosylated biantennary glycan, core fucosylated); and G2F (asialo-, digalactosylated biantennary glycan, core fucosylated) species.

[0406] Size exclusion chromatography (SEC) was used to determine the purity of vedolizumab. Reference standard and test samples (75 μg) were analyzed using two tandemly connected G3000 SWxl columns (Tosoh Bioscience, King of Prussia, PA, USA) in an isocratic phosphate-sodium chloride buffer, pH 6.8. This method separates antibody monomer from high molecular weight (HMW) species and low molecular weight (LMW) degradation products. Elution of protein species was monitored at 280 nm. Purity was determined by assessing the main peak (monomer) and total peak area. Sample purity (%) (calculated as % monomer) and aggregate % were reported.

[0407] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims. The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference. The present invention includes the following embodiments. [1] 1. A method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising: Culturing mammalian host cells in a production medium; adding supplements to the production medium, including uridine, manganese, and galactose, thereby producing a composition comprising the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and comprising a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6. [2] The method of [1] above, wherein the composition comprises a reduced amount of the basic isoform of the humanized anti-α4β7 antibody (as determined by cationic chromatography (CEX)) compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement but under substantially similar conditions. [3] The method according to [1] above, wherein the composition contains about 16% or less of the basic isoform of the humanized anti-α4β7 antibody. [4] The method according to [3] above, wherein the composition contains about 14% or less of the basic isoform of the humanized anti-α4β7 antibody. [5] The method according to [3] above, wherein the composition contains about 13% or less of the basic isoform of the humanized anti-α4β7 antibody. [6] The method according to any one of [1] to [5] above, wherein the supplementary components are added to the production medium, or added to a feed medium, and then the feed medium is added to the production medium. [7] The method according to any one of [1] to [6] above, wherein the cumulative concentration of uridine added to the production medium between supplementation and recovery is about 1 to about 7 mM, the cumulative concentration of manganese in the production medium between supplementation and recovery is about 0.002 to about 0.015 mM, and / or the cumulative concentration of galactose in the production medium between supplementation and recovery is about 3 to about 20 mM. [8] The method according to any one of [1] to [7] above, wherein manganese is added to the production medium as a supplementary component multiple times, with each addition being about 0.1 to 10 μM, about 0.2 to 1.5 μM, about 0.2 to 5 μM, about 0.25 to 2 μM, about 0.3 to 1.2 μM, or about 0.3 to 0.8 μM. [9] The method according to [8] above, wherein manganese is added to the production medium multiple times as a supplement, with each addition being about 0.2 to 1.5 μM.

[10] The method according to any one of [1] to [9] above, wherein uridine is added multiple times to the production medium as a supplementary component, with each addition being about 25 to 1000 μM, about 75 to 750 μM, about 55 to 620 μM, about 100 to 600 μM, about 150 to 450 μM, about 100 to 700 μM, about 100 to 600 μM, or about 170 to 630 μM.

[11] The method according to

[10] above, wherein uridine is added multiple times to the production medium as a supplement in amounts of about 100 to 700 μM each.

[12] The method according to any one of [1] to

[11] above, wherein galactose is added multiple times to the production medium as a supplementary component, with each addition being approximately 0.1 to 10 mM, 0.2 to 7.5 mM, 0.5 to 5 mM, 0.4 to 2.8 mM, 0.5 to 3.5 mM, 0.7 to 2.9 mM, 0.75 to 2.5 mM, or approximately 1.2 mM or 1.4 mM.

[13] The method according to

[12] above, wherein the galactose is added to the production medium multiple times as a supplement in amounts of about 0.5 to 3.5 mM.

[14] The method according to any one of the above [8] to

[13] , wherein the supplement is added every day or every two days.

[15] The method according to any one of the above [8] to

[14] , wherein the supplementary components are added from the fourth day of the culture in the production stage.

[16] The method according to any one of the above [1] to

[15] , wherein the supply medium further contains zinc.

[17] The method according to

[16] above, wherein the cumulative concentration of zinc added to the production medium between replenishment and recovery is about 0.005 mM to about 0.045 mM.

[18] The method described in any of [1] to

[17] above, wherein the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplementary component but under substantially similar conditions.

[19] The method according to any one of the above [1] to

[18] , wherein the method is a fed-batch culture method.

[20] The method according to

[19] above, wherein the method is a production stage and the supplementary components are added to the production medium from about day 4 of the production stage.

[21] 1. A method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising culturing mammalian host cells in a production medium comprising zinc, thereby producing a composition comprising the humanized anti-α4β7 antibody; The method, wherein the mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, wherein the humanized anti-α4β7 antibody is an IgG1 and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[22] The method of

[21] above, wherein the composition comprises a reduced amount of the basic isoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured under the same conditions in the absence of zinc.

[23] The method according to

[22] above, wherein the composition comprises about 16% or less of the basic isoform of the humanized anti-α4β7 antibody.

[24] The method according to

[23] above, wherein the composition comprises about 14% or less of the basic isoform of the humanized anti-α4β7 antibody.

[25] The method according to

[23] above, wherein the composition comprises about 13% or less of the basic isoform of the humanized anti-α4β7 antibody.

[26] The method according to any one of the above

[21] to

[25] , wherein the concentration of zinc in the production medium is 2 μM to 60 μM.

[27] The method according to any one of the above

[21] to

[26] , wherein the method comprises supplementing the production medium with zinc by adding a feed medium containing zinc to the production medium.

[28] 28. The method according to claim 27, wherein the feed medium is added to the production medium from about day 4 of the production stage.

[29] The method according to any one of the above-mentioned [1] to

[28] , wherein the production medium contains 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine.

[30] The method according to

[29] above, wherein the production medium contains 4.5 to 5.5 g / L of lysine.

[31] The method according to

[29] above, wherein the production medium contains 5.5 to 8.8 g / L of lysine.

[32] The method according to any one of the above

[29] to

[31] , wherein the production medium contains 5.4 to 7.4 g / L of arginine.

[33] The method according to any one of the above

[29] to

[31] , wherein the production medium contains 7.4 to 12 g / L of arginine.

[34] 1. A method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising: The method includes culturing mammalian host cells in a production medium in a production stage to produce a composition comprising the humanized anti-α4β7 antibody, the production medium having an average temperature of about 37 degrees Celsius, and the host cells are genetically engineered to express a humanized IgG1 anti-α4β7 antibody, the humanized anti-α4β7 comprising a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6.

[35] The method according to

[34] above, wherein the method is a method for producing a composition containing 2.5% or less HMW species of the humanized anti-α4β7 antibody (as determined by SEC).

[36] 1. A method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising: culturing mammalian host cells in a growth medium during an expansion stage, wherein the mammalian host cells are genetically engineered to express a humanized anti-α4β7 antibody; and culturing the mammalian host cells in a production medium in a production stage so as to produce a composition comprising the humanized anti-α4β7 antibody; the mammalian host cells are cultured at about the same temperature during both the expansion step and the production step; The method, wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and comprises a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[37] The method according to

[36] above, wherein the method is a method for producing a composition containing a higher level of monomer of the humanized anti-α4β7 antibody (as determined by SEC) compared to a control culture cultured under substantially similar conditions but at a different temperature between the expansion step and the production step.

[38] The method according to

[36] or

[37] above, wherein the temperature is 36 to 38 degrees Celsius.

[39] The method according to any one of the above

[36] to

[38] , wherein the average temperature is 36.5 to 37.5 degrees Celsius.

[40] The method according to any one of claims 36 to 37, wherein the temperature is an average temperature of about 37 degrees Celsius.

[41] The method according to any one of the above [1] to

[40] , wherein the production medium has a temperature in the range of 36 to 38 degrees Celsius.

[42] The method according to

[41] above, wherein the temperature is in the range of 36.5 to 37.5 degrees Celsius.

[43]

[42] The method according to claim 42, wherein the temperature is an average temperature of about 37 degrees Celsius.

[44] The method according to any one of the above [1] to

[43] , wherein the production medium has a pH in the range of 6.5 to 7.

[45] The method according to

[44] above, wherein the production medium has a pH in the range of 6.8 to 7.0.

[46] The method according to any one of the above [1] to

[45] , wherein the production medium has a glucose level maintained at about 7 g / L or less during the production stage.

[47] The method according to any one of the above [1] to

[46] , wherein the production stage is carried out within 14 days.

[48] The method according to any one of the above [1] to

[46] , wherein the production stage lasts for 10 to 17 days.

[49] The method according to any one of [1] to

[48] above, carried out in a large-scale bioreactor.

[50] 49. The method according to claim 49, wherein the large-scale bioreactor is selected from the group consisting of a 200 liter (L) bioreactor, a 2000 L bioreactor, a 3000 L bioreactor, and a 6000 L bioreactor.

[51] The method according to any one of [1] to

[50] above, wherein the production step results in a titer of the humanized anti-α4β7 antibody of greater than 3 g / L.

[52] The method according to

[51] above, wherein the titer of the humanized anti-α4β7 antibody is about 3 to about 8 g / L.

[53] The method according to

[52] above, wherein the titer of the humanized anti-α4β7 antibody is about 5 to about 7 g / L.

[54] The method according to any one of [1] to

[53] above, wherein the mammalian host cells are Chinese hamster ovary (CHO) cells.

[55] The method according to

[54] above, wherein the CHO cells are GS-CHO cells.

[56] The method described in any of [1] to

[55] above, wherein the humanized anti-α4β7 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.

[57] The method according to any one of [1] to

[55] above, wherein the humanized anti-α4β7 antibody is vedolizumab.

[58] The method according to any one of [1] to

[57] above, wherein the method comprises recovering and purifying the antibody.

[59] 58. The method of claim 58, wherein the purification comprises (i) a purification step to remove cellular debris, undesired proteins, salts, minerals, or other undesired elements, and (ii) purification of the antibody from contaminating soluble proteins and polypeptides.

[60] The method according to

[58] or

[59] above, further comprising preparing a pharmaceutical formulation of the purified antibody suitable for human therapeutic use.

[61] The method according to

[60] above, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

[62] The method according to

[61] above, wherein the liquid pharmaceutical formulation is prepared by ultrafiltration / diafiltration.

[63] The method according to

[60] above, wherein the pharmaceutical preparation is a lyophilized antibody preparation.

[64] 63. The method according to claim 63, wherein the pharmaceutical formulation of the antibody is a dry antibody formulation lyophilized from a liquid pharmaceutical antibody formulation prepared by ultrafiltration / diafiltration following the purification.

[65] A composition comprising a humanized anti-α4β7 antibody produced by the method according to any one of [1] to

[64] above.

[66] A composition comprising a humanized anti-α4β7 antibody, obtainable by the method described in any one of [1] to

[64] above.

[67] The composition of any one of

[65] and

[66] above, comprising a population of humanized anti-α4β7 antibodies having glycosylation variants of (i) 90% or more, or (ii) 92-95% total asialo-, agalactic-, core-fucosylated biantennary glycans (G0F), asialo-, monogalactic-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalactic-, core-fucosylated biantennary glycans (G2F).

[68] The method described in any of [1] to

[20] above, wherein the method is a method for producing a composition having a reduced amount of the G0F glycoform of the humanized anti-α4β7 antibody (as determined by hydrophilic interaction chromatography (HILIC)) compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions but in the absence of the supplementary component.

[69] The method of

[68] above, wherein the composition comprises a level of the G0F glycoform of the humanized anti-α4β7 antibody that is reduced by at least about 15% compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplement.

[70] 69. The method of claim 69, wherein the composition comprises at least about a 20% reduction in the G0F glycoform of the humanized anti-α4β7 antibody compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplement.

[71] The method according to any one of [1] to

[20] above, wherein the method is a method for producing a composition having about 65% or less of the G0F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC).

[72] The method according to

[71] above, wherein the composition comprises about 60% or less of the G0F glycoform of the humanized anti-α4β7 antibody.

[73] The method according to

[71] above, wherein the composition comprises about 55% or less of the G0F glycoform of the humanized anti-α4β7 antibody.

[74] The method described in any of [1] to

[20] above, wherein the method is a method for producing a composition having an increased amount of the G1F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC) compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplementary component.

[75] The method of

[74] above, wherein the composition comprises at least about a two-fold increase in the G1F glycoform of the humanized anti-α4β7 antibody compared to the control.

[76] The method of

[74] above, wherein the composition comprises at least about a three-fold increase in the G1F glycoform of the humanized anti-α4β7 antibody compared to the control.

[77] The method according to any one of [1] to

[20] above, wherein the method is a method for producing a composition having about 25% or more of the G1F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC).

[78] The method according to

[77] above, wherein the composition comprises about 30% or more of the G1F glycoform of the humanized anti-α4β7 antibody.

[79] The method according to any one of [1] to

[20] above, wherein the method is a method for producing a composition having an increased amount of the G2F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC) compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplementary component.

[80] 79. The method of claim 79, wherein the composition comprises at least about a three-fold increase in the G2F glycoform of the humanized anti-α4β7 antibody compared to the control.

[81] 79. The method of claim 79, wherein the composition comprises at least about a four-fold increase in the G2F glycoform of the humanized anti-α4β7 antibody compared to the control.

[82] The method according to any one of [1] to

[20] above, wherein the method is a method for producing a composition having about 3% or more of the G2F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC).

[83] The method according to

[82] above, wherein the composition comprises about 4% or more of the G2F glycoform of the humanized anti-α4β7 antibody.

[84] The method described in any of

[68] to

[83] above, wherein the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplementary component.

[85] The method described in any of

[68] to

[83] above, wherein the method further increases the percentage of the major isoform species of the humanized anti-α4β7 antibody compared to the percentage of the major isoform species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of a supply medium comprising uridine, manganese, and galactose added to the production medium.

[86] The method according to any one of the above

[68] to

[85] , wherein the method is a fed-batch culture method.

[87] The method according to any one of the above

[68] to

[86] , wherein the feeding medium is added to the production medium from about day 4 of the production stage.

[88] The method according to any one of

[68] to

[87] above, wherein the production medium has a pH of about 6.8 to about 7.1.

[89] The method according to any one of

[68] to

[88] above, wherein the method comprises recovering and purifying the antibody.

[90] 89. The method according to claim 89, wherein the purification comprises (i) a purification step to remove cellular debris, undesired proteins, salts, minerals, or other undesired elements, and (ii) purification of the antibody from contaminating soluble proteins and polypeptides.

[91] The method according to

[89] or

[90] above, further comprising preparing a pharmaceutical formulation of the purified antibody suitable for human therapeutic use.

[92] The method according to

[91] above, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

[93] The method according to

[92] above, wherein the liquid pharmaceutical formulation is prepared by ultrafiltration / diafiltration.

[94] The method according to

[91] above, wherein the pharmaceutical preparation is a lyophilized antibody preparation.

[95] The method according to

[94] above, wherein the pharmaceutical formulation of the antibody is a dry antibody formulation lyophilized from a liquid pharmaceutical antibody formulation prepared by ultrafiltration / diafiltration following the purification.

[96] A composition comprising a humanized anti-α4β7 antibody, obtainable by the method according to any one of

[68] to

[88] above.

[97] A cell culture comprising host cells genetically engineered to express a humanized anti-α4β7 antibody and a production medium supplemented with uridine, manganese, and galactose (UMG), wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5.

[98] 97. The cell culture of claim 97, wherein the production medium comprises, on the day of harvest, supplemented uridine at a concentration of about 1 to about 7 mM, supplemented manganese at a concentration of about 0.002 to about 0.015 mM, and supplemented galactose at a concentration of about 3 to about 20 mM.

[99] The cell culture according to

[97] or

[98] above, wherein the production medium further comprises zinc.

[0100] 99. The cell culture of claim 99, wherein the production medium comprises zinc supplemented at a concentration of about 0.005 mM to 0.045 mM on the day of harvest.

[0101] The cell culture described in any one of

[97] to

[0100] above, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0102] A cell culture comprising host cells genetically engineered to express a humanized anti-α4β7 antibody and a production medium supplemented with zinc, wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0103] The cell culture described in

[0102] above, wherein the production medium contains zinc supplemented at a concentration of about 0.005 mM to 0.045 mM on the day of harvest.

[0104] A cell culture described in

[0102] or

[0103] above, wherein the production medium further contains uridine, manganese, and galactose (UMG).

[0105] The cell culture described in

[0100] above, wherein the production medium contains, on the day of harvest, supplemented uridine at a concentration of about 1 to about 7 mM, supplemented manganese at a concentration of about 0.002 to about 0.015 mM, and supplemented galactose at a concentration of about 3 to about 20 mM.

[0106] The cell culture described in

[0100] above, wherein the production medium contains, on the day of harvest, supplemented uridine at a concentration of about 2 to about 5 mM, supplemented manganese at a concentration of about 0.001 to about 0.01 mM, and supplemented galactose at a concentration of about 10 to about 15 mM.

[0107] the expressed humanized anti-α4β7 antibody a. 16% or less, 15% or less, 14% or less, 13% or less, or 12% or less basic isoforms; and / or b. A cell culture described in any one of

[97] to

[0106] above, having an isoform distribution comprising at least 65%, at least 68%, at least 70%, at least 72%, or at least 75% of the major isoform.

[0108] the expressed humanized anti-α4β7 antibody c. 65% or less, 60% or less, or 55% or less G0F; d. 25% or more, 27% or more, or 30% or more G1F; and / or e. A cell culture described in any one of

[97] to

[0107] above, having a fucosylated N-glycan content containing G2F of 2.5% or more, 3% or more, 3.5% or more, 4% or more, or 4.5% or more.

[0109] A cell culture described in any of

[97] to

[0108] above, wherein the expressed humanized anti-α4β7 antibody has a total fucosylated N-glycan (G0F + G1F + G2F) content of at least 92%, at least 93%, at least 94%, or at least 95%.

[0110] A cell culture described in any of

[97] to

[0108] above, wherein the expressed humanized anti-α4β7 antibody has a total fucosylated N-glycan (G0F+G1F+G2F) content of 92 to 95%.

[0111] The cell culture described in any of

[97] to

[0108] above, wherein the expressed humanized anti-α4β7 antibody has a total fucosylated N-glycan (G0F + G1F + G2F) content of 91 to 92%, 91 to 92.5%, or 91 to 93%.

[0112] The cell culture described in any one of

[97] to

[0111] above, wherein the cell culture further contains arginine and / or lysine.

[0113] The cell culture described in any one of

[97] to

[0112] above, wherein the host cells are CHO cells.

[0114] The cell culture described in

[0113] above, wherein the CHO cells are deficient in the gene encoding glutamine synthetase (GS).

[0115] A humanized anti-α4β7 antibody produced by the cell culture described in any one of

[97] to

[0114] above.

[0116] 1. A method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising: Culturing mammalian host cells genetically engineered to express the humanized anti-α4β7 antibody in a first production medium having a first pH; culturing the mammalian host cells in a second production medium having a second pH; the second pH is lower than the first pH; The method, wherein the humanized anti-α4β7 antibody is an IgG1 antibody and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and comprises a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[0117] The method according to

[0116] above, wherein the second pH is 0.1 to 0.5 pH units lower than the first pH.

[0118] The method according to

[0116] or

[0117] above, wherein the first pH is in the range of pH 6.8 to 7.2, and the second pH is in the range of pH 6.7 to 6.95.

[0119] The method according to any one of

[0116] to

[0118] above, wherein the mammalian host cells are cultured at the first pH for 120 hours or less.

[0120] The method according to any one of

[0116] to

[0118] above, wherein the mammalian host cells are cultured at the first pH for 85 to 110 hours.

[0121] The method according to any one of

[0116] to

[0118] above, wherein the mammalian host cells are cultured at the first pH for 90 to 100 hours.

[0122] The method described in any one of

[0116] to

[0121] above, further comprising recovering the anti-α4β7 antibody from the second production medium.

[0123] The method described in

[0122] above, wherein the anti-α4β7 antibody is recovered following culturing the mammalian host cells in the first production medium and the second production medium for a period of 13 to 15 days.

[0124] A method described in any of

[0116] to

[0123] above, wherein the composition has an increased level of the major isoform of the anti-α4β7 antibody compared to a control composition in which the mammalian host cells are cultured at the first pH without a pH shift.

[0408]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Claims

1. 1. A method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising: Culturing mammalian host cells in a production medium having a temperature in the range of 36-38 degrees Celsius and a pH of 6.7-7.0, wherein the mammalian host cells are Chinese hamster ovary (CHO) cells; adding supplements comprising uridine, manganese, and galactose to the production medium, thereby producing a composition comprising the humanized anti-α4β7 antibody; the mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being vedolizumab; the composition comprises about 16% or less of the basic isoform of the humanized anti-α4β7 antibody (as determined by CEX); The method.

2. the composition comprises about 14% or less, or about 13% or less, of the basic isoform of the humanized anti-α4β7 antibody (as determined by CEX); The method of claim 1.

3. 3. The method of claim 1 or 2, wherein the supplemental components are added to the production medium or to a feed medium which is then added to the production medium.

4. 4. The method of claim 1, wherein the cumulative concentration of uridine added to the production medium between feeding and harvesting is from about 1 to about 7 mM, the cumulative concentration of manganese in the production medium between feeding and harvesting is from about 0.002 to about 0.015 mM, and / or the cumulative concentration of galactose in the production medium between feeding and harvesting is from about 3 to about 20 mM.

5. manganese is added to the production medium as a supplement multiple times, with each addition being about 0.1-10 μM, about 0.2-1.5 μM, about 0.2-5 μM, about 0.25-2 μM, about 0.3-1.2 μM, or about 0.3-0.8 μM; Uridine is added multiple times to the production medium as a supplement, with each addition being about 25-1000 μM, about 75-750 μM, about 55-620 μM, about 100-600 μM, about 150-450 μM, about 100-700 μM, about 100-600 μM, or about 170-630 μM; and / or 5. The method according to claim 1, wherein galactose is added to the production medium as a supplement multiple times, with each addition being about 0.1 to 10 mM, 0.2 to 7.5 mM, 0.5 to 5 mM, 0.4 to 2.8 mM, 0.5 to 3.5 mM, 0.7 to 2.9 mM, 0.75 to 2.5 mM, or about 1.2 mM or 1.4 mM.

6. The supplement is added every day or every two days, and / or The supplementary components are added from the fourth day of the production stage culture. The method of claim 5.

7. 4. The method of claim 3, wherein the feed medium further comprises zinc.

8. 8. The method of any one of claims 1 to 7, wherein the method further reduces the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement but under substantially similar conditions.

9. 9. The method according to claim 1, wherein the production medium comprises 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine.

10. the production medium has a temperature in the range of 36.5 to 37.5 degrees Celsius or about 37 degrees Celsius; the production medium has a pH in the range of 6.8 to 7.0, and / or 7. The method of claim 6, wherein the production medium has a glucose level maintained at about 7 g / L or less during the production stage.

11. 7. The method of claim 6, wherein the production stage is within 14 days or in the range of 10 to 17 days.

12. The method of any one of claims 1 to 11, carried out in a large-scale bioreactor.

13. 7. The method of claim 6, wherein the production step results in a titer of the humanized anti-α4β7 antibody of greater than 3 g / L, or from about 3 to about 8 g / L, or from about 5 to about 7 g / L.

14. 9. The method of any one of claims 1 to 8, wherein the method is a method for producing a composition having a reduced amount of the G0F glycoform of the humanized anti-α4β7 antibody (as determined by hydrophilic interaction chromatography (HILIC)) compared to control mammalian host cells expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement but under substantially similar conditions.

15. 15. The method of claim 14, wherein the composition comprises a level of the G0F glycoform of the humanized anti-a4p7 antibody that is reduced by at least about 15% compared to a control mammalian host cell expressing the humanized anti-a4p7 antibody cultured under substantially similar conditions in the absence of the supplement.

16. 9. The method of any one of claims 1 to 8, wherein the method is a method for producing a composition having about 65% or less, about 60% or less, or about 55% or less of the G0F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC).

17. the method is for producing a composition having an increased amount of the G1F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC) compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplement; the method is for producing a composition having about 25% or more of the G1F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC); the method is for producing a composition having an increased amount of the G2F glycoform of the humanized anti-α4β7 antibody (as determined by HILIC) compared to a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplement; the method is for producing a composition having about 3% or more G2F glycoforms of the humanized anti-α4β7 antibody (as determined by HILIC). The method according to any one of claims 1 to 8.

18. the method further comprises reducing the percentage of acidic species of the humanized anti-α4β7 antibody compared to the percentage of acidic species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of the supplement; or The method further comprises increasing the percentage of the major isoform species of the humanized anti-α4β7 antibody compared to the percentage of the major isoform species produced in a control mammalian host cell expressing the humanized anti-α4β7 antibody cultured under substantially similar conditions in the absence of a feed medium comprising uridine, manganese, and galactose added to the production medium. The method according to any one of claims 14 to 17.

19. 1. A cell culture comprising host cells genetically engineered to express a humanized anti-α4β7 antibody and a production medium supplemented with uridine, manganese, and galactose (UMG), wherein the humanized anti-α4β7 antibody is vedolizumab, the host cells are Chinese hamster ovary (CHO) cells, the production medium has a temperature of 36-38 degrees Celsius and a pH of 6.7-7.0, and the cell culture comprises about 16% or less of a basic isoform of the humanized anti-α4β7 antibody (as determined by CEX).

20. 20. The cell culture of claim 19, wherein the production medium comprises, on the day of harvest, supplemented uridine at a concentration of about 1 to about 7 mM, supplemented manganese at a concentration of about 0.002 to about 0.015 mM, and supplemented galactose at a concentration of about 3 to about 20 mM.

21. 21. The cell culture of claim 19 or 20, wherein the production medium further comprises zinc.

22. the expressed humanized anti-α4β7 antibody a. 15% or less, 14% or less, 13% or less, or 12% or less basic isoforms; and / or b. has an isoform distribution comprising at least 65%, at least 68%, at least 70%, at least 72%, or at least 75% of the major isoform; and / or the expressed humanized anti-α4β7 antibody c. 65% or less, 60% or less, or 55% or less G0F; d. 25% or more, 27% or more, or 30% or more G1F; and / or e. having a fucosylated N-glycan content containing G2F of 2.5% or more, 3% or more, 3.5% or more, 4% or more, or 4.5% or more; The cell culture according to any one of claims 19 to 21.

23. 23. The cell culture of any one of claims 19-22, wherein the expressed humanized anti-α4β7 antibody has a content of total fucosylated N-glycans (G0F+G1F+G2F) of at least 92%, at least 93%, at least 94%, at least 95%, 92-95%, 91-92%, 91-92.5%, or 91-93%.

24. 24. The cell culture of any one of claims 19 to 23, wherein the cell culture further comprises arginine and / or lysine.

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