Cell culture methods and compositions for antibody production

Abstract

To provide cell culture methods for producing anti-α4β7 antibodies, e.g., vedolizumab, and compositions thereof.SOLUTION: Provided is a method for producing a composition including a humanized anti-α4β7 antibody. The method includes: culturing mammalian host cells in a production medium; and adding supplements including uridine, manganese, and galactose to the production medium, thereby producing a composition including the humanized anti-α4β7 antibody, the mammalian host cells being genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and including a heavy chain variable region including CDR3, CDR2, and CDR1 domains having specific amino acid sequences, and a light chain variable region.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Sequence Listing This application contains a sequence listing submitted in ASCII format via EFS-Web. , which is incorporated herein by reference in its entirety. The ASCII copy of the above is named "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. Regarding. [Background technology]

[0004] Mammalian cell culture technology has been used to produce therapeutic biologics, including therapeutic monoclonal antibodies. Commonly used. Proteins produced in mammalian cells are generally Mammalian cells are usually used because they have more similar post-translational modifications to the proteins they produce. For protein production, other forms of eukaryotic cells (such as yeast) or prokaryotic cells (such as bacteria) are used. However, mammalian cell culture is often used because these cells are highly The development of therapeutic antibodies for human use presents many challenges, particularly in the manufacture of therapeutic antibodies on a commercial scale. Production methods must be designed to ensure the safety, efficiency, and cost-effectiveness of protein products. It is necessary to maximize the antibody yield from cells while maintaining the efficiency of the antibody synthesis. Production characteristics, including molecular weight profile, aggregation levels, charge heterogeneity, and amino acid sequence integrity, Production requirements are important because the desired quality characteristics of the product must be maintained (Li et al. al., 2010, mAbs, 2(5):466-477).

[0005] Given the complexity of the cell culture process, it is important to balance manufacturing demands with therapeutics while maintaining high drug quality. Production of therapeutic antibodies, including producing sufficient protein product to meet therapeutic needs. Identifying cell culture parameters that can address the challenges associated with Summary of the Invention

[0006] Mammalian cell culture processes have been the subject of research for the past several decades, but There remains a need for improvements in the large-scale commercial production of antibodies. Increased cell viability, life span, and specific productivity of the product, as well as the potency of the recombinant protein produced. The improvement in value will reduce the price of the recombinant protein produced and, in the case of therapeutic proteins, the medical Such an increase would have a real impact on the price and availability of drugs. This can be particularly challenging given the need to maintain consistency in quality of therapeutic antibodies.

[0007] The invention provided herein relates, inter alia, to the expression of vedolizumab in mammalian host cells. The present invention also discloses cell culture methods and compositions for producing anti-α4β7 antibodies, such as α4β7 antibodies. Provided herein are anti-α4β7 antibodies, such as vedolizumab, obtained using the method. A composition comprising:

[0008] In one aspect, the present invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody, the method comprising the steps of: The method includes culturing mammalian host cells in a production medium and adding uridine, manganese, and and adding a supplement containing galactose to the production medium, thereby producing a humanized antibody. A composition comprising an α4β7 antibody is produced, wherein the mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 antibody and having the sequence set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR3 domain set forth in SEQ ID NO: 2. and a heavy chain variable region comprising the CDR1 domain set forth in SEQ ID NO: 8 and the CDR3 domain set forth in SEQ ID NO: 9. the CDR2 domain set forth in SEQ ID NO:7, and the CDR1 domain set forth in SEQ ID NO:6. The method includes providing a light chain variable domain comprising:

[0009] In some embodiments of the foregoing aspects, the method further comprises the steps of: - (as determined by CEX) of the basic isoform of the humanized anti-α4β7 antibody 1. A method for producing a composition having a reduced amount of a mammalian host cell in a production medium. The cells are cultured in a production medium supplemented with uridine, manganese, and galactose. and adding said culture to a culture medium, thereby producing a humanized culture medium cultured in the absence of supplements. The salts of the humanized anti-α4β7 antibodies compared to control mammalian host cells expressing the anti-α4β7 antibodies The present invention provides a method for producing a composition having a reduced amount of a specific isoform.

[0010] In some embodiments of the above aspects, the present invention provides a method for determining whether a patient is at risk of developing a disease (as determined by CEX) Methods for Producing a Composition Having Less Than About 16% of the Basic Isoform of a Humanized Anti-α4β7 Antibody The method includes culturing mammalian host cells in a production medium and and adding to the production medium supplements including guanidine, guanidine, and galactose, A composition having about 16% or less basic isoform of a humanized anti-α4β7 antibody. The present invention is characterized by a method for producing the above-mentioned.

[0011] In one embodiment, the composition comprises less than or equal to about 14% basic isoforms of a humanized anti-α4β7 antibody. Includes the game.

[0012] In another embodiment, the composition comprises less than or equal to about 13% basic isoforms of a humanized anti-α4β7 antibody. Includes form.

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

[0014] In one embodiment, the cumulative concentration of uridine added to the production medium during the replenishment and harvest is about 1 ~7 mM, and the cumulative concentration of manganese added to the production medium during replenishment and harvest is ~0 0.002 to about 0.015 mM and / or added to the production medium during feeding and harvesting In a particular embodiment, the cumulative concentration of galactose in the feed medium is about 3 to about 20 mM. In one embodiment, the medium further comprises zinc. The cumulative concentration of is about 0.05 mM to about 0.045 mM.

[0015] In one embodiment, manganese is about 0.1-10 μM, about 0.2-1.5 μM per addition. M, about 0.2 to 5 μM, about 0.25 to 2 μM, about 0.3 to 1.2 μM, or about 0.3 to In certain embodiments, the 0.8 μM mAb is added to the production medium as a supplement multiple times. The antigen was added multiple times to the production medium as a supplement, at approximately 0.2-1.5 μM each time. will be added.

[0016] In one embodiment, uridine is about 25-1000 μM, about 75-750 μM for each addition. M, approx. 55~620μM, approx. 100~600μM, approx. 150~450μM, approx. 100~7 00μM, about 100-600μM, or about 170-630μM each, In a particular embodiment, uridine is added to the culture medium at about 100 to 700 μM each time. It is added multiple times to the production medium as a supplement.

[0017] In one embodiment, galactose is added at about 0.1-10 mM, 0.2-7. 5mM, 0.5~5mM, 0.4~2.8mM, 0.5~3.5mM, 0.7~2.9m M, 0.75 to 2.5 mM, or about 1.2 mM or 1.4 mM each as supplement components. In a particular embodiment, galactose is added to the production medium multiple times at a concentration of about 0.5 to 3. It is added multiple times at 5 mM to the production medium as a supplement.

[0018] In one embodiment, the supplement is added every day or every two days. The supplementary components are added starting from the fourth day of the production stage culture.

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

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

[0021] In a further embodiment, galactose is present at a final concentration of about 85 mM to 600 mM. In one embodiment, galactose is added to the feed medium at a concentration of about 160-30%. It is added to the feed medium to a final concentration of 40 mM. is added to the feeding medium to a final concentration of approximately 50-150 mM.

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

[0023] In one embodiment, the method further comprises culturing a humanized anti-α4β antibody in the absence of supplementation. 7. Percentage of acidic species produced in control mammalian host cells expressing the antibody In comparison, the percentage of acidic species of the humanized anti-α4β7 antibody is reduced.

[0024] In one embodiment, the method further comprises the steps of adding uridine, manganese, and Percentage of major isoform species produced in the absence of a feed medium containing galactose Increased the percentage of major isoform species in humanized anti-α4β7 antibodies compared to Add.

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

[0026] In one embodiment, the feed medium is added to the production medium 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, comprising: The method includes culturing mammalian host cells in a production medium that includes zinc, Thus, a method is provided for producing a composition comprising a humanized anti-α4β7 antibody, wherein a mammalian host cell has been genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 antibody 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.

[0028] In some embodiments of the foregoing aspects, the method further comprises the steps of: - (as determined by CEX) of the basic isoform of the humanized anti-α4β7 antibody 2. A method for producing a composition having a reduced amount of zinc, the method comprising: and culturing a mammalian host cell in the absence of zinc, thereby of humanized anti-α4β7 antibody compared to control mammalian host cells expressing humanized anti-α4β7 antibody. A method for producing a composition having a reduced amount of the basic isoform.

[0029] In some embodiments of the above aspect, the method further comprises: ) A method for producing a composition having about 16% or less of the basic isoform of a humanized anti-α4β7 antibody 1. A method for producing a mammalian host cell comprising culturing said mammalian host cell in a production medium comprising zinc. Thus, about 16% or less of the humanized anti-α4β7 antibody has a basic isoform. The present invention relates to a method for producing a composition comprising the steps of:

[0030] In one embodiment, the composition comprises less than or equal to about 14% basic isoforms of a humanized anti-α4β7 antibody. Includes the game.

[0031] In one embodiment, the composition comprises less than or equal to about 13% basic isoforms of a humanized anti-α4β7 antibody. Includes the game.

[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 comprises adding a feed medium containing zinc to the production medium. Thus, supplementing the production medium with zinc. It is added to the production medium from about the fourth day of the incubation period.

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

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

[0036] In a further 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 during a production stage; The composition comprising the humanized anti-α4β7 antibody is produced, and the production medium is maintained at an average temperature of about 37 degrees Celsius. 3. Having the cultured cells at equilibrium temperature, the host cells are genetically engineered to express a humanized IgG1 anti-α4β7 antibody and the humanized anti-α4β7 has the CDR3 domain set forth in SEQ ID NO:4, the CDR3 domain set forth in SEQ ID NO:3 A heavy chain variable region comprising a CDR2 domain and a CDR1 domain as set forth in SEQ ID NO:2. 8, the CDR3 domain set forth in SEQ ID NO: 7, and the CDR2 domain set forth in SEQ ID NO: The method further features a light chain variable region comprising the CDR1 domain set forth in column number 6.

[0037] In some embodiments of the foregoing aspects, the method comprises: 2.) A method for producing a composition containing 2.5% or less HMW species of a humanized anti-α4β7 antibody, comprising the steps of: The method includes the step of producing a humanized anti-α4β7 antibody (as determined by SEC) at the production stage. 2. In a production medium, mammalian host cells are cultured to produce a composition containing 2.5% or less HMW species. The method further comprises culturing said culture medium.

[0038] In yet another aspect, the present invention is a method for producing a composition comprising a humanized anti-α4β7 antibody. The method includes culturing mammalian host cells in a growth medium during an expansion step. In a production step, mammalian host cells are genetically engineered to express a humanized anti-α4β7 antibody. In the second step, a mammalian cell is cultured in a production medium so as to produce a composition comprising a humanized anti-α4β7 antibody. The mammalian host cell is cultured in both the expansion and production stages. and incubated at approximately the same temperature, the humanized anti-α4β7 antibody is an IgG1 antibody, The CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the sequence A heavy chain variable region comprising the CDR1 domain set forth in SEQ ID NO: 2, and the CDR 3 domain, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6. The method further comprises the step of: providing a light chain variable region comprising the domain;

[0039] In some embodiments of the foregoing aspects, the method comprises: 2. A method for producing a composition containing a high level of humanized anti-α4β7 antibody monomer, comprising the steps of: The method includes culturing the mammalian host cells in a growth medium in an expansion step, The animal host cells are genetically engineered to express the humanized anti-α4β7 antibody and are then used in production. The production medium is then cultured in a manner that produces a composition containing a high level of humanized anti-α4β7 antibody monomer. said culturing of mammalian host cells in soil.

[0040] In one embodiment, the temperature is between 36 and 38 degrees Celsius. In yet another embodiment, the temperature is between 36.5 and 37.5 degrees Celsius. Temperature.

[0041] In one embodiment, the production medium of the methods disclosed herein is at a temperature in the range of 36-38 degrees Celsius. In one embodiment, the temperature is in the range of 36.5 to 37.5 degrees Celsius. In an embodiment, the temperature is an average temperature of about 37 degrees Celsius. It has a pH range of .5 to 7.

[0042] In one embodiment, the production medium of the method disclosed herein has a pH in the range of 6.8 to 7.0. It's H.

[0043] In one embodiment, the production medium of the methods disclosed herein has a production stage of about 7 g / L or less. The glucose levels are maintained at .

[0044] In one embodiment, the production phase lasts for 14 days or less. In another embodiment, the production phase lasts for 10 days or less. The range is ~17 days.

[0045] In some embodiments of the above aspects, the method is carried out in a large-scale bioreactor. In a particular embodiment, the large-scale bioreactor comprises a 200 liter (L) tank. Bioreactor, 2000L bioreactor, 3000L and 6000L bioreactor reactor.

[0046] In some embodiments, the production step produces greater than 3 g / L of humanized anti-α4β7 antibody. In certain embodiments, the titer of the humanized anti-α4β7 antibody is about 3 to about In another embodiment, the titer of the humanized anti-α4β7 antibody is about 5 to about 7 g / L. It is.

[0047] In some embodiments of the above aspects, the mammalian host cell is Chinese hamster ovarian cancer. In a particular embodiment, the CHO cells are GS-CHO cells. do.

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

[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, purification involves removing (i) cellular debris, undesired proteins, salts, (ii) a purification step to remove contaminants, minerals, or other undesirable elements; and purifying the antibodies from the soluble proteins and polypeptides of the substance. The method further comprises 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 embodiments, the liquid pharmaceutical formulation is prepared by ultrafiltration / diafiltration.

[0052] In other embodiments, the pharmaceutical formulation is a lyophilized dry antibody formulation. In such embodiments, pharmaceutical formulations of the antibodies are prepared by purification followed by UF / DF. The dried antibody formulation is a liquid pharmaceutical antibody formulation that has been lyophilized.

[0053] In some embodiments, the present invention provides a method for the detection of chromatographically active substances using hydrophilic interaction chromatography (HIL C) has a reduced amount of the G0F glycoform of the humanized anti-α4β7 antibody, as determined by A method for producing a composition, the method comprising culturing a mammalian host cell in a production medium. and adding supplements containing uridine, manganese, and galactose to the production medium. and thereby producing a humanized anti-α4β7 antibody cultured in the absence of supplementary components. Reduced G0F glycoforms of humanized anti-α4β7 antibodies compared to control mammalian host cells expressing The present invention provides a method for producing a composition having a quantity.

[0054] In one embodiment, the composition comprises a humanized anti-α4β7 antibody cultured in the absence of supplemental components. The G0F glycoform of the humanized anti-α4β7 antibody was reduced in comparison to the control mammalian host cells expressing the This includes levels that have decreased by at least about 15%.

[0055] In one embodiment, the composition comprises a humanized anti-α4β7 antibody cultured in the absence of supplemental components. The G0F glycoform of the humanized anti-α4β7 antibody was reduced in comparison to the control mammalian host cells expressing the This includes a reduction of at least about 20%.

[0056] In some embodiments, provided herein are methods for detecting chromatin-binding domains (as determined by HILIC). A method for producing a composition having about 65% or less of the G0F glycoform of a humanized anti-α4β7 antibody The method includes culturing mammalian host cells in a production medium and administering to the mammalian host cells a uridine, mannose, or guanidine-containing medium. and adding supplements to the production medium, the supplements including galactose and galactose, Thus, a composition having about 65% or less of the G0F glycoform of a humanized anti-α4β7 antibody is produced. This is the method.

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

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

[0059] In some embodiments, provided herein are methods for preparing chromatographically sensitive, high-resolution chromatographic (HPC) systems. Increase in G1F glycoform of humanized anti-α4β7 antibody as determined by FTIR (HILIC) 20. A method for producing a composition having an increased amount of a mammalian host cell in a production medium, the method comprising: The cells are cultured and supplemented with uridine, manganese, and galactose to produce a production medium. and adding to said culture medium a humanized antibody that has been cultured in the absence of supplementary components, thereby G1 of humanized anti-α4β7 antibody compared to control mammalian host cells expressing α4β7 antibody A method for producing compositions having increased amounts of F glycoforms.

[0060] In one embodiment, the composition comprises a humanized anti-α4β7 antibody cultured in the absence of supplemental components. The G1F form of the humanized anti-α4β7 antibody was expressed less frequently compared to control mammalian host cells expressing the antibody. This includes at least a two-fold increase.

[0061] In one embodiment, the composition comprises a humanized anti-α4β7 antibody cultured in the absence of supplemental components. The G1F glycoform of the humanized anti-α4β7 antibody is reduced compared to control mammalian host cells expressing the This includes at least a three-fold increase.

[0062] Further, in some embodiments, provided herein are methods for detecting chromatin-dependent changes in chromatin (determined by HILIC). Production of a composition having about 25% or more of the G1F glycoform of a humanized anti-α4β7 antibody 1. A method, comprising culturing a mammalian host cell in a production medium and and adding supplements to the production medium, the supplements including arginine, manganese, and galactose; This allows the production of compositions having about 25% or more of the G1F glycoform of the humanized anti-α4β7 antibody. This is a method.

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

[0064] In some embodiments, provided herein are methods for preparing chromatographically sensitive, high-resolution chromatographic (HPC) systems. Increased G2F glycoforms of humanized anti-α4β7 antibodies as determined by FTIR (HILIC) 20. A method for producing a composition having an increased amount of a mammalian host cell in a production medium, the method comprising: The cells are cultured and supplemented with uridine, manganese, and galactose to produce a production medium. and adding to said culture medium a humanized antibody that has been cultured in the absence of supplementary components, thereby G2 of humanized anti-α4β7 antibody compared to control mammalian host cells expressing α4β7 antibody A method for producing compositions having increased amounts of F glycoforms.

[0065] In one embodiment, the composition comprises a humanized anti-α4β7 antibody cultured in the absence of supplemental components. The number of G2F glycoforms of the humanized anti-α4β7 antibody is reduced compared to control mammalian host cells expressing the This includes at least a three-fold increase.

[0066] In one embodiment, the composition comprises a humanized anti-α4β7 antibody cultured in the absence of supplemental components. The number of G2F glycoforms of the humanized anti-α4β7 antibody is reduced compared to control mammalian host cells expressing the This includes at least a four-fold increase.

[0067] In some embodiments, provided herein are methods for detecting chromatin-binding domains (as determined by HILIC). When the composition is a humanized anti-α4β7 antibody, the composition has a G2F glycoform of about 3% or more. The method includes culturing mammalian host cells in a production medium and administering uridine, manganese, or the like to the host cells. and adding supplements to the production medium, the supplements including glycerol, glycerol, and galactose, thereby This produces a composition having about 3% or more of the G2F glycoform of humanized anti-α4β7 antibody 6. It is law.

[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 the feed medium. The feed medium is then added to the production medium.

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

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

[0072] In one embodiment, the feed medium contains between 85 mM and 600 mM galactose. In one embodiment, the feed medium comprises about 85-100 mM galactose. The medium contains about 50 to 150 mM galactose.

[0073] In yet another embodiment, the production medium further comprises zinc. The zinc concentration is about 50 μM to 150 μM.

[0074] In one embodiment, the method further comprises culturing a humanized anti-α4β antibody in the absence of supplementation. 7. Percentage of acidic species produced in control mammalian host cells expressing the antibody In comparison, the percentage of acidic species of the humanized anti-α4β7 antibody is reduced.

[0075] In one embodiment, the method further comprises the steps of adding uridine, manganese, and Percentage of major isoform species produced in the absence of a feed medium containing galactose Increased the percentage of major isoform species in humanized anti-α4β7 antibodies compared to Add.

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

[0077] In one embodiment, the feed medium is added to the production medium from about day 4 of the production phase. In this 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. In one embodiment, a large scale bioreactor is a 200 liter (L) bioreactor. Bioreactor, 2000L Bioreactor, 3000L and 6000L Bioreactor is selected from the group consisting of:

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

[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 polypeptide 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 such embodiments, purification involves removing (i) cellular debris, undesirable proteins, salts, minerals, or other undesirable elements; and (ii) a purification step to remove the soluble contaminants. and purifying the antibodies from the proteins and polypeptides. 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 embodiments, the liquid pharmaceutical formulation is prepared by ultrafiltration / diafiltration.

[0086] In other embodiments, the pharmaceutical formulation is a lyophilized dry antibody formulation. In such embodiments, pharmaceutical formulations of the antibodies are prepared by purification followed by UF / DF. The dried antibody formulation is a liquid pharmaceutical antibody formulation that has been lyophilized.

[0087] In another aspect, provided herein is a method for producing a humanized anti-α4β7 antibody comprising administering to said animal an isolated humanized anti-α4β7 antibody. Genetically engineered host cells and supplemented with uridine, manganese, and galactose (UMG) and a production medium comprising the cell culture, wherein the humanized anti-α4β7 antibody is an IgG1 antibody; A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and an amino acid sequence set forth in SEQ ID NO:5. and a light chain variable region comprising an LAT domain.

[0088] In some embodiments of the above aspects, the production medium contains about 15-100 mM of cucumber. gin at a concentration of about 20 to 200 nM, manganese at a concentration of about 85 to 500 mM, and galactosamine at a concentration of about 85 to 500 mM. Includes base.

[0089] In some embodiments of the above aspects, the production medium contains, on the day of harvest, about 1-7 mM supplemented uridine at a concentration of about 2-15 μM, supplemented manganese at a concentration of about 3-2 In some embodiments, the production medium comprises supplemented galactose at a concentration of 0 mM. On the day of harvest, it further contains 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 contains about 1-7 mM uridine at a concentration of about 2 to 15 μM, manganese at a concentration of about 3 to 20 mM, and galactose at a concentration of about 3 to 20 mM. In some embodiments, the production medium contains about 5-45 μM tRNA on the day of harvest. The composition further comprises zinc at a concentration of

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

[0092] In other embodiments, the expressed humanized anti-α4β7 antibody has a (a) humanized antibody sequence of 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 has a fucosylated N-glycan content containing 4.5% or more G2F.

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

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

[0095] In alternative embodiments, the expressed humanized anti-α4β7 antibody has a 91-92%, .5%, or 91-93% total fucosylated N-glycans (G0F+G1F+G2F) It has a content of.

[0096] In some embodiments of the above aspects, the cell culture further comprises zinc. wherein 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 this state, CHO cells are deficient in the gene encoding glutamine synthetase (GS). There are.

[0098] In another aspect, the present disclosure provides a method for the production of a medicament for the treatment of a cancer, comprising the steps of: A humanized anti-α4β7 antibody is provided.

[0099] In yet another aspect, the present disclosure provides a composition comprising a humanized anti-α4β7 antibody, The method comprises producing a humanized anti-α4β7 antibody in a first production medium having a first pH. and culturing the engineered mammalian host cell to produce a second production solution having a second pH. and culturing the mammalian host cells in the medium, wherein the second pH is lower than the first pH. The humanized anti-α4β7 antibody is an IgG1 antibody and has the CDR3 domain set forth in SEQ ID NO:4. the CDR1 domain as set forth in SEQ ID NO:2; and a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8 and the CDR4 domain set forth in SEQ ID NO: 7. and a light chain variable region comprising the CDR2 domain of SEQ ID NO:6 and the CDR1 domain of SEQ ID NO:7. nothing.

[0100] In some embodiments of the foregoing aspects, the second pH is 0.1 to 0.5 lower than the first pH. 5 pH units lower. In certain embodiments, the first pH is in the range of pH 6.8 to 7.2; 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 to 110 hours. In another embodiment, the mammalian host cells are cultured at the first pH for 90 to 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 administered to a patient in need of treatment for a period of 13 to 15 days. Cultivation of the mammalian host cells in the production medium and the second production medium is followed by harvesting.

[0103] In some embodiments, the composition inhibits the mammalian host cell from undergoing a first pH without a pH shift. Increased expression of the major isoform of anti-α4β7 antibodies compared to a control composition cultured with It has a level.

[0104] In another aspect, the present invention provides a method for the preparation of a method for the treatment of a cancer using any one of the methods disclosed herein. In one embodiment, the composition comprises the humanized anti-α4β7 antibody produced. The methodology used involves the identification of >92% total asialo-, agalacto-, and core-fucosylated biantennary glycans ( G0F), asialo, monogalactic, core fucosylated biantennary glycans (G1F), and / or or asialo, digalacto, and core fucosylated biantennary glycans (G2F), The present invention provides a population of humanized anti-α4β7 antibodies having humanized variants.

[0105] Furthermore, the present invention also includes the following embodiments: 1. Humanized anti-α (as determined by cation exchange chromatography (CEX) A method for producing a composition having a reduced amount of a basic isoform of 4β7 antibody, comprising the steps of: The method is as follows: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, A control expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement by A reduction in the basic isoform of said humanized anti-α4β7 antibody compared to mammalian host cells. and producing a composition having a determined amount of The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0106] 2. A basicity of the humanized anti-α4β7 antibody of about 16% or less (as determined by CEX) 1. A method for producing a composition having an isoform, the method comprising: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, A composition having about 16% or less of the basic isoform of the humanized anti-α4β7 antibody. Producing things, The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ ID NO:16, a CDR13 domain as set forth in SEQ ID NO:17, a CDR13 domain as set forth in SEQ ID NO:18, a CDR13 domain as set forth in SEQ ID NO:19 ... A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A chain variable region, comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

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

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

[0109] 5. After the supplement is added to the production medium or to the feed medium, 5. The method of any one of clauses 1-4, wherein said feed medium is added to said production medium.

[0110] 6. The method of claim 5, wherein uridine is added to the feeding medium at a final concentration of about 15 to 120 mM. How to.

[0111] 7. Add the uridine to the feed medium to a final concentration of about 20 to 70 mM uridine. The method of addition to the land, Article 6.

[0112] 8. Manganese is added to the feeding medium at a final concentration of about 0.02 to 0.3 mM. The method of item 5.

[0113] 9. Manganese is added to the feed medium to a final concentration of approximately 0.04 to 0.15 mM. The method of adding the information in accordance with Article 8.

[0114] 10. Add galactose to the supply medium to a final concentration of approximately 85 mM to 600 mM. The method of clause 5 is added to the above.

[0115] 11. Add galactose to the feeding medium to a final concentration of approximately 160-340 mM. The method of clause 10 is added.

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

[0117] 13. The method of claim 12, wherein the concentration of zinc in the supply medium is about 90 μM to 120 μM. Law.

[0118] 14. The method further comprises culturing the humanized anti-α4β7 antibody in the absence of the supplement. Compared to the percentage of acidic species produced in control mammalian host cells expressing the antibody. and reducing the percentage of acidic species in the humanized anti-α4β7 antibody. Either one of the methods.

[0119] 15. The method further comprises adding uridine, manganese, and galactose to the production medium. Percentage of major isoform species produced in the absence of lactose-containing feed medium Increase the percentage of the major isoform species of said humanized anti-α4β7 antibody compared to or (b) cause the user to do so by any of the methods set forth in clauses 1 to 14.

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

[0121] 17. The method of claim 16, wherein the feeding medium is added to the production medium from about day 4 of the production stage. How to.

[0122] 18. Humanized (as determined by cation exchange chromatography (CEX)) A method for producing a composition having a reduced amount of the basic isoform of an anti-α4β7 antibody. The method includes culturing mammalian host cells in a production medium that includes zinc, Thus, a control mammal expressing the humanized anti-α4β7 antibody cultured in the absence of zinc a reduced amount of the basic isoform of the humanized anti-α4β7 antibody compared to a host cell; and producing a composition comprising: The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0123] 19. A humanized anti-α4β7 antibody having a base content of about 16% or less (as determined by CEX) 2. A method for producing a composition having a sexual isoform, the method comprising the steps of: and culturing the mammalian host cell in a culture medium, thereby obtaining no more than about 16% of said humanized polypeptide. Producing a composition having a basic isoform of an anti-α4β7 antibody; The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

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

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

[0126] 22. The method according to any one of claims 18 to 21, wherein the concentration of zinc in the production medium is 2 μM to 60 μM. Either one of the methods.

[0127] 23. The method further comprises adding a feed medium containing zinc to the production medium to produce the fermentation product. 23. The method of any one of clauses 18-22, comprising supplementing the production medium with zinc.

[0128] 24. The method of claim 23, wherein the feeding medium is added to the production medium from about day 4 of the production stage. How to.

[0129] 25. The method of claim 24, wherein the concentration of zinc in the supply medium is about 90 μM to 120 μM. How to.

[0130] 26. The production medium contains 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine.

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

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

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

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

[0135] 31. HM of humanized anti-α4β7 antibody of 2.5% or less (as determined by SEC) 1. A method for producing a composition comprising a W species, the method comprising: 2.5% or less of said humanized anti-α4 in the production stage (as determined by SEC) The mammalian host cells are cultured in a production medium so as to produce a composition comprising the HMW species of the β7 antibody. This includes nurturing The production medium has an average temperature of about 37 degrees Celsius, and the host cells are human IgG1 anti-α genetically engineered to express the 4β7 antibody, The humanized anti-α4β7 antibody comprises a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:3, and a heavy chain variable region comprising the CDR2 domain described above and the CDR1 domain described in SEQ ID NO:2. The present invention further comprises a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, and and a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6.

[0136] 32. High levels of monomeric humanized anti-α4β7 antibodies (as determined by SEC) 1. A method for producing a composition comprising: Cultivating mammalian host cells in a growth medium during an expansion stage, said mammalian host cells being The host cells are genetically engineered to express a humanized anti-α4β7 antibody. And, The monomeric amount of the humanized anti-α4β7 antibody during production (as determined by SEC) The mammalian host cells are cultured in a production medium such that a composition containing high levels of the and The mammalian host cells are cultured at approximately the same temperature during both the expansion and production steps. R, The humanized anti-α4β7 antibody is an IgG1 antibody and has a CDR3 domain as 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 a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8 and the CDR4 domain set forth in SEQ ID NO: 7. A light chain variable region comprising a CDR2 domain and a CDR1 domain set forth in SEQ ID NO:6, The method.

[0137] 33. The method of clause 31 or 32, wherein the temperature is between 36 and 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. Any of clauses 1 to 35, wherein the production medium has a temperature in the range of 36 to 38 degrees Celsius. Or one way.

[0141] 37. The method of clause 36, wherein the temperature is in the range of 36.5 degrees Celsius 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. Any one of clauses 1 to 38, wherein the production medium has a pH in the range of 6.5 to 7. How to.

[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 glucose level of the production medium is maintained at about 7 g / L or less during the production stage. 41. The method according to any one of clauses 1 to 40, comprising:

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

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

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

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

[0150] 46. ​​The production step results in a titer of the humanized anti-α4β7 antibody of greater than 3 g / L. , any one of the methods set forth in clauses 1 to 45.

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

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

[0153] 49. The mammalian host cell is a Chinese Hamster Ovary (CHO) cell. Any one of the methods set forth in clauses 1 to 48.

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

[0155] 51. The humanized anti-α4β7 antibody comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:1. Clauses 1 to 50, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5. Either one of the methods.

[0156] 52. Any one of clauses 1 to 50, wherein the humanized anti-α4β7 antibody is vedolizumab. How to.

[0157] 53. A humanized anti-α4β7 antibody produced by any one of the methods of clauses 1 to 52. A composition comprising:

[0158] More than 54.92% of total asialo-, agalacto-, and core fucosylated biantennary glycans (G0F ), asialo, monogalactic, core fucosylated biantennary glycans (G1F), and / or Glycosylation of asialo-, digalacto-, and core fucosylated biantennary glycans (G2F) The composition of clause 53, comprising a population of humanized anti-α4β7 antibodies having an ant.

[0159] 55. (As determined by Hydrophilic Interaction Chromatography (HILIC)) A method for producing a composition having a reduced amount of the G0F glycoform of a humanized anti-α4β7 antibody, comprising the steps of: The method is as follows: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, Thus, a control mammal expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement was having a reduced amount of the G0F glycoform of said humanized anti-α4β7 antibody compared to mammalian host cells. and producing a composition comprising The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ ID NO:16, a CDR13 domain as set forth in SEQ ID NO:17, a CDR13 domain as set forth in SEQ ID NO:18, a CDR13 domain as set forth in SEQ ID NO:19 ... A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A chain variable region, comprising a CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0160] 56. The composition is a humanized anti-α4β7 antibody cultured in the absence of the supplement. at least about 15% reduced levels of said gene, as compared to a control mammalian host cell expressing said gene 56. The method of clause 55, comprising the G0F glycoform of the humanized anti-α4β7 antibody.

[0161] 57. The composition is the humanized anti-α4β7 antibody cultured in the absence of the supplement. the G0F glycotype of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing Article 55 methods, including a reduction of at least about 20%.

[0162] 58. Approximately 65% ​​or less of the humanized anti-α4β7 antibody (as determined by HILIC) 1. A method for producing a composition having a G0F glycoform, the method comprising: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, and producing a composition having about 65% or less of the G0F glycoform of the humanized anti-α4β7 antibody. and The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0163] 59. The composition comprises about 60% or less of the G0F glycoform of the humanized anti-α4β7 antibody. Article 58 Method.

[0164] 60. The composition comprises about 55% or less of the G0F glycoform of the humanized anti-α4β7 antibody. Article 58 Method.

[0165] 61. Increase in G1F glycoform of humanized anti-α4β7 antibody (as determined by HILIC) 2. A method for producing a composition having an increased amount of glycerol, the method comprising: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, A control expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement by The humanized anti-α4β7 antibody has an increased amount of the G1F glycoform compared to a mammalian host cell. and producing a composition comprising: The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0166] 62. The composition is the humanized anti-α4β7 antibody cultured in the absence of the supplement. the G1F glycotype of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing Clause 61 methods, including at least about a two-fold increase.

[0167] 63. The composition is a humanized anti-α4β7 antibody cultured in the absence of the supplement. the G1F glycotype of the humanized anti-α4β7 antibody compared to a control mammalian host cell expressing Article 61 methods, including at least about a three-fold increase.

[0168] 64. Approximately 25% or more of the humanized anti-α4β7 antibody is 1. A method for producing a composition having a G1F glycoform, the method comprising: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, and producing a composition having about 25% or more of the G1F glycoform of the humanized anti-α4β7 antibody. and The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0169] 65. The composition comprises about 30% or more of the G1F glycoform of the humanized anti-α4β7 antibody. Article 64 Method.

[0170] 66. Increased G2F glycoforms of humanized anti-α4β7 antibodies (as determined by HILIC) 2. A method for producing a composition having an increased amount of glycerol, the method comprising: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, Thus, a control mammal expressing the humanized anti-α4β7 antibody cultured in the absence of the supplement was having increased amounts of the G2F glycoform of said humanized anti-α4β7 antibody compared to mammalian host cells. and producing a composition comprising The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

[0171] 67. The composition is a humanized anti-α4β7 antibody cultured in the absence of the supplement. The G2F glycoform of the humanized anti-α4β7 antibody is compared to a control mammalian host cell expressing Article 66 methods, including at least about a three-fold increase.

[0172] 68. The composition comprises the humanized anti-α4β7 antibody cultured in the absence of the supplement. The G2F glycoform of the humanized anti-α4β7 antibody is compared to a control mammalian host cell expressing Article 66 methods, including at least about a four-fold increase.

[0173] 69. The G value of humanized anti-α4β7 antibodies is greater than or equal to about 3% (as determined by HILIC). 1. A method for producing a composition having a 2F glycoform, the method comprising: Cultivating mammalian host cells in a production medium; Supplements including uridine, manganese, and galactose are added to the production medium, Therefore, a composition having about 3% or more of the G2F glycoform of the humanized anti-α4β7 antibody can be produced. Including, The mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, the humanized anti-α4β7 antibody being an IgG1 and having a CDR3 domain as set forth in SEQ ID NO:4, a CDR4 domain as set forth in SEQ ID NO:5, a CDR5 domain as set forth in SEQ ID NO:6, a CDR6 domain as set forth in SEQ ID NO:7, a CDR7 domain as set forth in SEQ ID NO:8, a CDR8 domain as set forth in SEQ ID NO:9, a CDR9 domain as set forth in SEQ ID NO:10, a CDR10 domain as set forth in SEQ ID NO:11, a CDR11 domain as set forth in SEQ ID NO:12, a CDR12 domain as set forth in SEQ ID NO:13, a CDR13 domain as set forth in SEQ ID NO:14, a CDR13 domain as set forth in SEQ ID NO:15, a CDR13 domain as set forth in SEQ A duplicated sequence including the CDR2 domain shown in column number 3 and the CDR1 domain shown in SEQ ID NO:2. A CDR3 domain as set forth in SEQ ID NO: 8, a CDR2 domain as set forth in SEQ ID NO: 7, The method comprises a light chain variable region comprising the CDR1 domain set forth in SEQ ID NO:6, and a CDR2 domain set forth in SEQ ID NO:7.

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

[0175] 71. After the supplement is added to the production medium or to the feed medium, 71. The method of any one of clauses 55 to 70, wherein said feed medium is added to said 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 contains 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 claim 71, wherein the feed medium comprises 85 mM to 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 claim 78, wherein the concentration of zinc in the production medium is about 50 μM to 150 μM. How to.

[0184] 80. The method further comprises culturing the humanized anti-α4β7 antibody in the absence of the supplement. Compared to the percentage of acidic species produced in control mammalian host cells expressing the antibody. and reducing the percentage of acidic species in the humanized anti-α4β7 antibody. One of 9 ways.

[0185] 81. The method further comprises adding uridine, manganese, and galactose to the production medium. Percentage of major isoform species produced in the absence of lactose-containing feed medium Increase the percentage of the major isoform species of said humanized anti-α4β7 antibody compared to or any one of the methods set forth in Articles 55 to 80.

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

[0187] 83. The method of claim 82, wherein the feeding medium is added to the production medium from about day 4 of the production stage. How to.

[0188] 84. Any one of clauses 1 to 83, wherein the production medium has a pH of about 6.8 to about 7.1. There are two ways. [Brief description of the drawings]

[0189] [Figure 1] We provide results from a predictive profiler based on experiments testing different culture conditions including pH, temperature, galactose Gal+ addition, UMG addition, and feeding strategy conditions in production cell cultures. [Figure 2A] Graphical representation of the effect 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] Graphical representation of the effect 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]Graphical representation of the effect 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] Graphical representation of the effect 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] Graphical representation of the effect 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] Graphical representation of the effect 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]Graphical representation of the effect 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] Graphical representation of the effect 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. [Diagram 3] A graph comparing the effect of various arginine and lysine concentrations on the percentage of basic species (A) and antibody titers (B) is shown. 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 results of maximum desirability 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). Corresponding concentrations for high (H), medium (M), or low (L) concentrations of lysine and arginine are outlined in Table 5. [Figure 4B] Predicted results of maximum desirability 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). Corresponding concentrations for high (H), medium (M), or low (L) concentrations of lysine and arginine are outlined in Table 5. [Figure 4C] Predicted results of maximum desirability 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). Corresponding concentrations for high (H), medium (M), or low (L) concentrations of lysine and arginine are outlined in Table 5. [Figure 5A] Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5B] Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5C] Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5D]Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5E] Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5F] Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5G] Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 5H]Graphical representation of 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. Numbers on the x-axis correspond to zinc concentrations as outlined in Table 6. [Figure 6A] Graphs show 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). The solid black lines in A, C, and E represent the top process criteria for each attribute, while the solid black lines in B represent the bottom acceptance criteria. Numbers on the x-axis correspond to zinc concentrations outlined in Table 6. [Figure 6B] Graphs show 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). The solid black lines in A, C, and E represent the top process criteria for each attribute, while the solid black lines in B represent the bottom acceptance criteria. Numbers on the x-axis correspond to zinc concentrations outlined in Table 6. [Figure 6C] Graphs show 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). The solid black lines in A, C, and E represent the top process criteria for each attribute, while the solid black lines in B represent the bottom acceptance criteria. Numbers on the x-axis correspond to zinc concentrations outlined in Table 6. [Figure 6D]Graphs show 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). The solid black lines in A, C, and E represent the top process criteria for each attribute, while the solid black lines in B represent the bottom acceptance criteria. Numbers on the x-axis correspond to zinc concentrations outlined in Table 6. [Figure 6E] Graphs show 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). The solid black lines in A, C, and E represent the top process criteria for each attribute, while the solid black lines in B represent the bottom acceptance criteria. Numbers on the x-axis correspond to zinc concentrations outlined in Table 6. [Figure 7A] Graphical representation of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), percentage of basic species (B), 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 representation of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), percentage of basic species (B), 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 representation of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), percentage of basic species (B), 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 representation of time course data comparing the effect of days of vedolizumab culture on the percentage of acidic species (A), percentage of basic species (B), 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] A graphical depiction of the correlation between isoform distribution and pH shift parameters shows the correlation between final cell culture pH (after pH shift) and % acidic isoform species (left panel) or % major isoform (right panel). [Figure 8B] The correlation between isoform distribution and pH shift parameters is shown graphically, with pH shift duration correlated with % acidic isoform species (left panel) or % major isoform (right panel). [Figure 9] Figure 1 shows the N-glycan structures that may be present in a population of anti-α4β7 antibodies such as vedolizumab. The glycan legend is provided in the figure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0191] (used interchangeably throughout) a cell surface molecule, "α4β7 integrin " or "α4β7" refers to the α4 chain (CD49D, ITGA4) and the β7 chain (ITGB7). It is a heterodimer of the human α4 integrin and β7 integrin genes, GenB. ank (National Center for Biotechnology Information, Bethesda, Md.) fSeq accession numbers NM_000885 and NM_000889 are for B and T lymphocytes, especially memory CD4+ lymphocytes. Typical of α4β7, it can exist in either a resting or activated state. The ligands for 4β7 are vascular cell adhesion molecule (VCAM), fibronectin, and mucosal adhesion molecule (MAA). Resin (MAdCAM (e.g., MAdCAM-1)) The binding antibodies are referred to herein as "anti-α4β7 antibodies."

[0192] As used herein, an antibody having "binding specificity for the α4β7 complex" or or an antigen-binding fragment thereof binds to α4β7 but not to α4β1 or α E Does not bind to B7 Vedolizumab is an example of an antibody with binding specificity for the α4β7 complex.

[0193] The term "about" means that the value that follows is not an exact value, but is within + / - 5% of the value. This means the midpoint of a range. If the value is a relative value expressed as a percentage, The word "about" means that the value that follows is not an exact value, but rather a range of values ​​within + / - 5% of the value. The center point of the range, so that the upper limit of the range cannot exceed the 100% value. means.

[0194] As used herein, the term "aggregate" or "aggregates" refers to two The term "aggregate" refers to an association of antibodies or antibody fragments. For example, aggregates are antibodies and / or antibody fragments. The antibody aggregates may be dimers, trimers, tetramers, or multimers greater than tetramers. They can be soluble or insoluble. The association between the aggregating molecules is related to the mechanism by which they are associated. The association may be any covalent or non-covalent bond between the aggregated molecules. This may be directly or indirectly through other molecules that link them together. Examples of such molecules include disulfide bonds with other proteins, hydrophobic associations with lipids, and electrochemical associations with DNA. These include carboxyl-linked cleavage, affinity association with leached Protein A, or mixed-mode association with multiple components. Aggregates can occur during protein expression in cell culture, as well as downstream processes. They can be irreversibly formed during protein purification processes or during drug storage. The presence of aggregates in a solution can be detected, for example, by size exclusion chromatography (SEC) (e.g. For example, SEC with UV detection, SEC with light scattering detection (SEC-LSD), flow fractionation, analytical ultracentrifugation sedimentation velocity, or capillary electrophoresis-dodecyl sulfate The concentration of acetylcholine in the blood can be determined using sodium dodecyl sulfate (CE-SDS, reduced and non-reduced).

[0195] As used herein, the term "antibody" refers to an antibody that is an antibody that is linked to other antibodies by disulfide bonds. It consists of four polypeptide chains connected together: two heavy (H) chains and two light (L) chains. Each heavy chain is intended to refer to an immunoglobulin molecule that comprises a heavy chain variable region (as defined herein). The heavy chain constant region (CH) is abbreviated as HCVR or VH in the present specification. The constant region consists of three domains, CH1, CH2, and CH3. 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 in frame The complementarity determining regions (CDRs) are interspersed with more conserved regions called framework regions (FRs). Each VH and VL has three CDs. It is composed of R and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: There are: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In embodiments, the antibody has a fragment crystallizable (Fc) region. The body is of the IgG1 isotype and has kappa light chains.

[0196] As used herein, "charged species," "charged isoform," or "charged isoform" refers to a The term "isoform species" refers to an overall species that is distinct from the predominant species of an antibody or its antigen-binding portion. Antibodies or antigen-binding portions thereof (e.g., pedigree antibodies) characterized by a specific charge. izumab, or an antigen-binding portion thereof) and Charged isoform species can be identified by cation exchange chromatography (CEX), e.g. CEX-HPLC, CEX-Mass Spectrometry, or Detection can be achieved by a variety of methods known in the art, such as isoelectric focusing. For example, antibody preparations are typically prepared by CEX, CEX-HPLC, or CEX-mass spectrometry. When antibodies are isolated using ELISA, the majority of the antibodies are isolated as the predominant (major) isoform of that antibody. The eluate from the CEX resin with a retention time that is characteristic of the CEX resin retention time. This can be visualized by plotting the amount of antibody eluted from the resin as a function of the concentration. When visualized, the major isoform of an antibody or its antigen-binding portion is the largest peak. The fraction of antibodies or antigen-binding portions thereof that elutes from the CEX resin within 1 h of incubation. The charged isoform species were then identified by having a different retention time from the major isoform. For example, charged isoform species can be identified by CEX, CEX-HPLC, or CEX When detected by mass spectrometry, acidic isoform species are It can be eluted from the resin with a shorter retention time than the major isoform, and the basic isoform The species is retained off the resin with a longer retention time than the major isoform of the antibody or its antigen-binding portion. It can be dissolved.

[0197] As used herein, the terms "acidic species" or "acidic isoform species" refer to , antibodies or antigen-binding portions thereof characterized by an overall acidic charge (e.g., The term refers to a variant of the antibody or antigen-binding portion thereof. The acidic species can be separated by cation exchange chromatography (CEX), e.g., by using a cation exchange high performance liquid chromatography (HPLC). CEX-HPLC, CEX-mass spectrometry, or isoelectric focusing Detection can be accomplished by a variety of methods known in the art, such as: The acidic species of an antibody or antigen-binding portion thereof is the major isoform of the antibody or antigen-binding portion thereof. The acidic species of the antibody elute from the CEX resin with a shorter retention time than the elution time of the antibody. These may include, but are not limited to, structural variants, and / or fractional variants. In some embodiments, a composition comprising an antibody or antigen-binding portion thereof comprises more than one In some embodiments, the IL-16A-binding domain may include a plurality of acidic isoform species. Isoform species can be identified based on differences in retention times during CEX-HPLC separation. For example, when a composition containing an antibody, e.g., vedolizumab, is analyzed using CEX, , one or more acidic isoforms each representing one or more acidic isoform species of the antibody; The peaks can be identified.

[0198] As used herein, the terms "basic species" or "basic isoform species" The term refers to an antibody or antigen-binding portion thereof (e.g., The basic species of the antibody or antigen-binding portion thereof is a variant of the cationically linked agonist (Cyclohexanone). Cation exchange chromatography (CEX), e.g., cation exchange high performance liquid chromatography The relevant technical analysis, such as CEX-HPLC, CEX-mass spectrometry, or isoelectric focusing, Detection can be achieved by a variety of methods known in the art. The basic species of the original binding moiety is longer than the major isoform of the antibody or its antigen-binding portion. The antibody is eluted from the CEX resin at the retention time. The basic species of the antibody include charge variants, structural barriers, and These may include, but are not limited to, chromatin-specific variants, and / or fractionated variants. In one embodiment, the composition comprising an antibody or antigen-binding portion thereof contains more than one type of salt. In some embodiments, the nucleotide sequence may include multiple basic isoform species. Species can be identified based on differences in retention times during CEX-HPLC separation. For example, when a composition containing an antibody, e.g., vedolizumab, is analyzed using CEX, each One or more basic isoform peaks representing one or more basic isoform species of the antibody. In one embodiment, the basic isoform of vedolizumab can be identified. , and vedolizumab, which has a carboxyl-terminal lysine (C-Lys). , host cell impurities, or other impurities not associated with the antibody or antigen-binding portion thereof. are not considered to be "basic species" or "basic isoform species" of the body or its antigen-binding portion. I can't.

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

[0200] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody The terms Fab, Fab', F(ab')2, and Fv fragments, single chain antibodies, functional heavy chain antibodies (nanobodies), as well as at least Any portion of an antibody that has specificity for one desired epitope (e.g., An isolated complementarity determining region having sufficient framework sequence to specifically bind to Antigen-binding fragments are fragments of an antibody that are produced by recombinant techniques or by enzymatic or chemical synthesis of the antibody. It can be produced by selective cleavage.

[0201] As used herein, the term "humanized antibody" refers to an antibody that retains the antigen-binding properties of the parent antibody. Non-human antibodies (e.g., mouse) that retain or substantially retain the immunogenicity of the antibody in humans, but are less immunogenic in humans. It refers to an antibody derived from

[0202] Polypeptides such as antibodies produced by recombinant mammalian host cell lines using cell culture techniques. A peptide is a "recombinant polypeptide," a "protein," or, in the case of an antibody, a "recombinant antibody." The expressed protein is then either produced within the cell or harvested. In one embodiment, the recombinant antibody is an antibody, e.g., Recombinant anti-α4β7 antibodies, such as vedolizumab, that have binding specificity for the α4β7 complex. The methods and compositions described herein are for producing recombinant antibodies. With respect to the compositions and cell culture methods, unless otherwise specified, the term "antibody" refers to It is used interchangeably herein with the term "recombinant antibody."

[0203] The term "recombinant host cell" or "host cell" refers to a cell that is capable of expressing a recombinant polypeptide, e.g. For example, a recombinant host refers to a cell that has been genetically engineered to express an antibody. The cells contain an expression vector that includes a nucleic acid encoding the antibody heavy chain, light chain, or both. The term "host cell" refers not only to the particular subject cell but also to the progeny of such a cell. It is also to be understood that the present invention is intended to include the development of specific Such offspring are, in effect, the same as the parent cells, since certain modifications may occur in subsequent generations. Although these may not be identical to host cells, they are within the scope of the term "host cell" as used herein. Furthermore, unless otherwise specified, the term "cell" includes, for example, When host cells, mammalian cells, or mammalian host cells are used, the term refers to the cells. It should be understood that populations are intended to be included.

[0204] As used herein, the term "cell culture process" refers to the production of recombinant polypeptides. "Cell culture process" refers collectively to the cell culture steps involved in, for example, the production of antibodies. The term generally refers to the process of growing or maintaining cells under controlled conditions. The cell culture process can be carried out in vitro or ex vivo. In some embodiments, the cell culture process has both an expansion stage and a production stage. In the cerebellum, the expansion and production stages are separated by a transition or shift stage. "Culturing" refers to subjecting cells to cell culture under conditions suitable for proliferation or maintenance of the cells. In certain embodiments, the cell culture is a culture medium in which the recombinant polypeptide of interest is contacted with the medium. The generation or maintenance of a population of host cells capable of producing a peptide, e.g., an anti-α4β7 antibody. For example, the expression vector can be expressed in a suitable mammalian host cell, such as a mammalian host cell. Once integrated into Chinese hamster ovary (CHO) host cells, the host is able to express the relevant nucleotides. The cell culture may be cultured under conditions suitable for expression of the nucleotide coding sequence. It can also refer to a solution containing cells.

[0205] The terms "culture medium" and "cell culture medium" (plural, "culture medium") are used to grow or maintain cells. As will be appreciated by those skilled in the art, a nutrient source refers to a source of nutrition used to sustain a cell. It may contain components necessary for the proliferation and / or survival of cells, or may be involved in the proliferation and / or survival of cells. The enzymes may contain components that aid in survival. Vitamins, essential or non-essential amino acids (e.g., cysteine, Examples of medium components include nutrients (e.g., thymine and cystine), and trace elements (e.g., copper). Examples of culture media include growth media and production media.

[0206] The cell culture medium may also be used to, for example, increase the production of a recombinant polypeptide or to enhance cell survival. "Contains one or more components that aid in the cell culture process by improving the efficiency of the In one embodiment, the medium may be supplemented with "media supplements" or "supplements." In one embodiment, the supplements It is not formulated with the culture medium, e.g., it is not formulated with the production medium or the feed medium. Supplementary components are added in such a way that their combination with the feed solution or medium results in a final low concentration of the supplementary component. The supplement may be prepared in a concentrated form, such as a starting point, e.g., a stock or base. The supplement may include one or more components already present in the medium, and / or the supplement may be added to the medium. In one embodiment, a supplemental component is added to the feed solution. can be.

[0207] Supplementary ingredients can affect certain aspects of the cell culture, such as cell type, growth pattern, etc. Depending on the characteristics of the product (protein of interest) and the substrate, cell growth can be improved or recombinant potentiation can be achieved. Examples of substances that can be added as supplements include those that increase the production of polypeptides. The supplement contains one or more trace elements, one or more hormones, one or more amino acids, one or more vitamins, amine, one or more fatty acids, one or more non-ionic detergents, one or more nucleosides In some embodiments, the saccharide may be one or more saccharides, but is not limited to one or more saccharides. In the form, the supplement may comprise insulin, vegetable hydrolysates, and / or animal hydrolysates. The one or more supplemental components may be added at one or more stages of the cell culture process. may be added.

[0208] The cell culture medium and / or supplements may vary in accordance with the nature of the component(s), e.g., one or more origins. They may be supplied as known chemical compositions such as elements, inorganic salts or organic ions or sugars, or There is variability based on whether it is supplied as a mixture, e.g., a complex component such as a hydrolysate. is "defined" to a certain degree, in that its source may be known or unknown. " or "undefined." The presence of any complex components reduces the degree of definition.

[0209] The terms "growth stage," "growth stage," "expansion stage," and "expansion" are used interchangeably herein. The term "phase" refers to the period during which cultured host cells are rapidly dividing and increasing in number. During the expansion phase, cells are generally grown in a growth medium (or expansion medium) that maximizes cell proliferation. The growth stage can be, for example, in a batch culture, under conditions designed to grow the cells. It can precede the production phase in time, so that the two phases are separated by a transition phase. They may (or may not) be separated.

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

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

[0212] "Production medium" refers to a medium for producing a recombinant polypeptide, e.g., an antibody of interest, e.g., an anti-α4β7 antibody. It is a cell culture medium that promotes the production of

[0213] As used herein, a "feed solution" or "feed medium" refers to a growth medium or production medium. In order to improve or maintain the profile of the protein produced by the cells in the culture, the growth medium may be This refers to the cell culture medium that is added to the cell culture medium or production medium. A feed solution may be added to maintain a specific protein titer level produced by Feeding solutions are known in the art. In one embodiment, the feeding solution is It is supplemented with additional nutrients identified as beneficial to the production of proteins from animal cells.

[0214] Growth can also occur in a production medium, and production can occur in a growth medium, so that growth It will be appreciated that the culture medium and the production medium may be the same. However, in one embodiment In the case of a production medium, which promotes production of the polypeptide of interest more than if a growth medium were used, is selected.

[0215] As used herein, the term "batch culture" refers to any method for culturing cells. Components (including cells and all culture nutrients) are delivered to the culture vessel at the start of the culture process. This refers to the culture in which

[0216] As used herein, the term "fed-batch cell culture" refers to a method for culturing cells and medium that is initially cultured in a culture vessel. and additional supplemental components, e.g., nutrients, are continuously (via a feed solution) or Delivered in discrete increments, with periodic cell and / or product harvest prior to termination of the culture This refers to batch culture, with or without fermentation.

[0217] As used herein, the term "perfusion culture" refers to the initiation of the culture process of cells and A supplement is fed to the culture vessel and additional supplement(s) are continuously fed to the culture. refers to a culture in which product is continuously harvested from the medium during the culturing process.

[0218] As used herein, the term "vector" refers to a vector that contains another nucleic acid linked to it. Vector is intended to refer to a nucleic acid molecule capable of transporting a nucleic acid molecule. A "mid" is a circular double-stranded DNA segment to which additional DNA segments can be ligated. It refers to DNA. Another type of vector is a phage vector. Another type of vector are viral vectors, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., For example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors. Vectors (e.g., non-episomal mammalian vectors) are vectors that, upon introduction into a host cell, They can integrate into the genome of the host and thereby replicate together with the host genome. Vectors are capable of driving the expression of genes to which they are operably linked. These vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors of utility in recombinant DNA techniques are in the form of plasmids. There is often.

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

[0220] "Isolated nucleic acid" refers to a nucleic acid that is outside or separated from its normal context. Isolated means and includes non-naturally occurring, recombinant, or naturally occurring sequences. A nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules are distinguished from the nucleic acid molecule as it exists in natural cells. However, An isolated nucleic acid molecule means, for example, that the nucleic acid molecule is not present in a chromosomal location that is distinct from that of natural cells. This includes nucleic acid molecules contained in cells that normally express certain proteins.

[0221] As used herein, "purified" (or "isolated") refers to a substance that is free from other components. Substantially free of nucleic acid molecules (e.g., polynucleotides) or amino acid molecules (e.g., In some embodiments, a purified polynucleotide is A peptide or purified polypeptide is not isolated from other components present in the environment in which it is produced. For example, an isolated polypeptide is recovered or separated from the cell in which it was produced. Separated from other components (e.g., endoplasmic or cytoplasmic proteins and RNA) The isolated polynucleotide may be isolated from other nucleic acid components (e.g., histones) and / or upstream nucleic acid components. or separated from downstream nucleic acid sequences.

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

[0223] As used herein, the term "inoculation" or "seeding" refers to the initiation of a culture. Addition of cells to the medium or placing the cell culture in a bioreactor or separate vessel for incubation The process refers to the production of cells that have previously been grown in a separate bioreactor or vessel. Alternatively, the cells may be frozen and placed in a bioreactor or container. The term may refer to any number of cells, including a single cell. .

[0224] As used herein, the term "titer" refers to the amount of recombinant protein produced by cell culture. Titer refers to the total amount of expressed polypeptide, e.g., antibody, divided by a given amount of medium volume. is typically expressed as milligrams of antibody per milliliter or grams per liter of medium. The titer is expressed in units of gram. The titer is a comparison of the protein product obtained under different culture conditions. It can be expressed or evaluated in terms of a relative measurement, such as a percentage increase in titer. Cut.

[0225] As used herein, the term "recovered" refers to, for example, a compound secreted from a host cell. For expressed proteins, cell culture medium from cells and cell debris of the cell culture (the desired expression medium) (Recovering non-secreted proteins is a process that allows cells to The medium containing the protein of interest is called the "culture supernatant." Recovery methods include, but are not limited to, centrifugation, microfiltration, depth filtration, and filtration through absolute pore size membranes. This can be done using any of a number of techniques, including but not limited to: or subsequent steps to isolate the desired protein from the cells, including clarification. , is generally considered to be a purification step.

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

[0227] As used herein, in the context of recombinant polypeptides, e.g., antibodies, preparations, The term "upstream processing" refers to the production and synthesis of a polypeptide (e.g., an antibody) from a cell. Refers to an activity involving the collection (e.g., during cell culture) of a protein, e.g., an antibody of interest.

[0228] As used herein, the term "downstream processing" refers to the synthesis of a protein, e.g. This refers to one or more techniques used after upstream processing to purify an antibody. For example, Downstream processes include, for example, affinity chromatography including Protein A affinity chromatography. Concentration chromatography, size exclusion chromatography, anion or cation Ion exchange chromatography, hydrophobic interaction chromatography, etc. Purification of protein products using hybridization chromatography (HIC), or displacement chromatography include.

[0229] As used herein, the term "glycosylation profile" refers to the glycosylation of oligosaccharides. In the context of anti-α4β7 antibodies, the glycosylation profile of The IL describes the N-linked glycosylation in the Fc region of the antibody. In conjunction with this, the glycosylation profile is shown to be similar to that of SEQ ID NO: 13, but with a binding to asparagine 301 of the heavy chain. It refers to glycosylated species.

[0230] II. Methods and Compositions of the Invention Provided herein are methods for producing vedolizumab in mammalian, e.g., non-human, cell cultures. The present invention relates to methods and compositions for producing anti-α4β7 antibodies, such as mabs. Particularly, high anti-α4β7 antibody titer levels, i.e., 1 g / L, were achieved in mammalian cell cultures. to achieve, for example, 3-10 g / L, 4-8 g / L, or 5-7 g / L. Further provided herein are based on cell culture parameters that can be used to The authors report that anti-α4β7 antibodies, such as vedolizumab, achieve reduced levels of the basic isoform. and compositions for achieving low aggregation levels of anti-α4β7 antibodies such as vedolizumab; and methods and compositions for producing specific glial cell-specific antibodies, such as anti-α4β7 antibodies, such as vedolizumab. Further provided herein are methods and compositions for achieving the can form. reduced levels of sexual isoform species; low levels of high molecular weight aggregates; and / or is a composition comprising an anti-α4β7 antibody, such as vedolizumab, having a specific glycan type.

[0231] In particular, the methods and compositions disclosed herein include those comprising the anti-α4β7 antibody vedolizumab, or It can be used to produce antibodies having the antigen-binding region of vedolizumab. Lizumab is also sold under the trademark ENTYVIO® (Takeda Pharma Vedolizumab is a mutated human IgG1 A humanized antibody that contains the framework regions and antigen-binding CDRs from the mouse antibody Act-1. (as described in U.S. Pat. No. 7,147,851, which is incorporated herein by reference) (There are.)

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

[0233] α4β7 integrin is a distinct subset of memory T lymphocytes that preferentially traffic to the gastrointestinal tract. MAdCAM-1 is expressed on the surface of enterocytes and mediates T cell migration to enterocyte lymphoid tissues. Playing an important role in lymphocyte homing: α4β7 integrin and MAdCAM-1 The interaction of these two factors is important in mucosal inflammation, such as the chronic inflammation characteristic of ulcerative colitis and Crohn's disease. Vedolizumab is believed to be a major cause of inflammatory diseases, including Crohn's disease and ulcerative colitis. Enteropathy, HIV, pouchitis including chronic pouchitis, fistulizing Crohn's disease, graft-versus-host disease, and abdominal It may be used to treat cavity diseases.

[0234] The heavy chain variable region of vedolizumab is provided in SEQ ID NO: 1, and the light chain variable region of vedolizumab is Vedolizumab has a CDR1 as set forth in SEQ ID NO: 2, a CDR2 as set forth in SEQ ID NO: 3, a CDR3 as set forth in SEQ ID NO: 4, a CDR4 as set forth in SEQ ID NO: 5, and a CDR5 as set forth in SEQ ID NO: 6. The heavy chain variable region comprises a CDR2 as set forth in SEQ ID NO:3, and a CDR3 as set forth in SEQ ID NO:4. Lizumab has CDR1 set forth in SEQ ID NO: 6, CDR2 set forth in SEQ ID NO: 7, and CDR3 set forth in SEQ ID NO: The nucleic acid sequence encoding the light chain variable region is 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 described in U.S. Patent Publication No. 2010 / 003366. No. 297699, the entire contents of which are incorporated herein by reference. The sequence of tegafur is disclosed in U.S. Patent Publication 2014 / 0341885 and U.S. Patent Publication 2014 / 0341885. No. 0377251, the entire contents of each of which are expressly incorporated herein by reference. are incorporated into the system.

[0235] The methods and compositions provided herein include anti-α4β7 antibodies, particularly vedolizumab or vedolizumab. An antibody having a binding region, i.e., a CDR or variable region, of dolizumab, or a mammalian cell The present invention is useful for producing antigen-binding fragments of anti-α4β7 antibodies in cells.

[0236] The methods and compositions disclosed herein relate to mammalian cell culture processes. Human cells are primarily endowed with the ability to produce properly folded and assembled heterologous proteins, and Due to their ability to make post-translational modifications, such as modifications made by human cells, they are of clinical relevance. It has become a major system for the production of mammalian proteins for human therapeutic applications, Chinese hamster ovary (CHO) cells, as well as, for example, mouse bone marrow Neoplasms (NS0), baby hamster kidney (BHK), human fetal kidney (HEK-293), Cell lines derived from a variety of other mammalian sources, including human retinal cells, have been used to develop therapeutic antibody-containing vaccines. Among these, CHO cells have been approved by regulatory authorities for the manufacture of biopharmaceuticals. is the most commonly used industrial host widely used for the production of heterologous proteins. Therefore, dihydrofolate reductase negative (DHFR-) or glutathione Large-scale production of antibodies in CHO cells, including GS-negative CHO cells. Methods for production are well known in the art (e.g., Trill et al., urr.Opin.Biotechnol.6(5):553-60(1995), Bi rch 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 Examples include GS-CHO, CHO-K1 DUX B11 and DP-12 CHO cells. Suitable for use in the compositions and methods provided herein include, but are not limited to, CHO cells are described in the following documents: U.S. Pat. No. 4,766,075; , No. 853,330; No. 5,185,259; No. 5,122,464; No. 5, No. 591,639; No. 5,879,936; Lubiniecki et al., in Advances in Animal Cell Biology and T. technology for Bioprocesses,Spier et al., eds. (1989), pp. 442-451. Derivatives include, for example, CHO / -DHFR (Urlaub and Chasin. Proc.Natl.Acad.Sci.USA,77:4216 (1980)), C HO-K1 DUX B11(Simonsen and Levinson,Proc. .Natl.Acad.Sci.USA 80:2495-2499(1983);Ur Laub and Chasin, supra), and DP-12 CHO cells (March 1989 EP 307,247, published on the 15th of this month, or U.S. Patent No. 5,721,121 Some examples include:

[0237] Other examples of suitable mammalian cell lines include SV40 transformed monkey kidney C VI strain (COS-7, ATCC (商標) CRL1651); human embryonic kidney line 293S(G raham et al., J. Gen. Virolo., 36:59 (1977)); Baby hamster kidney cells (BHK, ATCC (商標) CCL 10); Mouse Sertoli Cell (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 cervix Cancer cells (HELA, ATCC (商標) CCL 2); canine kidney cells (MDCK, ATC C (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; 29 3T cells (Pear, WS, et al., Proc. Natl. Acad. Sci. USA, 90:8392-8396(1993); NS0 cells (Sato t al.Tissue Culture Association,24:1223( 1988);SP2 / 0(Sato et al.J.Exp.Med.,165:1 761 (1987)); and TR-1 cells (Mather et al., Annals NYAcad.Sci.,383:44(1982)), and hybridoma cells Examples include strains.

[0238] Many host cell types are capable of producing the encoded recombinant polypeptide, including The product encoded by a particular nucleic acid produced in one host cell can be expressed by that nucleic acid in another host cell. The difference may be in one or more biochemical properties. Examples of biochemical properties include basic properties such as primary, secondary, or tertiary structure. Protein structure, signal peptide processing, glycosylation, N-terminal acetylation, and Certain differences can be attributed to post-translational modifications such as silylations, lipidations, or phosphorylations. May depend on enzymatic machinery and / or medium or growth conditions. Recombinant therapeutic antibodies In the case of may affect one or more antibody characteristics, such as activity, solubility, or storage stability. do.

[0239] In some embodiments, a product having a difference from a reference product can be purified, e.g., further processed in a downstream process. In other embodiments, the difference between the reference product and the product may be reduced or eliminated by the process technique. The products that are produced can be reduced or eliminated by controlling the enzymatic machinery of the cell, e.g., by upstream process technology. In some embodiments, controlling the enzymatic machinery of a cell can include: Mutation of a cell to recombinantly modify its genetic background, e.g., mutating an enzyme or In some embodiments, regulating the enzymatic machinery of a cell includes modifying the expression of By providing a specific culture medium or adding one or more supplements, In some embodiments, the enzyme machinery of a cell is controlled. This 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. 5,313,633). See U.S. Pat. No. 7,402,410 and U.S. Patent Application Publication No. 20070122404. ). These publications describe specific properties of antibodies, such as binding affinity, effector functions, Their biochemical properties, such as charge profile, molecular weight, and glycosylation pattern, have been described. The antibodies showed certain properties when cultured with NS0 cells, but not when cultured with CHO cells. The properties of recombinant proteins, e.g. antibodies, have also been shown to be different in different variants of CHO cells. This can change when changing, for example, from DHFR- cells to GS- cells. Described herein is the use of this in, for example, the production of recombinant proteins, e.g., antibodies. These fluctuations are controlled, and GS-CHO cells (also referred to simply as "GS-CHO" cells in this specification) In particular, the present invention provides a method for limiting or minimizing the change in characteristics when expressing an antibody, e.g., vedolizumab, in a subject In certain embodiments, described herein are methods and media compositions for miniaturizing Methods and methods for producing anti-α4β7 antibodies, such as vedolizumab, in GS-CHO cells and a composition.

[0241] Potential changes when producing anti-α4β7 antibodies such as vedolizumab in cell culture Examples of properties that can be considered include their charge profile, glycosylation profile, and high molecular weight. (HMW) impurity species. Temperature, pH, shear stress, dissolved oxygen, medium composition, etc. Culture conditions may contribute to the change in properties. The charge of the enzyme is determined by the C-terminal lysine, the N-terminal Variation in the presence or absence of pyroglutamic acid or sialic acid at the terminal end, and / or deacetylation Glycosylation profiles may vary due to variations in amide or oxidation. The presence or absence of alkoxyl or terminal galactose, the processing of high mannose species, etc. (Hossler et al. (2009) Glycobi (See Biol. 19:936-949.) Media supplements may prevent such changes. Can be controlled.

[0242] In some embodiments, these include charge variation, glycan variation, and aggregate content. The properties of anti-α4β7 antibodies produced in cell culture can be influenced by, but are not limited to, the medium, e.g. Sugars (e.g., galactose), metal cofactors (e.g., manganese) in the production medium, and and / or by adjusting the amount of nucleosides (e.g., uridine). In some embodiments, the methods include, but are not limited to, charge variation, glycan variation, and aggregate content. The properties of the anti-α4β7 antibody produced in cell culture, which are not defined, may vary depending on the medium, e.g., the production It can be controlled by adjusting the amount of lysine and arginine in the stage medium. In embodiments, the following include, but are not limited to, charge variation, glycan variation, and aggregate content: The properties of the anti-α4β7 antibody produced in cell culture may vary depending on the medium, e.g., the production medium. In addition, temperature shifts during production can be controlled by adjusting the amount of zinc used in the Temperature shifts may prove beneficial for antibody production. It is known in the art that there are Ytotechnology 23:47-54), provided herein are cell culture A method based on maintaining the culture temperature, i.e., the culture conditions do not include substantial shifts; For example, a method that does not include a shift of more than 1 degree above or below 37 degrees Celsius.

[0243] A. Zinc supplementation In some embodiments, provided herein are methods for treating cancer in which zinc is supplemented during the production stage. Humanized anti-α4β7 antibodies, e.g., vedolizumab or its antigen, in CHO cell culture In some embodiments, zinc is C As a medium supplement to control charge fluctuations of anti-α4β7 antibodies in HO cell cultures In another embodiment, zinc is used in the manufacture of anti-α4β7 antibodies produced in CHO cell culture. As a medium supplement to control the level of high molecular weight (HMW) aggregates in the preparation of Metal ions are used as hydrochlorides, sulfates, nitrates, bromides, acetates, stearates, etc. The medium supplement can be in the form of salt, citrate, or phosphate. It may be provided to the culture in concentrated form along with the feed during the fermentation or production phase. The components can be provided in concentrated form in a feed solution that is also a concentrated replenishment component. In embodiments, the supplement may be diluted more than once at each stage of the supplement preparation.

[0244] In some embodiments, metal ions, such as zinc, are added to the production culture. The presence of zinc for the production of anti-α4β7 antibodies such as vedolizumab is essential for the basic agonist activity of the antibody. The levels of isoforms were reduced (compared to the control process, which was the same process except for the addition of zinc). In some embodiments, zinc may be added to the production culture more than once. In one embodiment, zinc can be added as a supplement to the starting production medium of the production stage culture. In one embodiment, zinc is added to the production stage culture, for example, as a biosynthetic agent after the start date. Zinc is added to the production culture directly or in a feed solution. In one embodiment, zinc is added to the starting production medium. Supplements to the production medium after the start date are added, e.g., zinc is added at the start of the production stage. In some embodiments, zinc is added to a feed solution that is added to the production stage culture. After the start of the incubation, supplements are added multiple times. For example, zinc is added twice daily. Added daily, 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 start date of the production stage culture is Not added on the first, second, third, fourth, fifth, or sixth day of feeding, but daily thereafter is added every 2 days. In one embodiment, zinc is added as a supplement to the starting medium and during the production phase. In another embodiment, zinc is added as a daily supplement to the starting medium. minutes and as a daily supplement to the production-stage medium from day 4 of the production-stage culture. Zinc can be supplemented 1 day prior to harvest, 2 days prior to harvest, or 3 days prior to harvest. In one embodiment, zinc is added as a supplement to the starter medium and after 4 days of the production stage culture. It is added as a daily supplement to the production stage medium from day 1 until one day before harvest.

[0245] In certain embodiments, zinc is present at about 16% or less of the human A method for producing a composition having a basic isoform of a humanized anti-α4β7 antibody, comprising: The host cell, e.g., vedolizumab, is grown in a mammalian host cell, e.g., GS-C, in a production medium that contains zinc. In certain embodiments, the production medium may include a supplement containing zinc. Adding a supplementary supply reduces the basic isoform of the humanized anti-α4β7 antibody by approximately 14% or less. In certain embodiments, the supplement for the production medium includes zinc. The composition comprises about 13% or less of the basic isoform of a humanized anti-α4β7 antibody. In certain embodiments, including zinc in the supplement for the production medium provides about 1 The present invention provides a composition comprising 2% or less of the basic isoform of a humanized anti-α4β7 antibody. In an embodiment, including zinc in the supplement for the production medium provides about 11% or less humanized Compositions comprising the basic isoform of an anti-α4β7 antibody are provided. In some embodiments, The levels of basic isoforms were measured on day 14 of cell culture, i.e., after the subculture of cell cultures. In another embodiment, the levels of the basic isoform can be measured after 14 days of inoculation. The activity can be measured on day 15 of cell culture.

[0246] In certain embodiments, zinc is present in greater than or equal to about 70% of human zinc (as determined by CEX). A method for producing a composition having a major isoform of a humanized anti-α4β7 antibody, comprising: , e.g., vedolizumab, is grown in mammalian host cells, e.g., GS-CH, in a production medium containing zinc. In certain embodiments, the production medium is supplemented with zinc. Adding the ingredient feed resulted in approximately 71% or more of the major isoform of the humanized anti-α4β7 antibody. In certain embodiments, the production medium is supplemented with a supplemental feed comprising zinc. The addition of the antibody to the composition provides a composition comprising at least about 72% of the major isoform of the humanized anti-α4β7 antibody. In certain embodiments, adding a supplemental feed comprising zinc to the production medium The present invention provides a composition comprising about 73% or more of the major isoform of a humanized anti-α4β7 antibody. In certain embodiments, adding a supplemental feed containing zinc to the production medium reduces the production of zinc by about 74% or more. The present invention provides a composition comprising the major isoform of the above humanized anti-α4β7 antibody. In an embodiment, the level of the major isoform can be measured on day 14 of cell culture. In another embodiment, the level of the major isoform is measured on day 15 of cell culture. It is possible.

[0247] In another embodiment, zinc supplementation is used to reduce HMW contamination in preparations containing anti-α4β7 antibodies. The level of contamination can be limited. In some embodiments, the level is about 10 to 200 μM. Add zinc to the culture medium at a concentration of about 50-150 μM or about 100-130 μM. and by reducing the level of HMW aggregates (as determined by SEC) to <5%. , <4%, <3%, <2.5%, <2%, <1.5%, or <1% can.

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

[0249] Zinc ions can be supplemented to the medium, e.g., the production stage medium, at a final concentration of 10 to 100. 200μM, 10~100μM, 15~90μM, 20~80μM, 10~80μM, 1 0~70μM, approx. 14~55μM, approx. 10~60μM, approx. 10~30μM, approx. 10~20 μM, about 14 μM, about 50 μM, about 55 μM, about 57 μM, or about 15 μM. As mentioned above, zinc ions can be added multiple times. In one embodiment, zinc is added during the production stage. The resulting production medium is added at approximately 2-60 μM, 5-57 μM M, 5~50μM, 5~40μM, 8~30μM, 10~20μM, 12~15μM, or about 14 μM zinc. In one embodiment, the cumulative zinc concentration in the production medium is , which is accounted for by supplementation up to the time of harvest, is approximately 15.5 μM, and each supplement component was added to the medium Zinc is added at about 1 to about 4 μM. In one embodiment, zinc is added at about 50 to 150 μM of the production medium. μM, 75-150 μM, 100-150 μM, 80-130 μM, or 100-13 0 μM zinc is added to the production medium of the production stage culture. In embodiments, zinc is present in the production medium at about 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, M, 60μM, 70μM, 80μM, 90μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 190μ or 200 µM zinc concentration is added to the production medium of the production stage cultures. In some cases, too much zinc in the starting culture can reduce cell viability and / or may reduce antibody titers.

[0250] In some embodiments, zinc is added to the culture medium (e.g., production stage culture medium) at a concentration of 10 to 1 The addition may be made over a period of 6 days, e.g., 10 to 17 days, 10 to 15 days, or 12 to 14 days. In some embodiments, zinc supplements are added to the cell culture, e.g., as part of a feed solution. For example, zinc may be added incrementally on days 0, 1, 2, 3, 4, 5th day, 6th day, 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day, or on day 14. In some embodiments, zinc can be added to the medium daily. In some embodiments, zinc can be added daily or every other day. Every other day additions can begin when the cells reach production stage. In some embodiments, zinc is administered daily starting on day 4, 5, or 6 of cell culture. Or, zinc can be added to the production medium every other day. In one embodiment, zinc is added at about day 4. In one embodiment, zinc can be added daily from about day 4 to about day 10. It can be added daily for up to four days. In one embodiment, zinc is added at a concentration of about 10-80 μM. or at a concentration of about 50-150 µM to replenish the production medium from the supply during the production phase. In one embodiment, zinc is added as a supplement after the start of the production medium. 50μM, 2~60μM, 5~57μM, 5~50μM, 5~40μM, 8~30μM, Production stages to have zinc concentrations of 10-20 µM, 12-15 µM, or approximately 14 µM. It is added to supplement the starting medium of the culture and also to supplement the production medium after the starting date. (For example, days 2-10, 2-8, 2-6, 3-6 of culture) or from day 4) for each addition to the production-stage cultures, 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 In another embodiment, zinc is added in multiple doses of 1.9 μM or 1.9 μM. Approximately 2-50 μM, 5-40 μM, 8-30 μM, 10-20 μM, 12-15 μM, or is added to supplement the starter medium of the production culture to have a zinc concentration of about 14 μM; and 0.1 for each addition to the production-stage medium from day 4 of the production-stage culture ~10μM, 0.5~5μM, 0.75~4μM, 0.9~3μM, 1.0~2.7μM 1.4 μM, or 1.9 μM each day. In some embodiments, the production medium has a total zinc concentration of about 10-20 μM (e.g., 15-17 μM). A feed solution is added to the production medium such that zinc is added to the production medium to have a In some embodiments, the zinc supplement described herein is added to a CHO cell culture medium, e.g. For example, the total amount of the β-glutamyltransferase that is added to the culture medium provided in International Patent Publication WO98 / 08934A1 is The contents of which are incorporated herein by reference. In some embodiments, In some embodiments, the zinc supplement described herein is added to the CD-CHO medium. The zinc supplement listed in is CD-CHO AGT (catalog number 12490-001(I It is added to the NIH (Non-vitrogen, Carlsbad, CA, USA).

[0251] In one embodiment, zinc is present in a feed solution of about 90-120 μM, about 95-120 μM, about 1 00-120 μM, about 105-120 μM, about 110-120 μM, or about 117 μM Such supplemental feed solution is then added to replenish the feed solution to have a concentration of Supplementary ingredients can be added to the production medium.

[0252] In some embodiments, provided herein are anti-α4β7 antibodies or antigens thereof. A host cell (or a population of host cells) expressing the binding moiety and a zinc-containing or supplemented In another embodiment, provided herein is a cell culture comprising a production medium in which The anti-α4β7 antibody or antigen-binding portion thereof is produced in a production medium containing or supplemented with zinc. It is a cell culture obtained by culturing expressing host cells.

[0253] The aforementioned cell cultures can incorporate any of the embodiments described herein. For example, in some embodiments, the host cell is a CHO cell, e.g., a GS-CHO cell. , or DHFR - In some embodiments, the host cell is a CHO cell. The present invention relates to an antibody or an 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 the CDR1 domain set forth in SEQ ID NO:2, Contains the CDR2 domain set forth in SEQ ID NO:3 and the CDR3 domain set forth in SEQ ID NO:4 A heavy chain variable region, a CDR1 domain as set forth in SEQ ID NO:6, a CDR2 domain as set forth in SEQ ID NO:7 1. An antibody or its derivative comprising a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO:8. In some embodiments, the host cell expresses vedolizumab, or In some embodiments, the host cell expresses an antigen-binding portion thereof. (encoding the light chain variable region of an anti-α4β7 antibody) and the nucleic acid set forth in SEQ ID NO: 10 ( In some embodiments, the host The cells were cultured using a nucleic acid sequence as set forth in SEQ ID NO: 11 (encoding the light chain of vedolizumab) and a nucleic acid sequence as set forth in SEQ ID NO: 2, and the nucleic acid according to claim 2 (encoding the heavy chain of vedolizumab).

[0254] In some embodiments, the cell culture is at about 10-100 μM, 10-100 μM, 15~90μM, approx. 20~80μM, approx. 10~80μM, approx. 10~70μM, approx. 14~5 5μM, about 10-60μM, about 10-30μM, about 10-20μM, about 14μM, about 50 In some embodiments, the zinc concentration is about 5 μM, about 55 μM, about 57 μM, or about 15 μM. In terms of morphology, cell cultures were approximately 2-60 μM, 5-57 μM, 5-50 μM, and 5-40 μM , 8-30 μM, 10-20 μM, 12-15 μM, or about 14 μM In some embodiments, the cell culture is treated with about 5-45 μM, 50-150 μM, 75 ~150μM, 100~150μM, 80~130μM, or 100~120μM In some embodiments, the cell culture contains about 1-10 μM zinc, 10 Contains zinc at concentrations of ~30μM, 30-50μM, 50-70μM or 70-90μM In some embodiments, the cell culture is treated with about 1-30 μM, 10-40 μM, 20-50 μM, Contains zinc at concentrations of 0 μM, 30-60 μM, 40-70 μM, or 60-90 μM. In some embodiments, the cell culture is treated with about 1-50 μM, 20-60 μM, 30-70 μM, In some embodiments, the composition includes a zinc concentration of 100-250 μM, 40-80 μM, or 50-100 μM. In the cell culture, the concentration of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM , 70μM, 80μM, 90μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 190μM, or 2 Contains zinc at a concentration of 100 μM.

[0255] In some embodiments, provided herein are anti-α4β7 antibodies or antigens thereof. GS-CHO host cells expressing the binding moiety were incubated with 50-150 μM, 100-120 μM, Or, cultivate in a production medium containing or supplemented with zinc at a concentration of 100-120 μM. In some embodiments, the antibody is a cell culture that can be obtained by vedolizumab, or an antigen-binding portion thereof.

[0256] In some embodiments, the cells of the cell culture are grown in medium lacking zinc or supplemented with zinc. Basic (as determined by CEX) compared to equivalent cell cultures containing no medium The present invention relates to an anti-α4β7 antibody having a reduced level of the isoform, or an antigen-binding portion thereof. In some embodiments, the expressed antibodies have about 16% or less basic isoforms. In some embodiments, the expressed antibodies contain no more than about 15% basic isoforms. In some embodiments, the expressed antibody contains no more than about 14% basic isoforms. In some embodiments, the expressed antibody contains no more than about 13% basic isoforms. In some embodiments, the expressed antibody contains no more than about 12% basic isomers. In some embodiments, the expressed antibody contains about 11% or less basic isoforms. Contains isoforms.

[0257] In some embodiments, provided herein are methods for producing monoclonal antibodies. (i) a host cell expressing an anti-α4β7 antibody or an antigen-binding portion thereof, and The cell cultures provided herein, which contain a production medium containing or supplemented with lead, are used to treat host cells that are susceptible to anti-α (ii) culturing for a period of time sufficient to express the 4β7 antibody or an antigen-binding portion thereof; and recovering the anti-α4β7 antibody or antigen-binding portion thereof from the cell culture. In some embodiments, the anti-α4β7 antibody recovered from the cell culture or The population of antigen-binding portions of the IgG1A antigen-binding domain is expressed in a zinc-deficient medium or in a medium not supplemented with zinc. As compared to the population of anti-α4β7 antibodies or antigen-binding portions thereof recovered from the culture (C Decreased levels of basic isoforms and / or increased levels of In some embodiments, the protein comprises a major isoform at a level that is greater than or equal to 100% of the total protein. The population of anti-α4β7 antibodies, or antigen-binding portions thereof, was incubated in a zinc-deficient or zinc-containing medium. Anti-α4β7 antibodies or their antigens recovered from equivalent cell cultures containing unsupplemented medium. Reduced levels of aggregates (as determined by SEC) compared to the bound moiety population and / or contain increased levels of monomers. In some embodiments, the cell culture comprises: 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 embodiments of the present invention, the cell culture comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, The cells are cultured for 15, 16, 17, 18, 19, or 20 days. Obtained or obtainable by the above-mentioned method provided in the specification It is an anti-α4β7 antibody.

[0258] In each of the embodiments described herein, the cell culture medium may, in some embodiments, contain sugars, nucleases, The enzyme may be further supplemented with cleosides, and / or metal cofactors. For example, cell culture The medium may be further supplemented with uridine, manganese, and zinc. 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 sugars, nucleosides, and / or Humanized anti-α4β7 antibody was produced in CHO cell cultures supplemented with α- or β-cofactors during the production phase. and methods and compositions for producing vedolizumab or an antigen-binding portion thereof. do.

[0260] In some embodiments, the glycosylation profile of an anti-α4β7 antibody is expressed in CHO cell culture. The medium supplement for controlling the growth of the cells contains sugar. For example, the sugar in the supplement is a glucose. It may be cose, fucose, or galactose.

[0261] In some embodiments, the glycosylation profile of an anti-α4β7 antibody is expressed in CHO cell culture. The medium supplement for controlling the file contains nucleosides. For example, Nucleosides include adenosine, uridine, cytidine, guanosine, thymidine, and / or can be inosine.

[0262] In some embodiments, the glycosylation profile of an anti-α4β7 antibody is expressed in CHO cell culture. The medium supplement for controlling the growth of the cells includes a metal cofactor. For example, the gold in the supplement The cofactor may be magnesium, manganese, iron, or copper.

[0263] In some embodiments, the glycosylation profile of an anti-α4β7 antibody is expressed in CHO cell culture. The medium supplement for controlling the file includes sugars and nucleosides. In this study, we controlled the glycosylation profile of an anti-α4β7 antibody in CHO cell culture. In some embodiments, the medium supplement for CHO cell culture includes sugars and metal cofactors. The medium supplement component for controlling the glycosylation profile of anti-α4β7 antibody in culture was , sugars, nucleosides, and metal cofactors.

[0264] In some embodiments, provided herein are oligosaccharides that contain galactose, uridine, and Humanized anti-α4β7 antibodies, e.g., in CHO cell cultures supplemented with manganese during the production phase For example, methods and compositions for producing vedolizumab or an antigen-binding portion thereof. 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 implementations, the different media supplement components are combined before being added to the cell culture. In the form of supplemental components for controlling the glycosylation profile of anti-α4β7 antibodies are added multiple times. For example, they may be added after the start date of the production stage culture. or is not added on the first, second, third, fourth, fifth, or sixth day of the production stage culture and then added every day or every two days thereafter. Components for controlling the production stage medium are added in daily supplements. In this study, the components for controlling glycosylation profile were added from day 4 of the production culture. The production stage medium is supplemented with daily supplements.

[0265] In some embodiments, a media supplement for controlling glycosylation is added during the expansion phase. In another embodiment, the antibody is provided to a CHO cell culture to produce anti-α4β7 antibodies. It is added to the production stage. In some embodiments, the method for controlling glycosylation The medium supplement components are diluted to 20-400 times, 25-300 times, or 30-250 times the final concentration in the medium. Available in 40-120x, 50x, 60x, 100x, or 200x concentrates In some embodiments, the amount of medium replenished is determined by ignoring consumption by the cells. may metabolize some of the supplemented ingredients to other chemical forms.

[0266] In one embodiment, a metal cofactor component, such as manganese, is added to a medium that contains a metal ion, such as zinc. In some embodiments, a metal cofactor, such as mannitol, is provided to the cell culture. The cancer concentrate was 10,000 to 50,000 times its final concentration in the medium, and 20,000-40,000 times the final concentration, or approximately 30,000 times its final concentration in the medium It may be 0 times.

[0267] In some embodiments, manganese is 0.1-100 μM, 0.5-50 μM, 1. 0~25μM, 2.0~15μM, 3~10μM, 1~50μM, 1~100μM, 20 ~50μM, 30~60μM, 40~70μM, 50~80μM, 70~100μM, 2 At concentrations of 0-70 μM, 30-80 μM, 40-90 μM, or 50-100 μM, e.g. It may be present in the medium, e.g., the production stage medium, or may be added to supplement the medium. In one embodiment, the concentration of manganese in the production stage medium is about 5.15 μM. Therefore, the production phase medium was replenished on a schedule with approximately 5.15 μM manganese. In some embodiments, an average manganese concentration of about 1 μM can be achieved. , about 5μM, about 10μM, about 20μM, about 30μM, about 40μM, about 50μM, about 60μ M, about 70 μM, about 80 μM, about 90 μM, or about 100 μM, for example, during the production stage. The supplement may be present in the medium, which is a culture medium, or may be added to supplement the medium.

[0268] In one embodiment, manganese is added at 0.1-10 μM, 0.2-1.5 μM, 0.2-5μM, 0.25-2μM, 0.3-1.2μM, 0.3μM-0.8μM, or approximately 0.5 μM, or 0.56 μM increments to replenish the production phase medium after the start date. In one embodiment, manganese is added in multiple doses as a supplemental component to the syrup. Add 2-1.5μM each time as a supplement component to replenish the production medium after the start date. In one embodiment, manganese is added at about 0.31-1.2 μM for each addition. It is added to the production medium as a supplement multiple times after the start date to replenish the production medium. In some embodiments, the manganese supplement is administered daily or biweekly, beginning on day 4 of the production stage culture. In some embodiments, supplemental ingredients are not added on the day of harvest.

[0269] In one embodiment, manganese is present in a feed medium having a manganese concentration of 0.02 mM to 0.2 mM. M, 0.03mM~0.15mM, 0.03mM~0.10mM, 0.03mM~0.0 5mM, 0.03mM~0.04mM, approx. 0.03mM, approx. 0.04mM, approx. 0.05mM M, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.1 mM, or about 0.1 It is added as a supplement to the feed medium to a final concentration of 4 mM. Manganese should be added so that the final concentration of manganese in the supply medium is 0.1 to 100 μM. In one embodiment, manganese is added as a supplement to the feed medium. Manganese was added as a supplement to the feeding medium to a final concentration of 39 μM. In one embodiment, the manganese-supplemented feed medium is added after the start of the production stage culture (e.g., , Days 2-10, 2-8, 2-6, 3-6 of production stage culture , or from day 4), added to the production medium (e.g., multiple times, e.g., daily or biweekly) In one embodiment, the manganese-supplemented feed medium is added from day 4 of the production stage culture. It is added to the production medium.

[0270] Uridine may be added for more than one reason: To support cell proliferation Uridine can be added to nutritional supplements along with other nucleosides to inhibit anti-alpha It can also be added as a supplement to control the glycosylation profile of the 4β7 antibody. In some embodiments, uridine is about 0.1 to 20 mM, about 0.9 to 3.0 mM. 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. 1~1.5mM, approx. 1~5mM, approx. 1~7mM, approx. 1~6mM, approx. 1~5mM, approx. 1~4 mM, approx. 2~4mM, approx. 2~5mM, approx. 2~3mM, approx. 1mM~10mM, approx. 10mM~ 15mM, approx. 10mM-20mM, approx. 10mM-30mM, approx. 1mM-40mM, approx. In a medium, for example a production medium, at a concentration of about 10 mM to 50 mM, or about 10 mM to 30 mM In some embodiments, the IL-10 receptor may be present in the medium or may be added to supplement the medium. , uridine was about 0.9 mM, 1.0 mM, about 2 mM, about 1.5 mM, about 2.0 mM, about 2.7mM, approx. 2.5mM, approx. 2.7mM, approx. 2.8mM, approx. 5mM, approx. 6mM, approx. 7m M, approx. 8mM, approx. 9mM, approx. 10mM, approx. 11mM, approx. 12mM, approx. 13mM, approx. 14m M, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM may be present in a medium, e.g., a production medium, or to supplement the medium, at a concentration of In some embodiments, the amounts listed in the production medium may be added to the basal medium or Describes the amount of supplement provided and consumed, metabolized, or produced by cells In some embodiments, the amount listed for the production medium is not taken into account until the harvest date. In one embodiment, uridine is added to the production stage medium in a cumulative amount as the sum of all the additions of uridine. The concentration is 0.1-20mM, 0.5-12mM, 1-8mM, 1.5-5mM, 1.6-4. It can be supplemented to 8 mM or to about 2.4 mM.

[0271] In one embodiment, uridine is added at 25-1000 μM, 75-750 μM, 55~620μM, 100~600μM, 150~450μM, 100~600μM, 1 To supplement the production medium, add 70 to 630 μM or approximately 250 or approximately 300 μM. In some embodiments, these supplements are added multiple times after the start date. Uridine supplements were added daily or every two days from the fourth day of production culture, and In one embodiment, the uridine supplement does not need to be added on the day of withdrawal. For example, supplements containing uridine may be added at 10 to 500 times the final concentration in the medium. 20-400 times, 25-300 times, 40-250 times, about 50 times, about 60 times the final concentration of About 100 times, or about 200 times.

[0272] In one embodiment, uridine is provided in a feed medium having a concentration of about 1-40 mM uridine, 15- 25mM, 15~100mM, 20~90mM, 15~70mM, 15~50mM, 15 ~30mM, approx. 18mM, approx. 19mM, approx. 19.3mM, approx. 20mM, approx. 33mM, approx. 5 It is added as a supplement to the feed medium to a final concentration of about 0 mM or about 66 mM. In one embodiment, the uridine-supplemented feed medium is added after the start of the production stage culture (e.g., For example, days 2 to 10, days 2 to 8, days 2 to 6, days 3 to 6 of production stage culture The 5'-5'-10 ... In certain embodiments, the uridine-supplemented feed medium is added to the production stage culture for 4-8 days. It is added to the production medium from the day.

[0273] In one embodiment, the sugar, e.g., galactose, containing supplemental component is an anti-α4β7 antibody. 10-500x the final concentration in the medium to control the glycosylation profile, 20-400x, 25-300x, 30-250x, 40-120x, and approximately 5x the final concentration of 0x, about 60x, about 100x, or about 200x. Intactose is available in concentrations of 0.1-100 mM, 1-75 mM, 2.5-50 mM, 3-20 mM, and 5 ~35mM, about 8~25mM, 0.1~10mM, 0.1~20mM, 0.1~30mM , 1~10mM, 1~20mM, 1~30mM, 1~40mM, 1~50mM, 1~60 mM, 1~70mM, 1~80mM, 1~90mM, 1~100mM, 20~40mM, 40~60mM, 60~80mM, 80~100mM, 20~50mM, 30~60mM , 40~70mM, 50~80mM, 70~100mM, 20~70mM, 30~80m M, 40-90 mM, 50-100 mM, or 50-150 mM concentration, e.g. It may be present in the medium, be a stage medium, or may be added to supplement the medium. In some embodiments, galactose is about 0.1 mM, about 0.2 mM, about 0.3 mM, approx. 0.4mM, approx. 0.5mM, approx. 0.6mM, approx. 0.7mM, approx. 0.8mM, approx. 0.9m M, approx. 1mM, approx. 2mM, approx. 3mM, approx. 5mM, approx. 6mM, approx. 7mM, approx. 8mM, approx. 9m M, approx. 10μM, approx. 12.5mM, approx. 12mM, approx. 12.8mM, approx. 13mM, approx. 15m M, approx. 20mM, approx. 30mM, approx. 40mM, approx. 50mM, approx. 60mM, approx. 70mM, approx. 8 In a medium, e.g., a production stage medium, at a concentration of about 0 mM, about 90 mM, or about 100 mM. In one embodiment, galactose may be present or may be added to supplement the medium. The concentrations of each addition were approximately 0.1-10 mM, 0.2-7.5 mM, 0.5-5 mM, and 0. 4~2.8mM, 0.5~3.5mM, 0.7~2.9mM, 0.75~2.5mM, or approximately 1.2 mM or 1.4 mM at a time after the start date to replenish the production medium. In some embodiments, these galactose supplements are added multiple times. The nutrients were added daily or every two days from the fourth day of the production phase culture, and then added on the harvest day. It does not have to be added.

[0274] In one embodiment, galactose is present in a supply medium having a concentration of 50 to 150 mM galactose. M, 85mM~500mM, 90mM~400mM, 90mM~300mM, 90mM~ 200mM, 90mM to 100mM, about 95mM, about 96mM, about 97, about 100mM, to the feed medium to a final concentration of about 165 mM, about 250 mM, or about 330 mM. In one embodiment, the galactose-supplemented feed medium is added to the After the start of production-stage culture (e.g., days 2 to 10, days 2 to 8, and days 2 of production-stage culture), From day 1 to day 6, day 3 to day 6, or day 4) is added to the production medium (e.g. or multiple times, e.g., every day or every two days). In certain embodiments, galactose is supplemented. The feed medium is added to the production medium starting from day 4 of the production stage culture.

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

[0276] In some embodiments, the supplement contains 0.1-0.7 mM uridine, 0.2-1. Provide 5 μM manganese, and 0.5 to 3.5 mM galactose in the production stage medium. In some embodiments, UMG is administered on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th day, or UMG can be added to the medium on day 14. In some embodiments, UMG can be added daily or can be added every other day. In some embodiments, UMG can be added daily or every other day. Intermittent additions can be started when the cells reach the production stage. In some embodiments, the UMG is added daily starting on day 4, 5, or 6 of cell culture. Or, it can be added to the production medium every other day. In one embodiment, UMG is added to the production medium at about 4 days. In one embodiment, UMG can be added daily from about day 4 to about day 10. It may be added daily for up to about day 14. In some embodiments, the production stage medium comprises: 1.0 to 25 μM, 2.0 to 15 μM, 3 to 10 μM, or 5 μM, for example, about 0.2 to 1.5 μM, or manganese at average daily doses of 0.3 to 1.2 μM; 1.5 to 5 mM, 1.6 to 4.8 mM, or about 2.4 mM, or 2.7 mM, for example , uridine at an average daily dose of about 100-700 μM; ~2.9mM, 2.5-50mM, 5-35mM, about 8-25mM, or about 12mM or 12.6 mM, e.g., galactose at an average daily dose of about 0.5 to 3.5 mM. In certain embodiments, the average daily addition begins on day 4 of the production stage culture. In some embodiments, the UMG supplements described herein are administered in CHO cell culture media. For example, the culture medium provided in International Patent Publication WO 98 / 08934 A1 may be added to the culture medium. The entire contents of which are incorporated herein by reference. In some embodiments, the UMG supplement described in is added to the CD-CHO medium. The UMG supplement described herein is CD-CHO AGT (catalog number 12490- 001 (Invitrogen, Carlsbad, CA, USA).

[0277] In one embodiment, the UMG is added at a cumulative concentration of about 1- 7 mM uridine, about 2 to 15 μM manganese, and about 3 to 20 mM galactose. In some embodiments, the production medium is supplemented with The cumulative concentration of added zinc is about 5-45 μM.

[0278] In one embodiment, uridine, manganese, and galactose (UMG) are present in the feed medium. The uridine concentration is 1-40mM, 15-100mM, 15-90mM, 15-70mM M, 15~50mM, 15~30mM, approx. 18mM, approx. 19mM, approx. 20mM, approx. 21m M, uridine at a final concentration of about 33 mM, about 50 mM, or about 66 mM; The concentration of manganese in the solution varies from about 0.0001 to 0.1 mM, 0.02 mM to 0.2 mM, and 0. 03mM~0.15mM, 0.03mM~0.10mM, 0.03mM~0.05mM, 0.03mM~0.04mM, about 0.03mM, about 0.04mM, about 0.05mM, about 0 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.1 mM, or about 0.14 mM The final concentration of manganese is 85 mM to 500 mM galactose in the feeding medium. , 90mM~400mM, 90mM~300mM, 90mM~200mM, 50mM~1 50mM, 90mM-100mM, about 95mM, about 96mM, about 97mM, about 100mM galactose at a final concentration of about 165 mM, about 250 mM, or about 330 mM. In one embodiment, uridine, manganese, and The feed medium supplemented with galactose was added after the start of the production phase culture (e.g., Days 2-10, 2-8, 2-6, 3-6, or 4 of The production medium is added (e.g., multiple times, e.g., every day or every two days) from 0.1 to 1.5 days. In certain embodiments, a feed medium supplemented with uridine, manganese, and galactose is used to From the fourth day of the step culture, it is added to the production medium, for example, every day.

[0279] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The replenishing component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, and is capable of reducing levels of To produce a composition having a basic antibody isoform, a production medium (or a subsequent culture medium) is added. In one embodiment, the basic isoform is added to the culture medium (feeding solution). The level is about 16% or less (as determined by CEX). The levels of the basal isoform are approximately 15% or less (as determined by CEX) In one embodiment, the level of the basic isoform is In one embodiment, the level of the basic isoform is about 14% or less (CEX). In one embodiment, the amount of the basic isoform is about 13% or less (as determined by The levels are approximately 12% or less (as determined by CEX).

[0280] The addition of UMG to the production medium also enhances the production of VEDOLIZUMAB, a cytotoxic T cells produced by mammalian cells. This may affect the levels of acidic and / or major species in a composition of anti-α4β7 antibodies.

[0281] The addition of UMG to the production medium also enhances the production of VEDOLIZUMAB, a cytotoxic T cells produced by mammalian cells. Influencing the levels of G0F, G1F and / or G2F glycoforms in a composition of anti-α4β7 antibodies N-glycosylation may be present in the population of anti-α4β7 antibodies such as vedolizumab. A representation of the structure of the amine is provided in FIG.

[0282] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The replenishing component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, and is capable of reducing levels of To produce a composition having a G0F glycoform, the production medium (or a compound subsequently added to the production medium) In one embodiment, the level of G0F glycoforms is increased by the addition of Approximately 70% or less as determined by high-intensity chromatography (HILIC). In an embodiment, the level of the G0F glycoform is about 69% (as determined by HILIC). In one embodiment, the level of G0F glycoforms is determined by HILIC. In one embodiment, the level of G0F glycoforms is about 68% or less (by HILIC). In one embodiment, the level of G0F glycoform is about 67% or less (as determined by In one embodiment, the G0F glycoform is about 66% or less (as determined by HILIC). In one embodiment, the level of is about 65% or less (as determined by HILIC). The level of G0F glycoforms is approximately 64% or less (as determined by HILIC). In one embodiment, the level of the G0F glycoform is about 6 (as determined by HILIC). In one embodiment, the level of G0F glycoforms is 3% or less (as determined by HILIC). In one embodiment, the level of G0F glycoforms is about 62% or less when (HILIC In one embodiment, the level of G0F glycoform is about 61% or less (as determined by , about 60% or less (as determined by HILIC). The level of glycoforms is about 59% or less (as determined by HILIC). In this state, the level of the G0F glycoform was approximately 58% or less (as determined by HILIC). In one embodiment, the level of G0F glycoform (as determined by HILIC) In one embodiment, the level of G0F glycoforms is about 57% or less (determined by HILIC). In one embodiment, the level of G0F glycoforms is about 56% or less (as determined by HIL In one embodiment, the level of GOF glycoforms is about 55% or less (as determined by IC In one embodiment, the level of G0F glycoforms is approximately 40-75%. In one embodiment, the level of G0F glycoform is about 45-65% (as determined by , approximately 50-60% (as determined by HILIC).

[0283] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The replenishing component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, in the absence of a supplementary component. Reduced levels compared to control mammalian host cells expressing anti-α4β7 cultured in the presence of To produce a composition having the G0F glycoform, the production medium (or In one embodiment, the composition is added to a feed solution in the absence of supplemental ingredients. Compared to control mammalian host cells expressing cultured humanized anti-α4β7 antibodies, At least about 20-40% (e.g., 20-40%, 20- In one embodiment, the composition comprises a reduction of 30%, 20-25% of the total protein in the absence of supplemental ingredients. Compared to control mammalian host cells expressing humanized anti-α4β7 antibodies cultured in the presence of In one embodiment, the antibody comprises at least about a 20% reduction in the G0F glycoform of the humanized anti-α4β7 antibody. The composition comprises a control mammalian cell expressing a humanized anti-α4β7 antibody cultured in the absence of supplementation. At least about 25% of the G0F glycoform of the humanized anti-α4β7 antibody compared to mammalian host cells. The comparator is a test performed under substantially similar conditions except for the parameter specified to be different. For example, in the absence of supplemental ingredients.

[0284] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The filling component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, and is capable of increasing levels of To produce a composition having a G1F glycoform, the production medium (or a compound subsequently added to the production medium) In one embodiment, the level of G1F glycoform is measured by (HILIC) In one embodiment, the level of the G1F glycoform is greater than or equal to about 20% (as determined by In one embodiment, the G1F glycoprotein is at least about 21% (as determined by HILIC). The level of the type is about 22% or greater (as determined by HILIC). In this study, the level of the G1F glycoform was approximately 23% or higher (as determined by HILIC). In one embodiment, the level of the G1F glycoform (as determined by HILIC) is about In one embodiment, the level of the G1F glycoform is greater than or equal to 24% (determined by HILIC). In one embodiment, the level of the G1F glycoform is greater than or equal to about 25% (when In one embodiment, the level of the G1F glycoform is greater than or equal to about 26% (as determined by is about 27% or more (as determined by HILIC). The level of the F glycoform is greater than or equal to about 28% (as determined by HILIC). In the morphology, the level of the G1F glycoform was approximately 29% or more (as determined by HILIC). In one embodiment, the level of the G1F glycoform (as determined by HILIC) is In one embodiment, the level of G1F glycoform is greater than or equal to about 30% (by HILIC). In one embodiment, the level of the G1F glycoform is greater than or equal to about 31% (as determined by HI In one embodiment, the level of G1F glycoforms is about 32% or more (as determined by LIC). The peak is about 33% or higher (as determined by HILIC). The level of the G1F glycoform is about 20-45%. In one embodiment, the level of the G1F glycoform is , about 25-45% (as determined by HILIC). The level of the F glycoform is approximately 30-40% (as determined by HILIC).

[0285] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The replenishing component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, in the absence of a supplementary component. Compared to control cell cultures containing mammalian host cells expressing anti-α4β7 cultured in the presence of In order to produce a composition having an increased amount of the G1F glycoform, the production medium (or In one embodiment, the composition is added to a supplemental solution (a feed solution added to a production medium). A control comprising mammalian host cells expressing a humanized anti-α4β7 antibody cultured in the absence of At least about 2-3.5 times higher G1F glycoform of humanized anti-α4β7 antibody compared to cell culture In one embodiment, the increase includes an increase of 2 to 3.5 times, 2 to 3.3 times, or 2 to 3 times. The composition comprises a control mammalian cell expressing a humanized anti-α4β7 antibody cultured in the absence of supplementation. Compared to cell cultures containing mammalian host cells, the G1F form of the humanized anti-α4β7 antibody was less In one embodiment, the composition comprises about a two-fold increase in the expression of the β-glucose agonist (GlcNAc) in the absence of supplementation. compared to cell cultures containing control mammalian host cells expressing the humanized anti-α4β7 antibody , including at least about a three-fold increase in the G1F glycoform of the humanized anti-α4β7 antibody. Control cell cultures are cultured under substantially the same conditions, except for certain parameters, such as supplementation.

[0286] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The filling component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, and is capable of increasing levels of To produce a composition having a G2F glycoform, the production medium (or a compound subsequently added to the production medium) In one embodiment, the levels of G2F glycoforms are measured using a method In one embodiment, the level of G2F glycoforms is greater than or equal to about 2% (as determined by In one embodiment, the G2F glycoprotein is at least about 2.5% (as determined by HILIC). The level of the type is about 3% or more (as determined by HILIC). In the present study, the level of G2F glycoforms was approximately 3.5% or higher (as determined by HILIC). In one embodiment, the level of G2F glycoforms (as determined by HILIC) is about In one embodiment, the level of G2F glycoforms is greater than or equal to 4% (as determined by HILIC). In one embodiment, the level of G2F glycoforms is greater than or equal to about 4.5% (when measured by HILI). C) is about 5% or more. In one embodiment, the level of G2F glycoforms is , or greater than about 5.5% (as determined by HILIC). The level of F glycoform is greater than or equal to about 6% (as determined by HILIC). In this state, the level of the G2F glycoform was approximately 6.5% or higher (as determined by HILIC). In one embodiment, the level of G2F glycoforms (as determined by HILIC) is In one embodiment, the level of G2F glycoform is 10% or less. In the morphological analysis, the level of G2F glycoforms was approximately 2-4% (as determined by HILIC). In one embodiment, the level of G2F glycoforms (as determined by HILIC) In one embodiment, the level of G2F glycoforms is approximately 3-5% (determined by HILIC). When this is done, the average is about 2-7%.

[0287] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The filling component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, and is capable of increasing levels of To produce a composition having a G2F glycoform, the production medium (or a compound subsequently added to the production medium) In one embodiment, the levels of G2F glycoforms are measured using a method In one embodiment, the level of G2F glycoforms is greater than or equal to about 2% (as determined by In one embodiment, the G2F glycoform is about 3% or more (as determined by HILIC). The levels are approximately 4% or higher (as determined by HILIC).

[0288] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The replenishing component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, in the absence of a supplementary component. Increased levels compared to control mammalian host cells expressing anti-α4β7 cultured in the presence of To produce a composition having a G2F glycoform of In one embodiment, the composition is added to a feed solution in the absence of supplemental ingredients. Compared to cell cultures containing control mammalian host cells expressing cultured humanized anti-α4β7 antibodies. In comparison, the G2F glycoform of the humanized anti-α4β7 antibody is at least about 2- to 5-fold (e.g., 2- to 5-fold) In one embodiment, the composition comprises a non-additive increase in the amount of the supplement. A cell culture medium containing control mammalian host cells expressing a humanized anti-α4β7 antibody cultured in the presence of The humanized anti-α4β7 antibody comprises at least about a three-fold increase in the G2F glycoform compared to the control. In one embodiment, the composition is a humanized anti-α4β7 antibody cultured in the absence of supplementation. of the humanized anti-α4β7 antibody compared to cell cultures containing control mammalian host cells expressing The control cell cultures included at least about a four-fold increase in certain parameters, For example, they are cultured under substantially the same conditions, except for supplements.

[0289] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The replenishing component is a composition comprising an anti-α4β7 antibody, e.g., vedolizumab, in the absence of a supplementary component. Compared to control cell cultures containing mammalian host cells expressing anti-α4β7 cultured in the presence of To produce a composition having increased amounts of G1F and G2F glycoforms, a production medium (or a feed solution that is then added to the production medium). The composition is a control mammalian animal expressing a humanized anti-α4β7 antibody cultured in the absence of supplements. At least one of the G1F glycoforms of the humanized anti-α4β7 antibody is increased in a cell culture containing the host cell compared to the cell culture containing the host cell. Approximately a 2- to 5-fold (e.g., 2- to 5-fold, 2- to 4-fold, 3- to 4-fold) increase, and a small increase in G2F glycoforms In one embodiment, the increase includes an increase of at least about 2-fold to 5-fold (e.g., 2-5-fold, 2-4-fold, 3-4-fold). In this embodiment, the composition comprises a humanized anti-α4β7 antibody expressed in culture in the absence of supplementary components. The G1F glycosylation of the humanized anti-α4β7 antibody was compared to that of a cell culture containing a control mammalian host cell. In one embodiment, the composition comprises at least about a three-fold increase in G2F and G3F glycoforms. Control mammalian host cells expressing a humanized anti-α4β7 antibody cultured in the absence of adipocytes At least about two-fold increase in the G1F form of the humanized anti-α4β7 antibody compared to cell cultures containing The control cell cultures showed at least about a four-fold increase in specific parameters. -, for example, cultured under substantially the same conditions, except for supplementation.

[0290] In certain embodiments, a complex complement comprising uridine, manganese, and galactose (UMG) is used. The composition of the filling component is 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-, and core fucosylated biantennary glycans asialo, monogalacto, core fucosylated biantennary glycans (G1F), and and / or asialo-, digalacto-, and core fucosylated biantennary glycans (G2F) In order to produce a composition comprising an anti-α4β7 antibody having a sylated variant, a production medium ( or a feed solution which is then added to the production medium. The compositions and methods described in are 91-96%, 92-95%, 91-92%, 91-92. 5%, 91-93%, or 91-95% total asialo-, agalacto-, and core fucosylated dimers Branched glycans (G0F), asialo, monogalactic, core fucosylated biantennary glycans (G 1F), and / or asialo-, digalacto-, and core fucosylated biantennary glycans (G2F) It is possible to produce a population of humanized anti-α4β7 antibodies having glycosylation variants of In one embodiment, the compositions and methods described herein provide a 92-98%, 92-97%, %, 92-96%, 92-95% total asialo-, agalacto-, and core fucosylated biantennary glycans can (G0F), asialo, monogalacto, core fucosylated biantennary glycans (G1F), and / or asialo-, digalacto-, and core fucosylated biantennary glycans (G2F) A population of humanized anti-α4β7 antibodies can be produced that have cosylation variants.

[0291] The medium supplement consists of water, basal medium, or ascorbate, citrate, carbonate, (4 -(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), histidine Din, glutamate, acetate, succinate, gluconate, histidine, phosphate, malate Leynate, cacodylate, 2-[N-morpholino]ethanesulfonic acid (MES), biphenylsulfate Bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tri s), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine, and The phosphate buffer may be provided in a buffer such as an organic acid or other organic acid or zwitterionic buffer. In embodiments, the medium supplement has a pH of 5.5 to 7.0, 6.0 to 7.5, or 5.9 to 6.1. In other embodiments, the medium supplement has a pH of 1.5 to 5.5, 1.8 to 3.0 , 3.2 to 4.5, or 1.9 to 2.1. The ingredients have a pH of 7.5 to 9.0.

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

[0293] In certain embodiments, the supplemental component containing a metal, e.g., zinc or manganese, is low pH buffers such as phosphate or acetate buffers. Citrate also removes metal ions. In one embodiment, the metal ion supplement component may function to chelate the metal ion to limit toxicity. The metal-containing supplement may be zinc or manganese, for example, at 100 to 140 mM or 115 mM. In one embodiment, the supplement contains zinc and manganese in a citrate buffer of 125 mM. Buffers for metals containing include 118-122 mM citric acid, pH 1.9-2.1. Thus, in some embodiments, provided herein are anti-α4β7 antibodies or The GS-CHO host cells expressing the antigen-binding portion were incubated in 115-125 mM citrate buffer. 50-150 μM zinc and 10-50 mM manganese in a buffer solution pH 1.9-2.1 It is a cell culture obtained by culturing in a production medium supplemented with a solution. In embodiments, provided herein are anti-α4β7 antibodies, or antigen-binding portions thereof. GS-CHO host cells expressing GS-CHO were cultured in 115-125 mM citrate buffer supply supplement, p 10-100μM zinc by adding zinc and manganese to H1.9-2.1 and by culturing in a production medium supplemented with manganese concentrations of 0.1 to 100 μM. The resulting cell culture. In some embodiments, the citrate buffered metal supplement component comprises: For example, the feed replenisher is added daily from day 4 of the production culture.

[0294] In some embodiments, provided herein are anti-α4β7 antibodies or antigens thereof. A host cell (or a population of host cells) expressing a binding moiety, and a metal ion, a nucleoside a cell culture comprising a production medium containing or supplemented with a cofactor, sugar, and / or metal cofactor. In other embodiments, provided herein are metal ions, nucleosides, sugars, and Anti-α4β7 antibodies or their derivatives in a production medium containing or supplemented with and / or metal cofactors. The cell culture is obtained by culturing a host cell expressing an antigen-binding portion of the antibody.

[0295] In some embodiments, provided herein are anti-α4β7 antibodies or antigens thereof. A host cell (or a population of host cells) expressing a binding moiety, and a sugar, a nucleoside, and / or or a cell culture comprising a production medium that contains or is supplemented with a metal cofactor. In some embodiments, provided herein are nucleic acid sequences that include a sugar, a nucleoside, and / or a metal cofactor. A host cell expressing an anti-α4β7 antibody or an antigen-binding portion thereof in a production medium supplemented with or without the use of a β- It is a cell culture obtained by culturing the main cell.

[0296] In some embodiments, provided herein are anti-α4β7 antibodies or antigens thereof. A host cell (or a population of host cells) expressing a binding moiety, and uridine, manganese, and A cell culture comprising a production medium that contains or is supplemented with galactose (UMG). In an embodiment, provided herein is a method for the preparation of a guanidine-containing guanidine derivative comprising the steps of: In a production medium containing or supplemented with UMG, an anti-α4β7 antibody or an antigen-binding portion thereof is A cell culture is obtained by culturing a host cell expressing a polypeptide.

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

[0298] In some embodiments, the cell culture includes uridine at a concentration of 0.1 to 20 mM. For example, in some embodiments, the cell culture is supplemented with 0.1-20 mM, 1-20 mM, 0 .5~12mM, 1~8mM, 1.5~4mM, 0.1~1.5mM, 0.1~5mM, 5mM~10mM, 10mM~15mM, 15mM~20mM, 0.1mM~10mM, Other examples include 10 mM to 20 mM, 1 to 7 mM, 7 to 14 mM, or 14 to 20 mM. In embodiments, the cell culture is at 10-50 mM, 20-60 mM, 30-70 mM, 40- Uri at concentrations of 80mM, 50-90mM, 60-100mM, or 0.1-100mM In some embodiments, the cell culture comprises about 10 mM, about 15 mM, about 16 mM mM, approx. 17mM, approx. 18mM, approx. 19mM, approx. 20mM, approx. 21mM, approx. 22mM, approx. 25mM, approx. 27mM, approx. 30mM, approx. 33mM, approx. 35mM, approx. 40mM, approx. 45mM uridipine at a concentration of about 50 mM, about 55 mM, about 60 mM, about 66 mM, or about 70 mM Includes

[0299] In some embodiments, the cell culture includes manganese at a concentration of 0.1 to 100 μl. For example, in some embodiments, the cell cultures are treated with 0.1-100 μM, 0.5-50 μM M, 1.0~25μM, 2.0~15μM, 3~10μM, 0.1~10μM, 0.1~ 20μM, 0.1~30μM, 1~10μM, 1~20μM, 1~30μM, 1~40μM M, 1~50μM, 1~60μM, 1~70μM, 1~80μM, 1~90μM, 1~1 00μM, 20~40μM, 40~60μM, 60~80μ, 80~100μM, 20~ 50μM, 30~60μM, 40~70μM, 50~80μM, 70~100μM, 20 Manganese at concentrations of ~70 μM, 30–80 μM, 40–90 μM, or 50–100 μM In other embodiments, the cell culture comprises about 0.1 μM, about 0.2 μM, about 0.3 μM M, approx. 0.4 μM, approx. 0.5 μM, approx. 0.6 μM, approx. 0.7 μM, approx. 0.8 μM, approx. 0. 9μM, about 1μM, about 2μM, about 3μM, about 5μM, about 10μM, about 20μM, about 30μ M, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, contains manganese at a concentration of about 100 μM.

[0300] In some embodiments, the cell culture is supplemented with galactose at a concentration of about 0.1 to 100 mM. For example, in some embodiments, the cell culture comprises 1 to 75 mM, 2.5 to 50 mM mM, 5~35mM, approx. 8~25mM, 0.1~10mM, 0.1~20mM, 0.1~ 30mM, 1~10mM, 1~20mM, 1~30mM, 1~40mM, 1~50mM, 1~60mM, 1~70mM, 1~80mM, 1~90mM, 1~100mM, 20~4 0mM, 40~60mM, 60~80mM, 80~100mM, 20~50mM, 30~ 60mM, 40~70mM, 50~80mM, 70~100mM, 20~70mM, 30 Galactose is present at a concentration of 80 mM, 40 mM, 90 mM, or 50 mM, 100 mM, or 150 mM. In some embodiments, the cell culture comprises 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 1mM, about 2mM, about 3mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM , about 10μM, about 12.5mM, about 12mM, about 15mM, about 20mM, about 30mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 1 Contains galactose at a concentration of 100 mM.

[0301] In some embodiments, provided herein are uridine, manganese, and galactosamine hydrochloride. Anti-α4β7 antibodies or their derivatives in a production medium containing or supplemented with unsaturated glucose (UMG). by culturing a host cell (or a population of host cells) that expresses an antigen-binding portion For example, in some embodiments, the cell culture is a 0. 1-20 mM uridine (and ranges therein), 0.1-100 μM manganese (and ranges therein) 0.1 to 100 mM galactose (and ranges therein) In some embodiments, the cell culture is supplemented with zinc as described herein. In some embodiments, the cell culture can include any of the cell culture methods described herein. In some embodiments, the lysine and / or arginine may be further included. In some cases, the cell culture may further include zinc, lysine, and arginine. In some embodiments, provided herein is an anti-α4β7 antibody or antigen-binding portion thereof. A host cell (or a population of host cells) expressing (e.g., vedolizumab), and a production medium. The production medium comprises 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 A cumulative concentration of Zn in µM was added during the production phase, e.g., from day 4 to harvest. can be.

[0302] In some embodiments, provided herein are uridine, manganese, galactosamine, arginine ... and in a production medium containing or supplemented with phosphate and zinc, an anti-α4β7 antibody or its antigen. by culturing a host cell (or a population of host cells) that expresses the binding moiety For example, in some embodiments, the cell culture is capable of detecting 0.1 to 10% of the cell population. 20 mM uridine (and range therein), 0.1 to 100 μM manganese (and range therein) 0.1 to 100 mM galactose (and ranges therein), and 10 to 1 00 μM zinc (and ranges therein).

[0303] In some embodiments, provided herein are uridine, manganese, galactosamine, arginine ... In a production medium containing or supplemented with glycerol, zinc, lysine, and / or arginine, Cultivating a host cell (or a population of host cells) expressing the α4β7 antibody or an antigen-binding portion thereof. For example, in some embodiments, a cell culture can be obtained by culturing the The cell cultures were treated with 0.1 to 20 mM uridine (and ranges therein), 0.1 to 100 mM 0.1 to 100 mM manganese (and ranges therein), 0.1 to 100 mM galactose (and ranges therein) range), 10-100 μM zinc (and ranges therein), 5.0-8.8 g / L and / or arginine (and ranges therein) from 3.0 to 12.0 g / L The range may include.

[0304] In some embodiments, the cells of the cell culture are treated with uridine, manganese, and galactose. The same medium was used containing medium lacking uridine, manganese, and galactose, or medium without supplementation with uridine, manganese, and galactose. of the basic isoform (as determined by CEX) compared to cell cultures such as expressing reduced levels of anti-α4β7 antibodies, or antigen-binding portions thereof. In an embodiment, the expressed antibody has about 16% or less salt (as determined by CEX). In some embodiments, the expressed antibody includes a specific isoform (e.g., by CEX). In some embodiments, the total number of isoforms is about 15% or less (as determined by a quantification method). In the present study, the expressed antibody contained less than about 14% basic isoforms (as determined by CEX). In some embodiments, the expressed antibody comprises a In some embodiments, the expressed The antibody contains less than about 12% basic isoforms (as determined by CEX). In some embodiments, the expressed antibody has an affinity for about 11 Contains less than % basic isoforms.

[0305] In some embodiments, the cells of the cell culture are treated with uridine, manganese, and galactose. The same medium was used containing medium lacking uridine, manganese, and galactose, or medium without supplementation with uridine, manganese, and galactose. Decreased G0F glycoforms (as determined by HILIC) compared to cell cultures In some embodiments, the subject expresses an anti-α4β7 antibody, or an antigen-binding portion thereof, having a specific level. In terms of morphology, the cells in the cell cultures were 70% or less (as determined by HILIC), 6 Expressing anti-α4β7 antibodies with 5% or less, 60% or less, or 55% or less G0F content In some embodiments, the cells of the cell culture are 85% or less, 80% or less, 75% or less, 70% or less, 69% or less, 68% or less, 67% or less % 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 G In some embodiments, the cell culture expresses an anti-α4β7 antibody having an 0F content. The cells express anti-α4β7 antibodies with a G0F content of 45–65%. In an embodiment, the cells of the cell culture are cultured with an anti-α4β7 antibody having a G0F content of 50-60%. In some embodiments, the cells of the cell culture express 45-85% GF. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody having an amount of In some embodiments, the antibody expresses an anti-α4β7 antibody having a G0F content of 45-82%. may contain or be supplemented with uridine, manganese, and / or galactose as described herein. The G0F content of anti-α4β7 antibodies produced by cell cultures containing perfused medium was Media lacking lysine, manganese, and / or galactose, or media lacking uridine, manganese, and / or produced by equivalent cell cultures containing media not supplemented with galactose. At least 20%, at least 21%, or less than the G0F content of the anti-α4β7 antibody 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 At least 35%, at least 36%, at least 37%, at least 38%, at least 39% , or at least 40% decreased.

[0306] In some embodiments, the cells of the cell culture are treated with uridine, manganese, and galactose. The same medium was used containing medium lacking uridine, manganese, and galactose, or medium without supplementation with uridine, manganese, and galactose. Increased G1F glycoforms (as determined by HILIC) compared to cell cultures In some embodiments, the subject expresses an anti-α4β7 antibody, or an antigen-binding portion thereof, having a specific level. In terms of morphology, cells in cell cultures exhibited morphological changes of 10% or more (as determined by HILIC), 1 5% 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 anti-α4β7 antibodies with a G1F content of 33% or more. In an embodiment, the cells of the cell culture are cultured with an anti-α4β7 antibody having a G1F content of 25-45%. In some embodiments, the cells of the cell culture express 30-40% G1F. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody having a specific amount of In some embodiments, the cells express an anti-α4β7 antibody having a G1F content of 10-45%. or containing uridine, manganese, and / or galactose as described herein. The G1F content of anti-α4β7 antibodies produced by cell cultures supplemented with uridine, Media lacking manganese and / or galactose, or media lacking uridine, manganese and / or Antibodies produced by equivalent cell cultures containing medium supplemented with or without galactose were At least 2-fold, at least 2.25-fold, or less G1F content compared to the α4β7 antibody at least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, or increases by at least 3.5 times.

[0307] In some embodiments, the cells of the cell culture are treated with uridine, manganese, and galactose. The same medium was used containing medium lacking uridine, manganese, and galactose, or medium without supplementation with uridine, manganese, and galactose. Increased G2F glycoforms (as determined by HILIC) compared to cell cultures In some embodiments, the subject expresses an anti-α4β7 antibody, or an antigen-binding portion thereof, having a specific level. In terms of morphology, the cells in the cell cultures had morphology greater than 0.5% (as determined by HILIC); 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 In some embodiments, the cells express an anti-α4β7 antibody with a G2F content of 8% or greater. The cells in the cell culture express anti-α4β7 antibodies with a G2F content of 2-4%. In some embodiments, the cells of the cell culture are treated with anti-α4β7 having a G2F content of 3-5%. In some embodiments, the cells of the cell culture contain 2-7% G2F. In some embodiments, the cells of the cell culture express an anti-α4β7 antibody having an amount of Expressing anti-α4β7 antibodies with a G2F content of 0.5-7.5%. In some embodiments, the uridine, manganese, and / or galactose described herein may be included or may be present. The G2F content of anti-α4β7 antibodies produced by cell cultures supplemented with uridine diamine phosphate phosphate (PPP) was significantly increased by 100 mg / mL. , manganese, and / or galactose, or media lacking uridine, manganese, and / or or produced by equivalent cell cultures containing medium not supplemented with galactose. At least 2-fold, at least 2.25-fold, or less than the G1F content of the anti-α4β7 antibody At least 2.5 times, at least 2.75 times, at least 3 times, at least 3.25 times, at least At least 3.5 times, at least 3.75 times, at least 4 times, at least 4.25 times, at least at least 4.5 times, at least 4.75 times, or at least 5 times.

[0308] The cell cultures provided herein, in some embodiments, are characterized in that the population is a (HILIC When determined by the above, 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-, and core fucosylated biantennary fragments glycan (G0F), asialo, monogalactic, core fucosylated biantennary glycan (G1F) and / or asialo, digalacto, and core fucosylated biantennary glycans (G2F) A population of humanized anti-α4β7 antibodies can be produced that have glycosylation variants. In one embodiment, the cell culture has a purity of 91-96% (as determined by HILIC), 92-95%, 91-92%, 91-92.5%, 91-93%, or 91-95% Total asialo-, agalacto-, core fucosylated biantennary glycans (G0F), asialo-, mono- lacto, core fucosylated biantennary glycan (G1F), and / or asialo, digalacto , human, having a glycosylation variant of the core fucosylated biantennary glycan (G2F) In one embodiment, the cell culture can produce a population of purified anti-α4β7 antibodies. ~98%, 92~97%, 92~96%, 92~95% total asialo-, agalacto-, and core Fucosylated biantennary glycan (G0F), asialo, monogalactic, core fucosylated biantennary Glycans (G1F) and / or asialo, digalacto, core fucosylated biantennary glycans The present study produces a population of humanized anti-α4β7 antibodies with glycosylation variants of the glycosyltransferase (G2F). It is possible.

[0309] In some embodiments, provided herein are methods for producing monoclonal antibodies. (i) a host cell expressing an anti-α4β7 antibody or an antigen-binding portion thereof; Cell culture comprising a production medium containing or supplemented with lysine, manganese, and / or galactose. The cells are cultured for a period of time sufficient for the host cells to express the anti-α4β7 antibody or an antigen-binding portion thereof. and (ii) recovering the anti-α4β7 antibody or antigen-binding portion thereof from the cell culture. In some embodiments, the method comprises: The population of 4β7 antibodies, or antigen-binding portions thereof, may be fused to uridine, manganese, and / or galactose. Media lacking uridine or not supplemented with manganese, uridine, and / or galactose of an anti-α4β7 antibody or antigen-binding portion thereof, recovered from an equivalent cell culture containing medium. Reduced levels of basic isoforms (as determined by CEX) compared to the population In some embodiments, the anti-α4β7 antibody recovered from the cell culture, The population of antigen-binding portions is grown in a medium lacking uridine, manganese, and / or galactose. or equivalent medium containing medium without supplementation of uridine, manganese, and / or galactose. compared to a population of anti-α4β7 antibodies or antigen-binding portions thereof recovered from the cell culture, In some embodiments, the production medium further comprises zinc. In some embodiments, the production medium is supplemented with lysine and / or In some embodiments, the cell culture medium further comprises or is further supplemented with arginine. The 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 , 14, 15, 16, 17, 18, 19, or 20 days. is obtained or obtainable by the aforementioned method provided herein. It is an anti-α4β7 antibody that can

[0310] In one embodiment, provided herein is a method for treating pulmonary circulation disorders comprising administering to a patient a therapeutically effective amount of at least 88%, at least 90%, at least 91%, 92% or more, 93% or more, 94% or more, or 95% or more of total asialo-, agalacto-, co- Afucosylated biantennary glycans (G0F), asialo, monogalactic, core fucosylated biantennary Branched glycans (G1F) and / or asialo, digalacto, core fucosylated biantennary glycans A composition comprising vedolizumab having a glycosylation variant of can (G2F). In one embodiment, provided herein is 91-96%, 92-95%, 91-9 2%, 91-92.5%, 91-93%, or 91-95% total asialo- and agalacto- , core fucosylated biantennary glycan (G0F), asialo, monogalactic, core fucosylated Biantennary glycans (G1F) and / or asialo, digalacto, core fucosylated biantennary A composition comprising vedolizumab having a glycosylation variant of glycan (G2F). In some embodiments, the composition comprises uridine, manganese, and galactose. The GS-CHO cells recombinantly expressing vedolizumab were cultured in a production medium supplemented with In some embodiments, the composition can be obtained by Recombinantly express vedolizumab in production medium supplemented with ribonuclease, galactose, and zinc In some embodiments, the GS-CHO cells can be cultured. The composition may further comprise uridine, manganese, galactose, zinc, arginine, and / or GS-CHO cells recombinantly expressing vedolizumab were cultured in a production medium supplemented with lysine. This can be obtained by:

[0311] In one embodiment, provided herein is a method for producing a medicament for the detection of fibronectin-related inflammatory bowel disease (as determined by HILIC): 85% or less, 80% or less, 75% or less, 70% or less, 69% or less, 68% or less, 67% or less Below, 66%, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, 60% or more below, 59% or less, 58% or less, 57% or less, 56% or less, or 55% or less Asialo; Compositions containing vedolizumab with agalacto, core fucosylated biantennary glycan (G0F) In one embodiment, provided herein is a method for the preparation of a medicament for the detection of chromatographically significant changes in a chromatographically significant region of ... When the fucosylated nucleotides are 45-65%, or 50-60% asialo-, agalacto-, and core fucosylated nucleotides, A composition comprising vedolizumab having branched glycans (G0F). The above composition is then grown in a production medium supplemented with uridine, manganese, and galactose. Vedolizumab can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab. In some embodiments, the composition further comprises uridine, manganese, galactose, and GS-CHO cells recombinantly expressing vedolizumab were cultured in a production medium supplemented with Zn and Zn. In some embodiments, the composition can be obtained by , manganese, galactose, zinc, arginine, and / or lysine. The antibody was obtained by culturing GS-CHO cells recombinantly expressing vedolizumab in can.

[0312] In one embodiment, provided herein is a method for producing a medicament for the detection of fibronectin-related inflammatory bowel disease (as determined by HILIC): 10% or more, 15% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more Above, 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-, monogalacto-, or core fucosylated bisecting In one embodiment, the present invention provides a composition comprising vedolizumab having a branched glycan (G1F). The subsection provides that 25-45% (as determined by HILIC) or 3 Contains 0–40% asialo, monogalactic, core fucosylated biantennary glycans (G1F) In some embodiments, the composition comprises uridine dinucleotide. Vedolizumab is recombinantly expressed in a production medium supplemented with phosphate, manganese, and galactose. In some embodiments, the GS-CHO cells can be cultured. The aforementioned compositions were grown in a production medium supplemented with uridine, manganese, galactose, and zinc. Vedolizumab can be obtained by culturing GS-CHO cells recombinantly expressing vedolizumab. In some embodiments, the composition comprises uridine, manganese, galactose, zinc. Recombinantly expressing vedolizumab in production medium supplemented with lead, arginine, and / or lysine The antibody can be obtained by culturing GS-CHO cells expressing the antibody.

[0313] In one embodiment, provided herein is a method for producing a medicament for the detection of fibronectin-related inflammatory bowel disease (as determined by HILIC): 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-, digalacto-, or core fucosylated biantennary glycans (G2 F). In one embodiment, the composition provided herein comprises vedolizumab having The concentrations were 2-4%, 3-5%, or 2-7% (as determined by HILIC). Vedolizumab with sialo-, digalactosyl, and core fucosylated biantennary glycans (G2F) In some embodiments, the composition comprises uridine, manganese, and GS-CHO cells recombinantly expressing vedolizumab in production medium supplemented with galactose In some embodiments, the composition can be obtained by culturing Vedolizumab was formulated in production medium supplemented with uridine, manganese, galactose, and zinc. The gene can be obtained by culturing recombinantly expressing GS-CHO cells. In an embodiment, the composition includes uridine, manganese, galactose, zinc, arginine, and / or GS-CHO recombinantly expressing vedolizumab in a production medium supplemented with lysine. It can be obtained by culturing cells.

[0314] In some embodiments, the method for producing an anti-α4β7 antibody in a CHO cell culture comprises culturing the cells in a culture medium. The nutrients include media supplements containing metal ions and metal cofactors, as well as nucleosides and sugars. In some embodiments, the CHO cell culture medium further comprises providing a separate media supplement comprising: The method for producing anti-α4β7 antibodies in a culture medium comprises adding to the culture a medium supplement containing a metal ion, and providing additional media supplements including nucleosides, sugars, and metal cofactors. In some embodiments, the method for producing an anti-α4β7 antibody in a CHO cell culture comprises: Providing the culture with media supplements including metal ions, nucleosides, sugars, and metal cofactors. In some embodiments, the growth of anti-α4β7 antibodies in CHO cell cultures is The method comprises adding to the culture a medium supplement containing metal ions, nucleosides, and metal cofactors. This includes providing.

[0315] Exemplary media supplements and their methods of use provided in the Examples are illustrative of embodiments of the present invention. It is considered that.

[0316] C. Lysine and / or arginine supplementation In some embodiments of the foregoing aspects, the cell culture medium, e.g., the production stage medium, is In some cases, the patient may be supplemented with arginine and / or arginine. In one embodiment, the methods and compositions provided herein provide a method for administering zinc, lysine, and / or arginine to a mammalian animal. In another embodiment, a cell culture medium, such as a production stage medium, may be used, which is supplemented with In one embodiment, the methods and compositions provided herein include, but are not limited to, uridine, manganese, galactose, A cell culture medium, e.g., a production stage medium, supplemented with lysine and / or arginine. In another aspect, the methods and compositions provided herein can be used to supplemented with glycerol, manganese, galactose, zinc, lysine, and / or arginine. A cell culture medium, such as a production stage medium, can be employed.

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

[0318] The medium was supplemented with uridine, manganese, galactose, and zinc as described above. For example, in some embodiments, the cell culture medium, e.g., production stage medium, can be 0.1-20mM uridine, 0.1-100μM manganese, 0.1-100mM gas Supplemented with lactose and 1-100 μM zinc, 5.0-8.8 g / L lysine, and and / or arginine at 3.0–12.0 g / L may be additionally supplemented.

[0319] III. Upstream production methods The present invention provides an anti-α4β7 antibody, such as vedolizumab, with a titer of greater than 3 g / L. Anti-α4 in mammalian host cells using the conditions and / or supplemental components identified herein. Large-scale recombinant production of antibodies, such as β7 antibodies, in mammalian cell culture systems High level recombinant antibody expression is a known challenge in the art.

[0320] The overall process involves the incubation of mammalian cells genetically engineered to express anti-α4β7 antibodies. The cells are then inoculated into cell culture medium, followed by a multiplication step, a production step, and finally, the harvesting of the recombinant antibody. Between the various stages, in certain embodiments, there may be transition stages. do.

[0321] Thus, as a first step, a nucleic acid encoding the desired recombinant anti-α4β7 antibody ( For example, the cDNA can be inserted into a replicable vector for expression. The vectors are publicly available and known to those skilled in the art. The components of the vector are: Typically, these include one or more of the following, but are not limited to: a signal sequence, an origin of replication, One or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Each is described below. Optional signal sequences, origins of replication, marker genes that can be used Genes, enhancer elements, and transcription terminator sequences are known in the art. and further described in PCT Publication WO97 / 25428 or U.S. Patent No. 7,053,202. It is described in detail.

[0322] Expression vectors usually contain a nucleic acid that is recognized by a host organism and encodes a protein. The sequence includes a promoter operably linked to the sequence. The promoter is a promoter that is a promoter for the start code of a structural gene. It is a non-coding sequence (generally within about 100 to 1000 bp) located upstream (5') of the don. They control the transcription and translation of specific nucleic acid sequences to which they are operably linked. Such promoters typically fall into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters that respond to some change in culture conditions, e.g., nutrient Increased from DNA under its control in response to the presence or absence of a substrate or to changes in temperature It initiates transcription at controlled levels. It has numerous promoters recognized by a variety of potential host cells. These promoters can be isolated from the source DNA by restriction enzyme digestion. The promoter is then recovered from the vector and the isolated promoter sequence is inserted into the vector. It is operably linked to DNA encoding the desired protein.

[0323] Expression vectors that provide for transient expression in mammalian cells can be used. Generally, transient expression involves the use of an expression vector that can efficiently replicate in a host cell. The use of , synthesizes high levels of the desired polypeptide encoded by the expression vector (Sam brook et al., supra). Transient expression comprising an appropriate expression vector and a host cell. The system provides a convenient means for positive identification of polypeptides encoded by cloned DNA. and rapid characterization of such polypeptides for desired biological or physiological properties. The mammalian host cells are transfected with the expression vectors described above. The vector is then transfected, preferably transformed, and the promoter is induced, transformants are selected, or the desired product is produced. The cells are grown in conventional nutrient media modified to be suitable for amplifying the gene encoding the desired sequence. Such cells are then propagated and eventually undergo several rounds of replication to produce the following It is transferred to a larger vessel for growth and ultimately production of the polypeptide of interest.

[0324] Mammalian cells, such as CHO cells, can be cultured in small scale, e.g., up to 5 L, e.g., 5 ml, 25 Culture in ml, 50ml, 100ml, 250ml, 1L, 3L, or 5L vessels Alternatively, the culture may be, for example, 10 L, 20 L, 100 L, or 200 L. Alternatively, the culture may be in a medium-sized vessel, such as a 500 L, 1000 L, 2000L, 3000L, 5000L, 10,000L, and 15,000L containers, etc. Large-scale culture, such as for the production of therapeutic antibodies, may be performed in a vessel of more than 200 L. The cell culture is typically incubated for a few days or until the cells produce the protein(s) of interest. It will last for several weeks.

[0325] For purposes of the present invention, cell culture medium refers to the culture medium for mammalian cells in in vitro cell culture. Examples of cell culture media include expanded cell culture media, These include culture media and production cell culture media.

[0326] Cell culture media formulations are well known in the art. Typically, cell culture media are It is composed of buffers, salts, carbohydrates, amino acids, vitamins, and trace amounts of essential elements. Cell culture media may contain serum, peptones, protein hydrolysates, and / or proteins. A variety of tissue culture media, including serum-free and defined media, may or may not be included. Cell culture media are commercially available, for example, using one or a combination of the following cell culture media: Among others, RPMI-1640 medium, RPMI-1641 medium, Da Rubecco's modified Eagle's medium (DMEM), Minimum essential medium Eagle, F-12K medium, Ham F12 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L -15 medium, and EX-CELL™ 300 series (JRH Biosciences Cell culture media is a serum-free medium that is used to culture cells. Depending on the requirements of the cell culture and / or the desired cell culture parameters, amino acids, salts, sugars, vitamins, etc. Addition or enrichment of components such as amines, hormones, growth factors, buffers, antibiotics, lipids, and trace elements CHO cell culture media are known in the art and can be supplemented with 0.1% ethanol at elevated concentrations. For example, CD-CHO (Invitrogen), CD-CHO-AGT™ medium ( ThermoFisher Scientific), HYCELL™ CHO medium (GE Healthcare Life Sciences), or CHOMACS CD medium (Militenyi Biotech). As mentioned above, the commercially available medium is suitable for the induction of anti-α4β7 antibodies, such as Bedolli, in GS-CHO cells. It can be used as a starting medium for production stage cultures to produce izumab. In one preferred embodiment, the antibody is produced by infecting GS-CHO cells grown in CD-CHO medium. and CD-CHO medium is supplemented as described herein.

[0327] Prior to the production stage, mammalian cells are first grown under environmental conditions that maximize cell growth and viability. Following the growth phase, a production phase is initiated, whereby the polypeptide Cell culture conditions are used that maximize peptide production. The growth and production phases may be performed using one or more It may precede or be separated by a transition phase. For example, in one embodiment, Prior to the production phase of the cell culture process, the parameters of the production phase of the cell culture are This is preceded by a transfer phase of culture.

[0328] In the proliferation phase, mammalian cells are cultured under conditions and for periods of time that are maximized for proliferation. Culture conditions such as temperature, pH, and dissolved oxygen (DO2) vary depending on the specific host used. Generally, pH is affected by the addition of acids (e.g., CO2) or salts. Using either a base (e.g., Na2CO3 or NaOH), The appropriate temperature range for culturing mammalian cells such as CHO cells is The temperature is between about 30 and 40 degrees Celsius, and preferably in the range of 36 to 38 degrees Celsius.

[0329] Commercial processes for the production of proteins by mammalian cells generally involve multiple, For example, different, e.g., growing culture vessels prior to the final production step. , there are at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth stages.

[0330] Once the cells have grown to sufficient numbers, they are transferred to large-scale production vessels, such as bioreactors. The mammalian host cell is then transferred to initiate the production step, whereby the mammalian host cell produces the polypeptide of interest, i.e. The cells are cultured under conditions that promote the production of antibodies. One of the cell culture media described herein developed for recombinant polypeptide production. Alternatively, methods and compositions according to the present invention may be used in combination with one or more of the above. can be used in combination with commercially available cell culture media.

[0331] Typically, the growth stage occurs at a higher temperature than the production stage. For example, the growth stage may occur at about 100° Celsius. The first temperature may be from about 35 degrees Celsius to about 38 degrees Celsius, and the production phase may be from about 30 degrees Celsius to about However, as described in the Examples, the present invention One of the improvements identified in the specification is the development of cellular markers for the production of anti-α4β7 antibodies, such as vedolizumab. Maintaining substantially similar temperatures during the growth and production phases of mammalian cells in cell culture This results in an increase in antibody titers from the cell culture. By maintaining a similar temperature, the antibody titer of vedolizumab is greater than 1g / L, e.g. The concentration was about 5 to 7 g / L.

[0332] Thus, in one embodiment, the present invention provides a method for producing a humanized anti-α4β antibody in a mammalian host cell. 7. A method for producing an antibody, comprising culturing mammalian host cells in a cell culture medium during an expansion step. The cells are then cultured in cell culture medium during the production phase, and both the expansion and production phases are The method is carried out at approximately the same average temperature for both stages, e.g., 36-38 degrees Celsius. In one embodiment, the average temperature of both the expansion stage and the production stage is 36.5 degrees Celsius. ~37.5 degrees, e.g., about 37 degrees Celsius.

[0333] Alternatively, the present invention provides a method for producing a humanized anti-α4β7 antibody in a mammalian host cell. The method includes culturing mammalian host cells in a cell culture medium during an expansion stage and then culturing the mammalian host cells in a cell culture medium during a production stage. The cells are cultured in cell culture medium at approximately the same average temperature range for both the expansion and production stages. , e.g., at any temperature in the range of 36-38 degrees Celsius, e.g., 36.5-37.5 degrees Celsius The present invention is characterized by a method that is carried out.

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

[0335] In one embodiment, the pH of the cell culture medium is 6.0 to 8.0, 6.5 to 7.5, 6.7 to The range is 7.0, 6.7 to 6.9, 6.95 to 7.05, or 7.1 to 7.2. Intermediate pH values, e.g. 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 the same as all other numbers described herein. Any combination of the above values ​​is also contemplated as being part of the present invention. Ranges of values ​​using as upper and / or lower limits are intended to be included within the scope of the invention. In some embodiments, the pH of the culture is changed from one pH to another, e.g. The pH may be shifted to a lower pH than at the time of inoculation. For example, the pH may be 6.9 to 7.1, 6.95 to 7. .05, or pH7.00 ±0.1, ±0.05 or ±0.02 pH range, 6 pH range: 7-7.0, 6.75-6.85 or pH6.8±0.1 or ±0.02 The timing of the shift may be after 2, 3, 4, or 5 days in 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 gene encoding an antibody that is genetically engineered to express an antibody. A method for producing a humanized anti-α4β7 antibody in a genetically engineered mammalian host cell, comprising: The animal host cells are cultured in a production medium at a first pH, which is then shifted to a second pH, and the second The pH of the second pH is lower than the first pH. For example, in some embodiments, The pH is 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 may range from pH 6.8 to pH 7.2. After the pH shift has occurred, the adjusted pH is 0.1 to 0.5 pH units, e.g. It can be decreased by 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. do.

[0337] As described in the Examples below, a pH shift during production can, for example, alter the basic acidic acid of an antibody. reducing the amount of isoforms, reducing the amount of acidic isoforms in the antibody, and / or Alternatively, the amount of the major isoform of the antibody can be increased.

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

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

[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 is fed with nutrients and acetate that are consumed during the production phase of the cell culture. Concentrated feed media can be supplemented with a concentrated feed medium that contains components such as amino acids. Such concentrated feed media can be based on any cell culture medium formulation. Most or a subset of the ingredients in the base can be added, e.g., at about 5x, 6x, 7x, 8x, or 10x the usual amount. 9x, 10x, 12x, 14x, 16x, 20x, 25-40x, 30x, 50x, 10 0x, 40-120x, 200x, 400x, 600x, 800x, and even around 1000 Concentrated feed media are often used in fed-batch processes.

[0342] In one embodiment, the production stage is a fed-batch culture. Fed-batch culture is a process for producing proteins from mammalian cells. This is a culture method that is widely used to produce proteins on a large scale. For example, Chu and d Robinson(2001),Current Opin.Biotechnol 12:180-87. Antibody production can be demanding on cells. However, the basal or starting medium is not capable of supporting high cell density and high levels of antibody production. Without fresh nutrients such as amino acids and energy sources, yields will decrease and cells will die. For example, a culture that consumes its supply of amino acids such as tyrosine may die. Fed-batch culture of mammalian cells involves the addition of a concentrated feed containing nutrients to the culture. A culture in which the medium is continuously or periodically fed. For example, feeding can be done daily, once every two days, This can be done on a set schedule, such as once every 3 days. one selected from the group consisting of glucose, zinc, manganese, uridine, and galactose; These additional nutrients can be added to the cells starting on or about the fourth day of the production phase, e.g., by using medium supplements. The feeding solution is added to the cell culture medium every day, every other day, every second day, and combinations thereof. In some embodiments, tyrosine is added at a schedule consistent with the production phase. For example, two boluses are added on days 4 and 11. In another embodiment, tyrosine is For example, in a feed replenisher, the components are added daily to the production culture. In some embodiments, glucose is added to the culture during the production phase. Course consumption can be measured, for example, by measuring glucose or its metabolic products, such as lactate. In some embodiments, a feed supplement containing glucose is added. and glucose levels are between 1 and 10 g / L, 2 and 7 g / L, 2.5 and 6 g / L, or about 7 It is controlled at a level of g / L.

[0343] In certain embodiments, fed-batch culture methods involve the use of mammalian cell culture media to replenish growing cells. It is used in the propagation stage of the cultivation process.

[0344] In a particular embodiment, the cell culture of the present invention is carried out in a large-scale bioreactor and is fed-batch culture. In one embodiment of fed-batch culture, the mammalian host cells and medium are first cultured. Additional culture nutrients are supplied to the culture vessel, either continuously or in discrete increments, to promote the growth of the culture. Fed-batch culture with or without periodic cell and / or product harvest before the end of the culture. For example, the entire culture (including cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture can include semi-continuous fed-batch culture, in which all components for cell culture are (containing cells and all culture nutrients) is delivered to the culture vessel at the start of the culture process. This is distinct from simple batch culture, which is

[0345] The methods described herein provide for the production of cells having a titer of humanized anti-α4β7 antibody greater than 1 g / L. In one embodiment, the methods described herein can be used to achieve cell culture. The method is 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. used to achieve a titer of humanized anti-α4β7 antibody of 1000 μg / L.

[0346] The methods disclosed herein allow for the achievement of antibody compositions with specific glycosylation patterns. In one embodiment, the methods described herein can be used to identify individuals with a population of 8 8% or more, 90% or more, or 91% or more total asialo-, agalacto-, or core fucosylated dimers Branched glycans (G0F), asialo, monogalactic, core fucosylated biantennary glycans (G 1F), and / or asialo-, digalacto-, and core fucosylated biantennary glycans (G2F) The present invention provides a population of humanized anti-α4β7 antibodies having glycosylation variants.

[0347] The methods disclosed herein also include the step of preparing an antibody composition having a constant amount of a major isoform of the antibody. In one embodiment, the methods disclosed herein can be used to achieve a positive More than 61% of the major Providing a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of an antibody isoform. In another embodiment, the methods disclosed herein provide a method for determining whether a patient is a 100% 15 ... , a composition having an amount of a major antibody isoform of 62% or more (e.g., vedolizumab In one embodiment, the method disclosed herein provides a method for producing a clarified product by CEX. A composition (e.g., a composition having an amount of a major antibody isoform of 63% or more as determined by In one embodiment, a clarified recovery comprising vedolizumab is provided. The method comprises: In another embodiment, a composition (e.g., a clarified recovery comprising vedolizumab) is provided comprising: The methods disclosed herein provide a method for detecting 65% or more of the major antibodies as determined by CEX. and providing a composition (e.g., a clarified recovery comprising vedolizumab) having an amount of an isoform. .

[0348] IV. Downstream Production Methods Compositions Comprising Anti-α4β7 Antibodies or Antigen-Binding Portions thereof, E.g., Vedolizumab of the Invention can be produced by the upstream cell culture methods and compositions provided herein. These upstream process technologies, optionally in combination with downstream production methods, can be used to produce antibodies or The antigen-binding portion can be isolated, purified, and / or formulated. Typically, mammalian cells are used to express the protein of interest and to recover recombinant antibodies. It has been engineered to secrete proteins into the cell culture medium, making it the first step in the purification process. The first step is to separate the cells from the medium. The harvested medium can then be further purified, for example by filtration. The medium, e.g., the clarified harvest, can then be clarified to remove cellular debris, Some additional ingredients remove unwanted proteins, salts, minerals, or other undesirable elements. The recombinant antibody may be subjected to additional purification steps to remove contaminating soluble proteins. and purifying the polypeptide, the procedure may include one or more of the following: Examples of suitable purification procedures include: Affinity chromatography using resins that react with cations; ion exchange columns or Resins for cation exchange chromatography (CEX), e.g. SP-Sepharose (商標) Ma or CM-Sepharose (商標) Fractionation on hydroxyapatite; anion exchange chromatography AEX; Hydrophobic Interaction Chromatography (HIC); Mixed-mode Chromatography (MIX) chromatography;ethanol precipitation;chromatofocusing;ammonium sulfate precipitation; For example, Sephadex G-75 (商標) Gel filtration using ultrafiltration and / or dialysis filtration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2 At the end of the purification process, the recombinant protein is highly pure and suitable for human therapeutic use, e.g., in pharmaceutical antibody formulations described below. After purification, the highly pure recombinant protein can be converted into a pharmaceutical formulation suitable for human administration. It may be ultrafiltered / diafiltered (UF / DF).

[0349] Following diafiltration and ultrafiltration, the antibody formulation may remain liquid or may be lyophilized. In one embodiment, the dry lyophilized antibody formulation can be 150 mg, 1 80mg, 240mg, 300mg, 360mg, 450mg, or 600mg of anti-alpha It will be provided in a single-dose vial containing the 4β7 antibody, which will be reconstituted with sterile water or other liquid for administration. In another embodiment, the anti-α4β7 antibody, e.g., vedolizumab, can be used to treat a patient in need thereof. The drug is stored in a container, e.g., a vial, syringe, or other container, at approximately 2-8°C until administration to the receiving subject. In some embodiments, the cartridge is a stable liquid pharmaceutical composition. The reconstituted lyophilized formulation or stable liquid pharmaceutical composition of 4β7 antibody is about 0% to 5.0% %, 0%-2%, ≦2%, ≦1%, ≦0.6%, or ≦0.5% aggregates.

[0350] Thus, in some embodiments, provided herein are humanized anti-α4β7 A reconstituted lyophilized antibody formulation or a stable liquid formulation comprising an antibody, or an antigen-binding portion thereof. In some embodiments, the reconstituted lyophilized form of the anti-α4β7 antibody is a pharmaceutical composition. The formulation or stable liquid pharmaceutical composition may be about 11%-16%, 12%-15%, <14%, < In some embodiments, the nucleotide sequence includes 13%, <12%, or <11% of the basic isoform species. In this embodiment, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody is 5%-75%, 66%-74%, 67%-73%, at least 65%, at least 66% , at least 67%, at least 68%, at least 69%, or at least 70% In some embodiments, the reconstituted lysate of an anti-α4β7 antibody comprises a major isoform. The lyophilized formulation or stable liquid pharmaceutical composition may have a viscosity of 92% to 98%, 92% to 97%, 92% to 96%, 92%-95%, at least 92%, at least 93%, at least 94%, or or at least 95% of the total asialo-, agalacto-, and core fucosylated biantennary glycans (G0 F), asialo, monogalactic, core fucosylated biantennary glycans (G1F), and / or asialo-, digalacto-, and core fucosylated biantennary glycans (G2F) glycosylation barrier In some embodiments, the anti-α4β7 antibody contains The reconstituted lyophilized formulation or stable liquid pharmaceutical composition may contain 45% to 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, 6 2% or less, 61% or less, 60% or less, 57% or less, 55% or less, 53% or less, 52% or less In some embodiments, the reconstitution of an anti-α4β7 antibody comprises a GOF content of 50% or less. The lyophilized formulation or stable liquid pharmaceutical composition may be 25% to 45%, 26% to 42%, 27%-40%, 30%-40%, 30%-45%, at least 25%, at least 26 %, at least 27%, at least 28%, at least 29%, at least 30%, at least at least 31%, at least 32%, at least 33%, at least 34%, at least 3 5%, 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% G1 In some embodiments, the reconstituted lyophilized formulation of an anti-α4β7 antibody comprises a F content. Alternatively, the stable liquid pharmaceutical composition may be 2% to 8%, 2.5% to 7.5%, 3% to 7%, 3.5 %~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% G2F content .

[0351] In some embodiments, a reconstituted lyophilized formulation or stable liquid formulation of an anti-α4β7 antibody is The pharmaceutical composition may contain amino acids (e.g., arginine, histidine, and / or histidine). monohydrochloride), sugars (e.g., sucrose), surfactants (e.g., polysorbate 80), and / or buffers (e.g., citrate, phosphate, etc.) In one embodiment, a reconstituted lyophilized formulation or a stable liquid pharmaceutical formulation of an anti-α4β7 antibody is The pharmaceutical composition comprises L-arginine, L-histidine, L-histidine monohydrochloride, sucrose, and / or polysorbate 80. In another embodiment, the reconstituted anti-α4β7 antibody The lyophilized formulation or stable liquid pharmaceutical composition may contain citrate, arginine, histidine, , and / or polysorbate 80.

[0352] The syringe or cartridge may contain 1 mL or 2 mL of drug (e.g., 160 mg / m L dose) or, for example, a higher dose (at least 320 m For volumes of 1000 g or 400 mg or more, the syringe or catheter may be larger than 2 ml. The cartridge may comprise 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, The composition may comprise at least about 400 mg, or at least about 500 mg, of an anti-α4β7 antibody. In some embodiments, the container, e.g., syringe or cartridge, can contain about 20 to 300 mg of the active ingredient. 120 mg, about 40 mg to 70 mg, about 45 mg to 65 mg, about 50 mg to 57 mg, or about 5 It can be manufactured to deliver 4 mg of an anti-α4β7 antibody, e.g., vedolizumab. In embodiments, the syringe or cartridge contains about 90-120 mg, about 95-115 mg, About 100 to 112 mg, or about 108 mg, of an anti-α4β7 antibody, e.g., vedolizumab In other embodiments, the syringe or cartridge may be manufactured to deliver about 14 0~250mg, approx. 150~200mg, approx. 160~170mg, approx. 160~250mg , about 175mg to 210mg, about 220mg to 260mg, or about 160mg, about 165mg g, about 180 mg, or about 200 mg of an anti-α4β7 antibody, e.g., vedolizumab. It can be manufactured to reach

[0353] The administration of the formulation can be by parenteral injection, such as intravenous, subcutaneous, or intramuscular. Intravenous injections are performed using sterile isotonic saline, a buffer solution such as phosphate buffered saline or Ringer's ( It can be further diluted with lactobacillus or dextrose solution and then injected. In some embodiments, the anti-α4β7 antibody is administered by subcutaneous injection, e.g., at the start of treatment. about 54 mg, about every 2, 3, or 4 weeks after the start of treatment, or after the third subsequent dose, It is administered in a dose of 108 mg, or about 165 mg or about 216 mg.

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

[0355] In various embodiments described herein, cation exchange chromatography (CEX) is used The antibody or antigen-binding portion thereof, e.g., vedolizumab, may be used to identify the major antigens present in the population. isoform(s), basic isoform(s), and acidic isoform(s). The CEX method fractionates antibody species according to their overall surface charge, allowing the determination of relative abundance. After dilution to low ionic strength using the mobile phase, the test sample is diluted in an appropriate buffer, e.g. For example, a CEX column equilibrated with 10 mM sodium phosphate, pH 6.6, e.g., Dio nex Pro-Pac (商標) WCX-10 column (Thermo Fisher Scientific The antibodies can be injected into the same buffer. Elution can be performed using a sodium chloride gradient in the buffer. Protein elution is monitored by spectroscopy at 280 nm. The peaks can be assigned to the classification of acidic, basic, or major isoforms. The basic peak elutes from the column at a shorter retention time than the major isoform peak. The peak elutes from the column with a longer retention time than the major isoform peak. Percent isoforms, total percent acidic species, and total percent basic species are reported. The retention times of the major isoforms in the sample were compared to those of the reference standard to determine conformational identity. Determine.

[0356] In one embodiment, the CEX assay involves diluting the test sample to low ionic strength and Inject this buffer into a CEX column equilibrated with 100 M sodium phosphate, pH 6.6. The column was eluted with a NaCl gradient of 1000 ng / ml and the peaks were monitored at 280 nm. The peaks were classified as acidic, predominantly The acidic peak elutes first with the shortest retention time and is assigned as the main peak. The peak elutes next, and the basic peak elutes with the longest retention time, and the peak areas are quantified. The amounts are calculated as a percentage of the total peak area.

[0357] In various embodiments described herein, hydrophilic interaction phase separation (HILIC) is used. to determine the glycoform profile of an antibody or antigen-binding portion thereof, e.g., vedolizumab. The HILIC method fractionates the free fluorescently labeled carbohydrates. The can was digested with N-glycosidase F to obtain fragments of the antibody or its antigen-binding portion. The released glycans can be released from the sample using Prozyme (Hayward, CA). (USA) GlykoPrep Rapid Glycoprotein Sample Standard methods such as those used in the Le Preparation System can be used to immediately label with a fluorescent tag, such as the InstantAB fluorescent tag. The glycans can be fractionated using ultra-high performance liquid chromatography. In some embodiments, the labeled glycans are selected from the group consisting of ACQUITY UPLC BEH amide capsids. Lam (Waters Corporation, Milford, MA (USA)) and The labeled glycans were fractionated using an acetonitrile / ammonium formate gradient system. It can be detected by fluorescence emission at 344 nm using an excitation wavelength of 278 nm. Thus, when used in the context of the present invention, HILIC fractionates free fluorescently labeled glycoforms. HILIC, preferably where intact glycoforms are purified by digestion with N-glycosidase F. The released glycoforms are then released from the sample of antibodies or antigen-binding portions thereof by For this purpose, standard labeling techniques are used, preferably Prozyme (Hayward, CA (USA) ) GlykoPrep Rapid Glycoprotein Sample Preparation Fluorescent tags, preferably those used in the separation system, InstantAB is immediately labeled with a fluorescent tag and is then subjected to high performance liquid chromatography, preferably ACQUITY UPLC BEH Amide Column(Waters Co poration, Milford, MA (USA) and acetonitrile / formic acid acetonitrile The labeled glycoforms were fractionated using an ammonium gradient system and excited at an excitation wavelength of 278 nm. The assay controls are detected by fluorescence emission at 344 nm using a commercially available standard, For example, InstantAB-labeled glucose homopolymer ladder (Agilent Technologies) A suitable solution from hnologies, Inc., Santa Clara, CA (USA) Quantitation can be achieved by determining the relative area percentage of the detected sugars. Based on. G0F (asialo-, agalactosylated biantennary glycan, core fucosylated); 1F (asialo, monogalactosylated biantennary glycan, core fucosylated); and G2F ( Percent peak area of ​​species (asialo, digalactosylated biantennary glycans, core fucosylated) The event is reported.

[0358] In various embodiments described herein, size exclusion chromatography (SEC) is used. using a monomer present in a population of antibodies or antigen-binding portions thereof, e.g., vedolizumab, To determine the relative levels of high molecular weight (HMW) aggregates and low molecular weight (LMW) degradation products The SEC method can be used to separate the size of antibody monomers from HMW species and LMW degradation products. Test samples and reference standards were prepared using commercially available S For example, in some preferred embodiments, SEC analysis can be performed using an EC column. The analysis was performed using a G3000 SWxl column (Tosoh Bioscience, King f Prussia, PA (USA)), or preferably two G3000 SWxl column and isocratic phosphate-sodium chloride buffer Elution of protein species can be performed using a HPLC system at pH 6.8. The main peak (monomer) and total peak areas are evaluated to determine purity. In one embodiment, SEC analysis was performed using two G3000 SWxl columns connected in tandem. The method involves injecting a sample into a column and monitoring the elution of protein species at 280 nm. Isocratic phosphate-chloride where the main peak (monomer) and total peak areas are measured The purification was performed in sodium buffer system, pH 6.8. Purity (%) of the sample (as monomer %) The percent of HMW aggregates, and / or percent LMW degradation products are reported.

[0359] Residual CHO host cell protein (HCP) impurities present in antibody preparations are If necessary, measured by enzyme-linked immunosorbent assay (ELISA) using standard techniques Cygnus Technologies, Southport, New Jersey For this purpose, we have developed a kit designed for this purpose, such as the CHO HCP ELISA Kit 3G from C (USA). Many ELISA kits that have been developed to detect host cell proteins in test samples are available on the market. The capture can then be performed using immobilized polyclonal anti-CHO HCP antibodies. The captured proteins are then detected using a suitable detection agent, e.g., horseradish peroxidase of the same antibody. In this exemplary embodiment, the CHO The amount of captured peroxidase, which is directly proportional to the concentration of HCP, was determined by peroxidase substrate 3. Colorimetry at 450 nm using 3',5,5'-tetramethylbenzidine (TMB) Therefore, the CHO HCP assay can be used to measure peroxidase activity. The enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB) was detected colorimetrically at 450 nm. The horseradish peroxidase activity of polyclonal anti-CHO HCP antibodies was converted to a substance that was quantified by ELISA. Polyclonal anti-CHO detected after binding to a peroxidase-labeled version The test involves capturing HCPs using HCP antibodies. The HCP concentration is determined by the The amount of TA in the antibody preparation can be determined by comparison with a CHO HCP standard curve, such as Reported as a percentage of total protein levels.

[0360] The following examples describe improved methods for producing antibodies in mammalian cell culture and The following examples are provided for illustrative purposes only and are not intended to be limiting of the scope of the present invention. The scope of the present invention is not intended to be limited in any way. Commercially available reagents mentioned in the examples include Used according to manufacturer's instructions unless otherwise indicated. EXAMPLES

[0361] Vedolizumab was previously used in dihydrofolate reductase-deficient (DHFR) Chinese hamsters. It was produced in a Chinese oocyte ovary (CHO) cell line (Urlaub and Chasin ( 1980)Proc.Natl.Acad.Sci.USA,77:4216-4220 (U.S. Patent Application Publication No. 20070122404). The selected clone was Although expression was stable, the production level was less than 2 g / L. Considering the high demand for materials, With this in mind, researchers have attempted to develop more highly productive cell lines.

[0362] Testing different selection systems on several thousand clones and culturing several clones in bioreactors After evaluating the effect of glutamine synthase-deficient (GS-) Chinese hamster ovary (G In one example, the GS CHO system produced 6.7 g / L antibody. Additional studies have demonstrated that culture conditions and media supplement components may affect certain quality attributes. In the following examples, it was shown that vedolizumab produced in GS-CHO cells can be used as a therapeutic agent. We describe our experiments to improve the quality of the

[0363] Example 1. Impact of cell culture production on product quality attributes To improve the quality characteristics of the crop, the Plackett-Burman method was used to screen the A leaning design was created and five process parameters were analyzed for eight bioreactor runs. The effects of regulator modifiers were evaluated. Cells were thawed and cultured using standard scale-up strategies. The cells were subcultured for 3 days and transferred from shake flasks to a 3 L production bioreactor with a working volume of 1.75 L. The 15-day bioreactor production included two feeds (unless otherwise specified). A bolus delivery strategy was used.

[0364] Design: Five different factors were selected for this screening study: 1) temperature 2) change in feeding strategy (2 g / L vs. 6 g / L glucose); 3) p H (6.85 vs. 7.05); 4) Change in uridine, manganese chloride, and galactose in the feed solution and 5) the addition of Sigma Gal+ / ExCell® glycerol. These five factors are listed in Table 1. Tested eight different bioreactor runs based on the Burman screening design It was done.

[0365] [Table 1]

[0366] [Table 2]

[0367] Feed: 3 x 10 to production bioreactor culture 5 Inoculate viable cells / mL and on day 4, Feed medium was added to the cultures based on cell growth and glucose consumption rates according to the study design. The feeding dose was set at the upper limit of 7 g / L glucose concentration. The temperature shift was Starting on day 7, patients were warmed from 37 degrees Celsius to either 33 degrees Celsius or 35 degrees Celsius, depending on the study design. All production bioreactor cultures were cultured at day 18 or after reaching 50% target cell viability. % or less was collected if it came first.

[0368] Product quality: The results of the conditions tested in Table 1 indicate (predict) conditions that improve product quality attributes. The data were analyzed using JMP software to investigate the effect of the variances in the variances (using the Profiler).

[0369] The results of the prediction profile are shown in Figure 1. In general, the increase in antibody titers, the base of vedolizumab, To achieve a reduction in toxic and acidic species and an increase in G2F isoforms of vedolizumab Conditions are generally favorable.

[0370] As shown in Figure 1, the model shows the rate of glucose consumption, the shift to 37 degrees Celsius and pH 6.85. The authors predicted that operation with a feed delivery based on the addition of UMG to the feed solution would be optimal. In contrast, the results in Figure 1 show that the G2 isoform, an acidic or basic species of vedolizumab, The addition of Gal+ was not essential because it had little effect on the antibody titer. Furthermore, a temperature shift from 37°C to 33°C had a negative effect on titer. However, it is advantageous to maintain cell production at 37 degrees, whereas a temperature below 7.05 (e.g., 6 A pH of .85 to <7) improves titer while maintaining low levels of the G2 isoform This suggests that this was the case.

[0371] The predictive profiler also uses UMG combinations to help you reach your carbohydrate goals. The advantages of using 1,2-diaminobutyric acid as a marker for the production of 1,2-diaminobutyric acid have been demonstrated, as well as higher titer levels of antibodies and lower levels of acidic species. The glucose consumption-based feeding strategy and the lower pH (6.85) compared to pH 7 resulted in better This shows advantages in achieving a lower ratio of basic species.

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

[0373] Design: GS-CHO cells were used in this experiment. The experiment was performed at 6.85 or 7.05. pH factors examined and supplement ingredients examined at 33x, 50x, and 66x concentrations Designed to accommodate UMG as (in production). Addition with pH shift on day 4 On the first day of the experiment, the titration pump of V01 was A loose connection caused a significant overpumping of the titrant, necessitating the reactor being shut down. The run template for V10 was the same as that for V01 because V10 had similar media and cells. The line template was quickly replaced to reflect the No temperature shift was used since no points were predicted.

[0374] Cells were fed using a consumption-based feeding method, where the current proliferation rate and Consumption rates are estimated to predict glucose requirements.

[0375] In the experiment, the amount of antibody titers, acidic species, basic species, major species, G0F species, G1F species, G2F species The feed medium (33 × (33 mM uridine, 0.066 mM manganese, and 165 mM galactose) , 50× (50 mM uridine, 0.1 mM manganese, and 250 mM galactose ), and 66× (66 mM uridine, 0.132 mM manganese, and 330 mM UMG supplementation with galactose (at various concentrations) and pH (adjusted at 7.05 and 6.85) were investigated. The effect of UMG supplementation on the CD-CHO production medium was evaluated. The experimental design is shown in Table 3.

[0376] [Table 3]

[0377] Results: The results of the experiment are used to generate predictive profiles and to assess the effect of different conditions on cell culture. The effect of conditions and the quality attributes of vedolizumab products were further investigated. The results of the predictive profile As shown in Figures 2A-2H (antibody titer vs. UMG (Figure 2A), acidic species % (CEX) vs. UMG (Figure 2H) B), percentage of basic species (CEX) vs. UMG ( Fig. 2C ), percentage of major species (CEX) vs. Percentage of UMG (Fig. 2D), G0F species vs. UMG (Fig. 2E), G1F species (Figure 2F), percentage of G2F species versus UMG (Figure 2G), and total glycans vs. UMG (Figure 2H). In each of Figures 2A-2H, vessels without UMG replenishment are indicated by a shaded This is indicated by a dot at the left edge of the region (the shaded region represents UMG replenishment). The two pH values ​​tested (pH 7.05 and pH 6.85) are shown in Figures 2A to 2H.

[0378] Cells with increased UMG replenishment, as illustrated in Figs. The cultures showed a higher number of primary, G1F, G2F, and IFN-γ species, respectively, compared to cultures without UMG supplementation. and the percentage of total glycans. Furthermore, cultures with UMG supplementation , lower titers (Figure 2A), lower acidic species (Figure 2B), and fewer soluble species (Figure 2C). B), less basic species (FIG. 2C), and less G0F species (FIG. 2E).

[0379] Across the range of UMG concentrations tested, the concentration of UMG had minimal effect on potency and acidic species. The effect on the percentage of basic species is low (i.e., basic species are more abundant at high UMG concentrations). The effect on the percentage of major species appeared to be high (although it decreased slightly when (i.e., the major species increased with increasing UMG concentration). There was no significant change in the carbohydrate profile.

[0380] Finally, regarding pH, as illustrated in Figures 2A-2H, low pH (6.85) Operation at pH 7.05 appears to perform better than operation at high pH. I saw it.

[0381] In a separate experiment, GS-CHO cells recombinantly expressing vedolizumab were treated with uridine (20. 91 mM), manganese (0.039 mM), galactose (96.69 CD-CH supplemented with feed medium containing 0.1 mM (0.1 mM concentration) and zinc (0.117 mM concentration). The culture was grown at a 3000 L scale in 1000 mL of O production medium. Feed was added daily to the culture starting on day 4. The amount of UMG added in each daily supplement was as follows: 0.17–0.63 mM uridine, 0.31-1.2 μM manganese, and 0.77-2.9 mM galactose. The uridine concentration in the production medium after daily supplementation from day 4 to day 13 of culture until the day of harvest. The average cumulative supplementation concentration of manganese was approximately 0.00515 mM. The mean cumulative supplemental concentration of zinc was approximately 12.8 mM, and that of galactose was approximately 12.8 mM. A filling component was added daily at a concentration of about 0.117 mM from the 4th to 13th days, and the viability was improved by the 14th day. The average cumulative supplement concentration in the production medium was approximately 0.0154 mM. The averages given refer to the average of the lots tested.

[0382] After 14 days of culture, the antibodies were harvested and purified, and the fucosylated glycans were analyzed 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 study was to characterize the quality attributes of vedolizumab, specifically its potency and percentage of basic species. The aim of this study was to test the effect of the levels of lysine and arginine in the feed medium on the expression of lysine and arginine in the culture medium.

[0385] Design: This study was conducted to evaluate antibody titers produced in GS-CHO cells and by CEX. The relationship between the level of basic species (C-terminal lysine level) and lysine and arginine as determined by The study was designed to evaluate the effect of nin concentration on the growth of cereals. was carried out.

[0386] Results: Effect of different arginine and lysine concentrations on the percentage of basic species The results of comparing the effects of 10-HT200 and 10-HT200 were shown in Figure 3A, and the effects on antibody titers were shown in Figure 3B. Both x-axes in Figure 3B are labeled as high concentration (H), medium concentration (M), or For example, "LM" corresponds to low levels of lysine and arginine. Refers to lysine (see Table 5) and moderate levels of arginine (see Table 5) These results suggest that low levels of lysine and arginine reduce basic species compared to controls. Although minimal, low levels of these amino acids had a negative effect on antibody titers (5–4 g, approximately 20%).

[0387] [Table 5]

[0388] A predictive analysis was performed based on the arg / lys experiment. The JMP analysis showed that the Thus, the optimal conditions for reducing basic species are low lysine and low arginine levels (LL). 4B and 4C show that "LM" (low lysine and medium arginine) and "LH (low lysine and high arginine) combination. However, low lysine (5g / L) and medium arginine (6.5g / L) are recommended because they affect titer production. / L) levels were used in Example 4.

[0389] Example 4: Effect of zinc on produce quality attributes The purpose of this experiment is to investigate the mechanism of VEDLIZ during cell culture, and more specifically, during the production phase of the culture system. The aim of this study was to test the effect of zinc levels on the quality attributes of the mab.

[0390] Three levels of zinc were tested, as listed in Table 6 below. Cultures were incubated for 14 The recovery ranged from 1 to 18 days. The zinc supplement consisted of 22x UMG and reduced lysine and arginine. The combination of lysine and arginine ("LM" as described in Example 3) in the feed was evaluated. The concentrations of nin were 5 g / L and 6.4 g / L, respectively.

[0391] [Table 6]

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

[0393] As shown in Figures 5B and 5C, with increasing zinc levels, the basic and acidic profiles Furthermore, a similar basic profile was achieved up to day 16 of culture. As shown in Figure 5D, the highest level of main (m ajor)) species was obtained at 57.2uM zinc (4Zn) on day 14 of recovery.

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

[0395] To determine whether there was an effect on the product quality of vedolizumab, various zinc Temperature was also tested in conjunction with concentration. Temperatures of 33, 35, and 37 degrees Celsius were tested on a scale of 0 to 4. The 37°C dietary supplement was tested in combination with 100 mg of zinc (see Table 6 above). It was most effective in maintaining the desired quality of the vedolizumab product. For example, Figure 6A shows The % basic isoform of the antibody under various zinc conditions and temperatures is shown. The percent levels of basic species after supplementation with 57.2uM zinc (4Zn) in Below the specified upper limit of the sex species (isoform) (indicated by the black line, i.e., 1 3% basic antibody isoforms (CEX), thus meeting the specification requirements. , supplemented with 57.2 uM zinc (4Zn) at 37°C but not at 33°C, as shown in Figure 6B. The sum of the glycans after the purification was above the lower acceptance threshold (shown by the black line). As shown in Figure 6C, protein aggregation (HMW species, the acceptance criteria is approximately 1.4% or less) at low temperatures. The data in Figure 6D show that increasing the incubation period increases the number of days of Titer production was generally faster at 37°C compared to 33°C and 35°C on day 14. The data in Figure 6E show that higher vedolizumab titers were generated. This suggests that the number of sexual species increased with increasing temperature and prolonged incubation. represents the upper tolerance limit for acidic species.

[0396] Overall, the data in Figures 6A-6E indicate that vedolizumab production in GS-CHO cells is They showed that it is best to stop fermentation by day 16 and maintain a production temperature of around 37°C. Furthermore, 4Zn offers benefits as a supplement.

[0397] Example 5: Effect of culture days on product quality attributes The purpose of this study was to test the effect of incubation time on the quality attributes of vedolizumab. there were.

[0398] 12, 13, 14, 15, 16, or 17 in GS-CHO cells after two separate runs After 1 day of incubation, the percentage of acidic antibody species, the percentage of basic antibody species, and the percentage of The percentage of major species and the titer of vedolizumab were assessed.

[0399] As shown in Figure 7D, an increase in titer was observed with increasing culture time. This increase in titer was due to an increase in acidic species and This was accompanied by a decrease in the major species. The basic species did not appear to be affected during run 1, but A trend towards increased basic species was observed during row 2. The data showed that the fermentation was stopped by day 16. This indicates that it is generally best to harvest the antibody by day 15. Occasionally, minimal levels of acidic species were obtained.

[0400] Example 6: Effect of pH on product quality attributes The objective of this study was to investigate the effect of pH during cell culture at the production stage on the quality attributes of vedolizumab. Specifically, the introduction of a pH shift in the production medium was examined using a bed The impact on the quality attributes of lizumab was evaluated.

[0401] The initial pH of the medium during the production stage was evaluated in the range of pH 6.8 to pH 7.2. During the experiment, the pH of the medium decreased to a final pH range of pH 6.6 to pH 7.0. The onset time of the pH shift ranged from 86 to 108 h, 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 36 hour interval describes the pH ramp time.

[0402] Highest % major antibody isoform (determined by CEX) and least acidic antibody isoform The foam % (determined by CEX) increased significantly at a final pH of ≥6.7 (Figure 8A) and ≥12 A pH shift completion time of less than 2 hours was observed (Figure 8B). pH shift during cell culture increases the levels of acidic antibody isoform species in vedolizumab preparations. This suggests that it may be possible to reduce

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

[0404] Cation exchange chromatography (CEX) is a method for isolating pedigree peptides according to their overall surface charge. Fractionate the mab antibody species (major isoforms, basic species, and acidic species). After dilution to low ionic strength with 10 mM sodium phosphate, pH 6.6, the test samples were DionexPro-Pac equilibrated with (商標) WCX-10 column (Thermo Fisher Scientific, Waltham, MA (USA) and Elute with sodium chloride in the same buffer. Protein elution is monitored at 280 nm. The peaks are then filtered and assigned to the classification of acidic, basic, or major isoforms. The percent isoforms, total percent acidic species, and total percent basic species are reported. The retention times of the major isoforms in the sample are compared to the retention times of the reference standards to determine conformation. Determine the seat.

[0405] Carbohydrate profile of vedolizumab by hydrophilic interaction phase separation (HILIC) The intact glycans are generated by fractionation of the released fluorescently labeled carbohydrates. Protein samples are released by digestion with Prozyme F and then GlykoPrep Rapid Glyc from me(Hayward, CA(USA)) Using the Polyprotein Sample Preparation System InstantAB fluorescent tag (Agilent Technologies, Inc., The labeled glycans were then immediately labeled with 100mM glycan ELISA kit (Santa Clara, CA, USA). , ACQUITY UPLC BEH Amide column (Waters Corporation) on, Milford, MA (USA)) and an acetonitrile / ammonium formate gradient system. Detection is by fluorescence emission at 344 nm using an excitation wavelength of 278 nm. The assay control was a commercially available InstantAB-labeled glucose homogenate. Polymer ladder (Agilent Technologies, Inc., Santa The determination of the appropriate resolution was performed by using a 30-μm NMR spectrometer (Microfluidic Devices, Inc., Clara, CA, USA). is based on the relative area percentage of the sugars detected. G0F (asialo-, agalactosyl G1F (asialo, monogalactosylated biantennary glycan, core fucosylated); G2F (asialo, monogalactosylated biantennary glycan, core fucosylated); glycan, core fucosylated); and G2F (asialo, digalactosylated biantennary glycan, The peak area percent of the core fucosylated (F) species is reported.

[0406] Size exclusion chromatography (SEC) was used to determine the purity of vedolizumab. The reference standard sample and the test sample (75 μg) were placed in two tandemly connected G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA (USA)) and isocratic phosphate-sodium chloride buffer The method is used to analyze high molecular weight (HMW) and low molecular weight (LMW) species. The antibody monomer is separated from the degradation products (MW). Elution of protein species is monitored at 280 nm. The purity is determined by evaluating the main peak (monomer) and total peak areas. The purity (%) of the sample (calculated as % monomer) and % aggregates are reported.

[0407] Equivalent Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. or may be ascertained using no more than routine experimentation. It is intended that this invention be covered by the following claims. The contents of all cited references, patents, and published patent applications are hereby incorporated by reference. is incorporated into.

[0408] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

Claims

Claim 1: A method for producing a composition comprising a humanized anti-α4β7 antibody, said method comprising culturing mammalian host cells in a production medium comprising zinc, thereby producing a composition comprising said humanized anti-α4β7 antibody; The method wherein the mammalian host cell is genetically engineered to express a humanized anti-α4β7 antibody, 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.

2. The method described in claim 1, wherein the composition contains 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.

3. The method described in claim 2, wherein the composition contains less than about 16% of basic isoforms of the humanized anti-α4β7 antibody, less than about 14% of basic isoforms of the humanized anti-α4β7 antibody, or less than about 13% of basic isoforms of the humanized anti-α4β7 antibody.

4. The concentration of zinc in the production medium is 2 μM to 60 μM, or the method comprising supplementing the production medium with zinc by adding a feed medium containing zinc to the production medium; The method according to any one of claims 1 to 3.

5. A method according to any one of claims 1 to 4, wherein the production medium contains 5.0 to 8.8 g / L of lysine and 3.0 to 12.0 g / L of arginine.

6. 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 that a composition comprising the humanized anti-α4β7 antibody is produced; the mammalian host cells are cultured at about the same temperature during both the expansion step and the production step; 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; The method.

7. The method described in claim 6, wherein the method is a method for producing a composition containing a high 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 stage and the production stage.

8. The temperature is 36 to 38 degrees Celsius, the average temperature is between 36.5 and 37.5 degrees Celsius; or 8. The method of claim 6 or 7, wherein the temperature is an average temperature of about 37 degrees Celsius.

9. The production medium has a temperature in the range of 36-38 degrees Celsius, 36.5-37.5 degrees Celsius, or about 37 degrees Celsius; and / or the production medium has a pH in the range of 6.5 to 7, or 6.8 to 7.0; and / or the production medium having a glucose level maintained at about 7 g / L or less during the production stage; The method according to any one of claims 1 to 8.

10. A method according to any one of claims 1 to 9, wherein the production stage lasts for 14 days or less or between 10 and 17 days.

11. The method of any one of claims 1 to 10, carried out in a large-scale bioreactor.

12. A method described in any one of claims 1 to 11, wherein the production step results in a titer of the humanized anti-α4β7 antibody greater than 3 g / L, or results in a titer of about 3 to about 8 g / L, or results in a titer of about 5 to about 7 g / L.

13. The method of claim 1, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

14. 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; or The method of any one of claims 1 to 13, wherein the humanized anti-α4β7 antibody is vedolizumab.

15. A composition comprising a humanized anti-α4β7 antibody produced or obtainable using a method according to any one of claims 1 to 14.

16. The composition of claim 15, comprising a population of humanized anti-α4β7 antibodies having glycosylation variants of (i) 90% or more or (ii) 92-95% of total asialo-, agalacto-, core-fucosylated biantennary glycans (G0F), asialo-, monogalacto-, core-fucosylated biantennary glycans (G1F), and / or asialo-, digalacto-, core-fucosylated biantennary glycans (G2F).

17. 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.

18. The cell culture of claim 17, wherein the production medium contains zinc supplemented at a concentration of about 0.005 mM to 0.045 mM on the day of harvest.

19. The cell culture of claim 17 or 18, wherein the production medium further contains uridine, manganese, and galactose (UMG).

20. The humanized anti-α4β7 antibody expressed a. 16% or less, 15% or less, 14% or less, 13% or less, or 12% or less basic isoforms; and / or b. has an isoform distribution that comprises 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 17 to 19.

21. The cell culture of any one of claims 17 to 20, 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%, 92-95%, 91-92%, 91-92.5%, or 91-93%.

22. A cell culture described in any one of claims 17 to 21, wherein the cell culture further contains arginine and / or lysine.

23. A cell culture described in any one of claims 17 to 22, wherein the host cells are CHO cells.

24. A humanized anti-α4β7 antibody produced by the cell culture described in any one of claims 17 to 23.

25. A method for producing a composition comprising a humanized anti-α4β7 antibody, said 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.

26. The method described in claim 25, wherein the second pH is 0.1 to 0.5 pH units lower than the first pH, and / or 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.

27. The method of claim 25 or 26, wherein the mammalian host cells are cultured at the first pH for 120 hours or less, 85 to 110 hours, or 90 to 100 hours.

28. A method described in any one of claims 25 to 27, further comprising recovering the anti-α4β7 antibody from the second production medium.

29. The method of claim 28, 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.

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