Method for producing anti α4β7 antibody

Abstract

To provide a method for purifying an anti α4β7 antibody.SOLUTION: A method for purifying an anti α4β7 integrin antibody such as vedolizumab from liquid solution derived from a clarified harvest of a mammal cell culture, for example, includes: (i) a step of providing a harvest of a clarified cell culture obtained from a culture of a recombinant host cell which expresses the anti α4β7 antibody or an antigen-binding portion thereof; and (ii) a step of purifying the anti α4β7 antibody or an antigen-binding portion thereof from the harvest of the cell culture, where the antibody is exposed to pH of 4.0 or less for within 24 hours.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for purifying an anti-α4β7 antibody or a fragment thereof.

[0002] Related Applications This application is a continuation of U.S. Provisional Application No. 62 / 859,499, filed June 10, 2019. No. 4, filed on Nov. 23, 2003, the entire contents of which are incorporated herein by reference. Be absorbed.

[0003] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference. The entire contents of the above ASCII text file was created on June 5, 2020. The file is named T103022_1090WO_SL.txt and has 10,009 The size in bytes. [Background technology]

[0004] Large-scale, economical purification of proteins is becoming increasingly important in the biotechnology industry. Generally, biological drugs are developed by producing large amounts of a therapeutic protein of interest. Prokaryotic organisms, e.g., bacteria, or eukaryotic organisms, e.g., mammalian or It is produced by cell culture using fungal cell lines. The cell lines used are viable organisms. Since the cells are large, they need to be supplied with a complex cell culture medium that contains sugars, amino acids, and growth factors. These may be derived from preparations of animal serum. Cellular proteins (HCPs), host nucleic acids and / or chromatographic materials, as well as coagulation Product-related impurities such as aggregates, misfolded species, or fragments of the protein of interest Separation of the desired recombinant therapeutic protein from process-related impurities, including those derived from ribosomal proteins, is essential for the development of therapeutic products for humans. Making it pure enough for therapeutic use presents formidable challenges.

[0005] Product-related and process-related impurities, including aggregates, can interfere with the purification process and cause may affect the protein and / or may have side effects upon administration of the antibody to a subject. This may be the cause of the effect (Shukla et al., J. Chromatog. rBAnalyt.Technol.Biomed.Life Sci.,848( 1), 28-39).

[0006] Therefore, it is possible to effectively remove impurities, improve protein recovery, and meet therapeutic requirements. There is a need in the art for improved methods for purifying therapeutic proteins, e.g., antibodies, while maintaining remains. Summary of the Invention

[0007] The present invention relates, at least in part, to a method for producing an anti-α4β7 antibody or antigen-binding portion thereof. In some embodiments, the method is based on the development of a process for the production of anti-α4β7 antibodies or The antigen-binding portion of the antibody is a humanized anti-α4β7 antibody or an antigen-binding portion thereof. In any of the embodiments, the humanized anti-α4β7 antibody, or antigen-binding portion thereof, has the sequence CDR1 as set forth in column number 2, CDR2 as set forth in SEQ ID NO: 3, and CDR3 as set forth in SEQ ID NO: 4. A heavy chain variable region comprising R3 and / or CDR1 of SEQ ID NO: 6, R4 of SEQ ID NO: 7 The light chain variable region may comprise a CDR2 and a CDR3 as set forth in SEQ ID NO:8. In embodiments, the humanized anti-α4β7 antibody, or antigen-binding portion thereof, comprises a polypeptide comprising SEQ ID NO:1. In some embodiments, the nucleic acid comprises a light chain variable region and / or a light chain variable region comprising SEQ ID NO:2. The humanized anti-α4β7 antibody, or antigen-binding portion thereof, may comprise a heavy chain comprising SEQ ID NO:9 and / or In an exemplary embodiment, the anti-α4β7 antibody comprises a light chain comprising SEQ ID NO: 10. A mab, or an antigen-binding portion thereof.

[0008] Thus, in one aspect, the present invention provides a humanized anti-α4β7 antibody or antigen-binding portion thereof. The present invention provides a method for producing a composition comprising: providing a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof; A composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof is incubated for at least 20 minutes to 10 hours. thereby producing a humanized anti-α4β7 antibody or antigen-binding portion thereof. Producing a composition.

[0009] In another aspect, the present invention provides a humanized anti-α4β antibody having reduced levels of basic isoform species. 7. A method for producing a composition comprising an antibody, or an antigen-binding portion thereof, comprising: Comprising: A composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, at a pH above 6.5 providing a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof at a pH that satisfies the requirements of the present invention; , the level of a basic isoform species of an anti-α4β7 antibody or an antigen-binding portion thereof in the composition Incubating for a time sufficient to reduce the amount of basic isoforms present; A composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, having reduced levels of . To generate.

[0010] In some embodiments of the foregoing aspects, the humanized anti-α4β7 antibody, or antigen-binding portion thereof, The fragment comprises CDR1 set forth in SEQ ID NO:2, CDR2 set forth in SEQ ID NO:3, and CDR3 set forth in SEQ ID NO:4. and / or a heavy chain variable region comprising the CDR1 set forth in SEQ ID NO: 6 and the CDR3 set forth in SEQ ID NO: 7. and a light chain variable region comprising the CDR2 set forth in SEQ ID NO: 8, and the CDR3 set forth in SEQ ID NO: 9. In some embodiments, the humanized anti-α4β7 antibody, or antigen-binding portion thereof, comprises SEQ ID NO:1. and / or a light chain variable region comprising SEQ ID NO:2. In the present invention, the humanized anti-α4β7 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising SEQ ID NO:9 and / or or a light chain comprising SEQ ID NO: 10. In an exemplary embodiment, the anti-α4β7 antibody comprises tibiotics, or an antigen-binding portion thereof.

[0011] In some embodiments, the method provides a method for determining whether a patient is at risk of developing a pulmonary embolism, such as <16%, <15%, <14%, <13%, < Humanized anti-α4β7 with 12%, <11% or <10% basic isoform species A composition comprising the antibody, or an antigen-binding portion thereof, is generated.

[0012] In some embodiments, the incubation is performed with a humanized anti-α4β7 antibody or an ... This incubation is carried out during purification of the original binding moiety and can be performed using (a) ultrafiltration / diafiltration of the antibody; (b) before ultrafiltration / desorption / filtration (UF / DF), or (b) in a pharma- ceutically acceptable buffer. This is done prior to formulation of the antibody in vivo.

[0013] In some embodiments, the incubation is carried out at ambient temperature. In some embodiments, the incubation is carried out at 15 to 30° C. In this embodiment, the incubation is carried out at 20 to 25°C.

[0014] In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is provided at a pH of about 6.5 to 8.5. In some embodiments, the humanized anti-α4β7 The composition comprising the antibody or antigen-binding portion thereof is provided at a pH of about 7.0 to 8.0. In some embodiments, a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, In another embodiment, the humanized anti-α4β7 antibody or The compositions containing the antigen-binding portions are provided at a pH of about 6.6 to 7.3. In embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof has a pH of about 6. .5, pH6.6, pH6.7, pH6.8, pH6.9, pH7.0, pH7.1, p H7.2, pH7.3, pH7.4, pH7.5, pH7.6, pH7.7, pH7.8 , pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, or It is provided at a pH of 8.5.

[0015] In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof The humanized antibody is incubated for a period of about 10 to 120 hours. The composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof is administered intravenously for a period of about 10 to 120 hours. In some embodiments, the humanized anti-α4β7 antibody or its antigen-binding The composition containing the moiety is incubated for a period of about 12 to 96 hours. In one embodiment, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is administered for about 12 to 72 hours. In some embodiments, the humanized anti-α4β7 antibody or The composition containing the antigen-binding portion is incubated for a period of about 12 to 48 hours. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises at least In some embodiments, the humanized anti-α4 The composition comprising a β7 antibody or an antigen-binding portion thereof is administered intravenously for a period of at least 15 to 36 hours. In some embodiments, the humanized anti-α4β7 antibody or its antigen-binding The composition containing the moiety is incubated for a period of about 24 to 120 hours. In one embodiment, the composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises about 24 to 96 In some embodiments, the humanized anti-α4β7 antibody or The composition containing the antigen-binding portion is incubated for a period of about 24 to 72 hours. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises: The antibody is incubated for a period of about 24 to 48 hours. The composition comprising a β7 antibody or antigen-binding portion thereof is Incubate for about 36 hours, about 48 hours, about 72 hours, about 96 hours, or about 120 hours. It will be.

[0016] In another aspect, provided herein is a humanized anti-α4β7 antibody or an antigen thereof. A method for purifying a binding moiety from a clarified cell culture harvest, the method comprising: ) obtained from a culture of recombinant host cells expressing an anti-α4β7 antibody or an antigen-binding portion thereof. and (ii) providing a clarified cell culture harvest comprising an anti-α4β7 antibody or and purifying the antigen-binding portion from the cell culture harvest, said antibody having a titer of 4.0 or greater. The anti-α4β7 antibody or antigen-binding portion thereof is exposed to a pH of 0.1 or lower for 24 hours. A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 5. The light chain variable region comprises a sequence.

[0017] In some embodiments, the anti-α4β7 antibody, or antigen-binding portion thereof, is , the levels of basic isoform species (determined by CEX) are decreased, The control included an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method. The composition has a pH of 4.0 or less (e.g., pH 3.6 to 4.0), Exposure is for a longer period, i.e., greater than 24 hours.

[0018] In some embodiments, the anti-α4β7 antibody, or antigen-binding portion thereof, is vedolizumab. or an antigen-binding portion thereof.

[0019] In some embodiments, a humanized anti-α4β7 antibody, or antigen-binding portion thereof, e.g., The composition comprising vedolizumab, or an antigen-binding portion thereof, comprises a first basic isoform peptide. A first basic isoform peak (BP1) and a second basic isoform peak (BP2), The method includes administering to a subject a subject, comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof, having reduced levels of BP2. In certain embodiments, the method produces a composition containing less than 2%, less than 1.5%, less than 1% or less than 0.7% BP2, A composition is formed that includes the combined moiety.

[0020] In some embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is derived from a mammalian cell culture expressing a humanized anti-α4β7 antibody or an antigen-binding portion thereof. In certain embodiments, the mammalian cell culture is Chinese Hamster Ovary (CH O) Cell culture. In certain embodiments, the CHO cell culture is In certain embodiments, the CHO cell culture includes a CHO cell lacking DHFR expression. The article includes CHO cells lacking glutamine synthetase (GS) expression.

[0021] In some embodiments, the method comprises affinity chromatography, cationic Exchange Chromatography, Anion Exchange Chromatography, Mixed Mode Chromatography Ceramic Hydroxyapatite (CHT) Chromatography and Hydrophobic Interaction One or more chromatographs selected from the group consisting of high-intensity interfacial chromatography (HIC), A protein separation step was used to isolate humanized anti-α4 from mammalian host cell proteins (HCPs). The method further includes purifying the composition comprising the β7 antibody or antigen-binding portion thereof.

[0022] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises: It is purified using an affinity chromatography resin containing Protein A.

[0023] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises: To reduce basic isoforms, affinity cloning was performed prior to incubation. In certain embodiments, the humanized anti-α4β7 antibody or The composition containing the antigen-binding portion is subjected to affinity chromatography after incubation. The product is purified using chromatography.

[0024] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises: In certain embodiments, the humanized antibody is purified using cation exchange chromatography. The composition comprising an α4β7 antibody or an antigen-binding portion thereof may be treated with a cationic antibody prior to incubation. In certain embodiments, the humanized anti-α4β antibody is purified using ion exchange chromatography. 7. The composition comprising the antibody or antigen-binding portion thereof may be subjected to cation exchange after incubation. It is purified using chromatography.

[0025] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises: In certain embodiments, the humanized antibody is purified using anion exchange chromatography. The composition comprising an α4β7 antibody or an antigen-binding portion thereof may be treated with an anion prior to incubation. In certain embodiments, the humanized anti-α4β antibody is purified using ion exchange chromatography. 7. The composition comprising the antibody or antigen-binding portion thereof may be subjected to anion exchange following incubation. It is purified using chromatography.

[0026] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or antigen-binding portion thereof comprises: Purified using CHT chromatography. In certain embodiments, the humanized anti-α4β 7. The composition comprising the antibody or antigen-binding portion thereof may be treated with CHT chromosome prior to incubation. In certain embodiments, the humanized anti-α4β7 antibody or The composition containing the antigen-binding portion is subjected to CHT chromatography after incubation. It is refined using.

[0027] In some embodiments, the method includes incorporating the composition into a pharmaceutical formulation.

[0028] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In some embodiments, the lyophilized pharmaceutical formulation is a dry, lyophilized pharmaceutical formulation. In some such embodiments, the method further comprises preparing the dried frozen sample so that it is suitable for administration. The method further comprises the step of reconstituting the lyophilized pharmaceutical formulation with a liquid.

[0029] In another embodiment, the pharmaceutical formulation is a liquid pharmaceutical formulation. In embodiments, the liquid pharmaceutical formulation is suitable for subcutaneous administration to humans.

[0030] In another embodiment, the present invention provides a humanized anti-α4β7 antibody, or antigen-binding portion thereof, The present invention provides a method for reducing and purifying the humanized anti-α4β7 isoform species. Antibodies or antigen-binding portions thereof are subjected to ultrafiltration / diafiltration (UF / DF). Thus, the preparation of a humanized anti-α4β7 antibody or an antigen-binding portion thereof in a pharma- ceutical acceptable carrier is 1. Primary incubation of a humanized anti-α4β7 antibody or antigen-binding portion thereof from a cell culture harvest against an agent At least 60%, at least 65%, at least 70%, or at least at least 75%, at least 80%, at least 85%, at least 90%, at least pH 6 for 95%, at least 97%, at least 98%, or at least 99% The purification process is carried out using humanized anti-α4β7 antibodies or their derivatives. By maintaining the antigen-binding portion of the antibody at a pH of 6.5 or higher, the antibody can be made humanized anti-α4β7 antibody or or the basic isoform species of its antigen-binding portion for an equivalent purification process, i.e. That is, the humanized anti-α4β7 antibody or an antigen-binding portion thereof is The cells were harvested from the cell culture in a pharma- ceutical acceptable carrier by UF / DF. The humanized anti-α4β7 antibody or its antigen-binding portion is primarily collected, and the humanized anti-α4β7 antibody or The antigen-binding moiety is formulated for a significant period of time, e.g., more than 5%, Between >0%, >15%, >20%, >25%, >30%, >35%, or >40%, p For an equivalent purification process, the pH may be reduced to below H6.5.

[0031] In another aspect, the present invention provides a humanized anti-α4β antibody having reduced levels of basic isoform species. 7. Compositions comprising the antibody, or antigen-binding portion thereof (e.g., vedolizumab) are provided. In some embodiments, the basic isoform of a humanized anti-α4β7 antibody, or antigen-binding portion thereof, The foam species may be any one of the humanized anti-α4β7 antibodies, or antigen-binding portions thereof, present in the composition. Less than 5%, Less than 14%, Less than 13%, Less than 12%, Less than 11%, Less than 10%, Less than 9%, Less than 8%, Less than 7%, Less than 6%, Less than 5%, Less than 4%, Less than 3%, Less than 2% or Less than 1% In some embodiments, a human having reduced levels of a basic isoform species is Compositions comprising modified anti-α4β7 antibodies or antigen-binding portions thereof can be prepared using the methods described herein. In some embodiments, a humanized anti-α4β7 antibody or antigen-binding portion thereof is produced. The compositions comprising the moieties are produced by a combination of methods of the present invention.

[0032] In another aspect, provided herein is a humanized anti-α4β7 antibody or an antigen thereof. A method for producing a composition comprising a binding moiety (e.g., vedolizumab), the method comprising: (a) Humanized anti-α4β7 antibody or its binding portion, and antigen and host cell protein (HCP contacting a sample containing the anion exchange resin in the presence of a loading buffer, The loading buffer has a conductivity of 11 mS / cm or less, so that the HCP is negatively charged. (b) contacting the flow-through material with an anion exchange resin; collecting the flow-through material from the resin, said flow-through material being a humanized anti-α4β7 antibody; or a collection comprising an antigen-binding portion thereof.

[0033] In another aspect, the present invention provides a humanized anti-α4β7 antibody or antigen-binding portion thereof (e.g., and vedolizumab), the method comprising: (a) producing a composition comprising a humanized anti-alpha A sample containing 4β7 antibody or its binding portion antigen and host cell protein (HCP) and an anion contacting the ion exchange resin in the presence of a loading buffer, wherein the loading buffer The mixing buffer is about 11 mS / cm or less (e.g., about 11 mS / cm or less, about 10 mS / cm Below, about 9mS / cm or less, about 8mS / cm or less, about 7mS / cm or less, about 6mS / cm or less, about 5 mS / cm or less, about 4 mS / cm or less, about 3 mS / cm or less, or about 2 mS / cm or less, resulting in a contact that binds the HCP to the anion exchange resin. (b) contacting the anion exchange resin with an elution buffer; and (c) separating the flow-through. and collecting the flow-through from the anion exchange resin, the flow-through being purified by humanized anti-α4β7 antibody. and a collection comprising the body or an antigen-binding portion thereof.

[0034] In some embodiments of the above aspects, the method comprises administering to a subject a humanized anti-α4β7 antibody, or and producing a composition comprising an antigen-binding portion of, for example, vedolizumab having a reduced amount of HCP. wherein the flow-through is a humanized anti-α4β7 antibody, or antigen-binding portion thereof, e.g. For example, vedolizumab and reduced amounts of HCP.

[0035] In some embodiments, the loading buffer has a pH between 9 mS / cm and 11 mS / cm (e.g. For example, about 9 mS / cm to about 11 mS / cm, or about 10 mS / cm to about 11 mS / cm )

[0036] In some embodiments, the loading buffer has a pH of less than 11 mS / cm (e.g., less than 11 Less than mS / cm, Less than 10mS / cm, Less than 9mS / cm, Less than 8mS / cm, 7mS / less than cm, less than 6mS / cm, less than 5mS / cm, less than 4mS / cm, less than 3mS / cm , or less than 2 mS / cm).

[0037] In some embodiments, the loading buffer is about 9 mS / cm, 9.5 mS / cm , 10 mS / cm, 10.5 mS / cm, or 11 mS / cm.

[0038] In some embodiments, the HCP is a Chinese hamster ovary (CHO) cell line. In certain embodiments, the HCP is a dihydrofolate reductase (DHFR) protein. In certain embodiments, the HCP is derived from a CHO cell lacking expression of glutamine synthetase. It is derived from CHO cells lacking GS gene expression.

[0039] In some embodiments, the anion exchange resin is washed with a wash buffer. In embodiments, the wash buffer has a conductivity of 11 mS / cm or less. In some embodiments, the wash buffer has a conductivity of 9 mS / cm to 11 mS / cm. In some embodiments, the wash buffer has a conductivity of less than 11 mS / cm. The wash buffer is approximately 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS In some embodiments, the wash buffer has a conductivity of 1 mS / cm, or 11 mS / cm. has the same conductivity as the loading buffer.

[0040] In some embodiments, the loading buffer contains sodium chloride and / or phosphate Contains sodium.

[0041] In some embodiments, the loading buffer contains 20-150 mM salt, 50-12 In one embodiment, the salt is sodium chloride. For example, the buffer may contain about 20 mM, about 30 mM , about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about 10 0 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, or about 150 mM Additionally or alternatively, the buffer may contain about 20 mM sodium chloride. approx. 30mM, approx. 40mM, approx. 50mM, approx. 60mM, approx. 70mM, approx. 80mM, approx. 90m M, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, or It may contain about 150 mM sodium phosphate.

[0042] In some embodiments, the wash buffer contains sodium chloride and / or sodium phosphate. This includes

[0043] In some embodiments, the wash buffer contains 20-150 mM salt, 50-125 mM In one embodiment, the salt comprises sodium chloride and For example, the buffer may contain about 20 mM, about 30 mM, about 40 mM, or about 50 mM sodium phosphate. 0 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM , about 110 mM, about 120 mM, about 130 mM, about 140 mM, or about 150 mM salt Additionally or alternatively, the buffer may contain about 20 mM, about 30 mM sodium chloride. mM, approx. 40mM, approx. 50mM, approx. 60mM, approx. 70mM, approx. 80mM, approx. 90mM, approx. 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, or about 15 0 mM sodium phosphate.

[0044] In some embodiments, the wash buffer has a conductivity equal to or lower than that of the loading buffer. In some embodiments, the wash buffer has the same conductivity as the loading buffer. It has the same conductivity as

[0045] In some embodiments, the anion exchange resin is an anion exchange column or It is formatted as a replacement membrane.

[0046] In some embodiments, the anion exchange resin comprises a quaternary amine functional group.

[0047] In some embodiments, a humanized anti-α4β7 antibody, or antigen-binding portion thereof (e.g., The sample containing vedolizumab and HCP was subjected to one or more chromatographic separation steps. In certain embodiments, the mammalian cell culture is The separation steps are affinity chromatography, cation exchange chromatography, and , mixed mode chromatography, hydrophobic interaction chromatography (HIC) and hydroxyapatite (CHT) chromatography It contains one or more steps.

[0048] In some embodiments, the amount of HCPs in the flow-through is 8 ppm or less, 7.5 ppm or less, m or less, 7ppm or less, 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm Below 4.5ppm. Below 4ppm, below 3.5ppm, below 3ppm, below 2.5ppm pm or less, or 2 ppm or less.

[0049] In some embodiments, the amount of HCP in the flow-through is greater than or equal to 12 mS / cm conductivity. generated when running this method using the same sample with a loading buffer exceeding At least 50% (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% or greater .

[0050] In some embodiments, the method further comprises administering to the subject the composition a medicament comprising one or more pharma- ceutically acceptable carriers or excipients. by a process involving ultrafiltration and / or diafiltration into a buffer containing the agent. The method further comprises treating the flow-through to exchange the buffer.

[0051] In some embodiments, the method includes incorporating the composition into a pharmaceutical formulation.

[0052] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In some embodiments, the lyophilized pharmaceutical formulation is a dry lyophilized pharmaceutical formulation. In such an embodiment, the method further comprises lyophilizing the dried lyophilized composition so that it is suitable for administration. The method further comprises the step of reconstituting the reconstituted pharmaceutical formulation with a liquid.

[0053] In another embodiment, the pharmaceutical formulation is a liquid pharmaceutical formulation. In an embodiment, the liquid pharmaceutical formulation is a pharmaceutical formulation suitable for subcutaneous administration to humans.

[0054] In another aspect, provided herein is a human ovarian tumor cell line produced by any of the methods of the invention. and a composition comprising a modified anti-α4β7 antibody, or an antigen-binding portion thereof (e.g., vedolizumab). Compositions comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab) are disclosed. Also provided herein are compositions obtainable by any of the methods of the invention. In some embodiments, the amount of HCPs in the composition is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, pm or less, 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5 ppm or less, 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, or In another embodiment, the humanized anti-α4β7 antibody, or antigen-binding The basic isoform species of the moiety (e.g., vedolizumab) is the antibody species present in the composition. Less than 16%, Less than 15%, Less than 14%, Less than 13%, Less than 12%, Less than 11%, 10% Less than, Less than 9%, Less than 8%, Less than 7%, Less than 6%, Less than 5%, Less than 4%, Less than 3%, Less than 2% Includes less than or less than 1%.

[0055] In some embodiments, a humanized anti-α4β7 antibody or antigen-binding portion thereof (e.g., vector Compositions comprising dolizumab) are produced by a combination of methods of the invention.

[0056] In another aspect, the present invention provides a method for recovering a soluble form of a compound after elution from a mixed-mode chromatography resin. The yield of the humanized anti-α4β7 antibody or antigen-binding portion thereof (e.g., vedolizumab) obtained by The present invention relates to a method for increasing the affinity of a mixed-mode chromatography resin to an equilibration buffer. and a solution containing the anti-α4β7 antibody or an antigen-binding portion thereof and a load The mixing buffer was added to the anti-α4β7 antibody or its antigen-binding moiety in a mixed-mode chromatograph. loading the mixture onto a mixed-mode chromatography resin such that it binds to the glycerol resin; The mixed-mode chromatography resin is washed with wash buffer and then incubated with anti-α4β7 antibody or its and eluting the originally bound moiety from the mixed-mode chromatography resin with an elution buffer. where the pH of the equilibration buffer, loading buffer and / or washing buffer is 7.0 or less. Below.

[0057] In one embodiment, the pH of the equilibration buffer, loading buffer, and / or wash buffer In another embodiment, the equilibration buffer is a loading buffer and / or the pH of the wash buffer is 6.5 to 7.0. In another embodiment, the equilibration buffer is The pH of the loading buffer and / or the washing buffer is 6.6 to 6.8.

[0058] In another embodiment, the salt of the equilibration buffer, loading buffer, and / or wash buffer The concentration is 30 mM to 70 mM. The salt concentration of the buffer and / or the washing buffer is 40 mM to 70 mM. The salt concentration of the equilibration buffer, loading buffer, and / or washing buffer should be between 50 mM and 100 mM. In another embodiment, the salt of the loading buffer and / or the wash buffer is 65 mM. In another embodiment, the salt is sodium chloride and / or or sodium phosphate.

[0059] In another embodiment, the salt of the equilibration buffer, loading buffer, and / or wash buffer In another embodiment, the loading buffer has a sodium chloride concentration of 30 mM to 70 mM. The sodium chloride concentration of the solution and / or washing buffer is 40 mM to 70 mM. In an embodiment, sodium chloride in the equilibration buffer, loading buffer, and / or wash buffer is In another embodiment, the equilibration buffer, the loader buffer, The sodium chloride concentration of the washing buffer and / or the washing buffer is 45 mM to 55 mM. In another embodiment, the equilibration buffer, loading buffer, and / or washing buffer may be The sodium chloride concentration is about 50 mM. The incubation buffer and / or the washing buffer further comprises sodium phosphate.

[0060] In some embodiments, the equilibration buffer, loading buffer, and / or washing buffer The pH of the solutions is the same. In some embodiments, the equilibration buffer, loading buffer, and / or the salt concentration of the wash buffer is the same. The loading buffer and / or the washing buffer may be the same buffer. In some embodiments, the mixed mode resin is a ceramic hydroxyapatite resin. For example, it may be CHT resin.

[0061] In another aspect, provided herein is a low basic species composition comprising an anti-α4β7 antibody. The composition comprises less than 16%, less than 15%, less than 14%, less than 1% of anti-α4β7 antibodies. Contains less than 3%, less than 12%, less than 11%, or less than 10% total basic isoform species This basic isoform species was positively associated with the predominant isoform of the anti-α4β7 antibody. The anti-α4β7 antibody has a heavy chain variable region comprising SEQ ID NO: 1 and a heavy chain variable region comprising SEQ ID NO: In some embodiments, the composition comprises a light chain variable region comprising a basic isoform The chromatographic species can be quantified by cation exchange (CEX) chromatography. For example, In some embodiments, the basic isoform species corresponds to a major isoform Determine the relative areas of the peaks that elute slower from the cation exchange (CEX) resin than the peaks that elute slower from the It can be quantified by determining

[0062] Thus, in another aspect, provided herein is a low-base antibody comprising an anti-α4β7 antibody. A species composition, the composition comprising less than 16%, less than 15%, less than 14% of anti-α4β7 antibodies. Less than 13%, less than 12%, less than 11%, or less than 10% total basic isoforms The basic isoform species is the major isoform of anti-α4β7 antibodies. has a net positive charge relative to the peak corresponding to this major isoform. Quantitation was performed by determining the relative areas of the peaks eluting slowly from cation exchange (CEX) resin. The anti-α4β7 antibody may comprise a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2. The composition comprises a chain variable region.

[0063] In some embodiments, the composition comprises the first basic isoform peak (BP1 ) and the second basic isoform peak (BP2). In some embodiments, The composition comprises less than 2% BP2. In some embodiments, the composition comprises less than 1.5% BP2. In some embodiments, the composition contains less than 1% BP2. In some embodiments, the composition comprises less than 0.7% BP2.

[0064] In some embodiments, the ratio of BP1 to BP2 is at least 3. In some embodiments, the ratio of BP1 to BP2 is at least 5. In some embodiments, the ratio of BP1 to BP2 is at least 7. , the ratio of BP1 to BP2 is at least 10.

[0065] In some embodiments, the composition comprises less than 8% total basic isoforms of the anti-α4β7 antibody. In some embodiments, the composition comprises less than 7% total anti-α4β7 antibodies. In some embodiments, the composition comprises an anti-α4β7 antibody. In some embodiments, the composition comprises less than 6% of the total basic isoform species. Contains less than 5% of the total basic isoform species of anti-α4β7 antibodies.

[0066] In another aspect, provided herein are compositions and pharma- ceuticals provided herein. In some embodiments, the composition is a pharmaceutical composition that includes an acceptable carrier or excipient. The pH of the pharmaceutical composition is between 6.0 and 7.0. In another embodiment, the pH of the pharmaceutical composition is about pH 6.3. H6.3~pH6.5.

[0067] In some embodiments, the pharmaceutical composition further comprises an amino acid. In embodiments, the amino acid is arginine or histidine.

[0068] In some embodiments, the pharmaceutical composition further comprises a sugar. , the sugar is sucrose or trehalose.

[0069] In some embodiments, the pharmaceutical composition comprises arginine, histidine, and sucrose. In some embodiments, the pharmaceutical composition comprises arginine, histidine, Contains sucrose and polysorbate 80.

[0070] In some embodiments, the pharmaceutical composition comprises at least 200 mg, at least 2 In some embodiments, the composition comprises at least 50 mg of an anti-α4β7 antibody, or at least 300 mg of an anti-α4β7 antibody. The pharmaceutical composition comprises about 300 mg of anti-α4β7 antibody.

[0071] In some embodiments, the anti-α4β7 antibody is vedolizumab, or an antigen-binding portion thereof. It is.

[0072] In another aspect, provided herein is a method for the treatment of a cancer comprising administering to the patient a therapeutically effective amount of an anti-cancer drug having less than 16% total basic isoform species, Less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% 2. A method for producing a low basic species composition comprising an anti-α4β7 antibody, the method comprising: providing a composition comprising an anti-α4β7 antibody at a pH greater than 0.3; incubating the composition for a period of more than 10 hours; thereby A low basic species composition, comprising less than 16%, 15%, 14%, or 20% total basic isoform species. %, less than 13%, less than 12%, less than 11%, or less than 10%. In some embodiments, the anti-α4β7 antibody comprises a heavy chain variable domain comprising SEQ ID NO:1. region, and a light chain variable region comprising SEQ ID NO:2.

[0073] In another aspect, provided herein is a method for the treatment of a cancer comprising administering to the patient a therapeutically effective amount of an anti-cancer drug having less than 16% total basic isoform species, Less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% 2. A method for producing a low basic species composition comprising an anti-α4β7 antibody, the method comprising: providing a composition comprising vedolizumab at a pH greater than 0.3; The composition is used to reduce the level of basic vedolizumab isoform species in the composition. and incubating for a sufficient period of time; thereby obtaining low-basic species including anti-α4β7 antibodies. A composition comprising less than 16%, less than 15%, less than 14%, less than 1 and producing a composition having less than 3%, less than 12%, less than 11%, or less than 10%. In some embodiments, the anti-α4β7 antibody comprises a heavy chain variable region comprising SEQ ID NO:1, and and a light chain variable region comprising SEQ ID NO:2.

[0074] In general, the basic isoform species are more abundant than the predominant isoform of anti-α4β7 antibodies. In some embodiments, the level of a basic isoform species is It can be quantified using cation exchange chromatography (CEX). In this state, the levels of basic isoform species are higher than the peaks corresponding to the major isoforms. To determine the relative areas of peaks eluting slower from a cation exchange (CEX) resin than from a cation exchange (CEX) resin, In some embodiments, the composition may be quantified by: The first isoform peak (BP1) and the second basic isoform peak (BP2). In some embodiments, the composition contains less than 2% BP2. In some embodiments, the composition contains less than 1.5% BP2. In some embodiments, the composition contains less than 0.7% BP2. include.

[0075] In some embodiments, the ratio of BP1 to BP2 is at least 3. In some embodiments, the ratio of BP1 to BP2 is at least 5. In some embodiments, the ratio of BP1 to BP2 is at least 7. , the ratio of BP1 to BP2 is at least 10.

[0076] In some embodiments, the composition comprises less than 8% total basic isoforms of the anti-α4β7 antibody. In some embodiments, the composition comprises less than 7% total anti-α4β7 antibodies. In some embodiments, the composition comprises an anti-α4β7 antibody. In some embodiments, the composition comprises less than 6% of the total basic isoform species. Contains less than 5% of the total basic isoform species of anti-α4β7 antibodies.

[0077] In some embodiments, provided herein is an antibody obtained by the aforementioned method. In some embodiments, the antibody is vedolizumab. or an antigen-binding portion thereof.

[0078] In some embodiments, provided herein is an antibody obtained by the aforementioned method. In some embodiments, the antibody is vedolizumab. or an antigen-binding portion thereof.

[0079] In another aspect, provided herein is a method for treating a disease or disorder in a human subject. The method includes administering to a human subject an amount of an anti-alpha antibody effective to treat a disease or disorder. For the pharmaceutical compositions provided herein comprising a 4β7 antibody (e.g., vedolizumab), In some embodiments, the pharmaceutical compositions provided herein include administering and / or produced according to the methods provided herein. Reduced levels of subisoform species and / or host cell protein levels In some embodiments, the composition comprises an anti-α4β7 antibody, comprises a heavy chain variable region comprising SEQ ID NO:1 and a light chain variable region comprising SEQ ID NO:2.

[0080] In one embodiment, the disease or disorder is inflammatory bowel disease (IBD). In embodiments, the IBD is ulcerative colitis, Crohn's disease, ileitis, celiac disease, non-tropical Sprue, enteropathy associated with seronegative arthropathy, microscopic colitis or collagenous colitis, These include eosinophilic gastroenteritis, or pouchitis following proctocolectomy and ileoanal anastomosis. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Additional diseases that may be treated include, for example, primary sclerosing cholangitis (PSC) and graft-versus-host disease. (GVHD) is one of the causes.

[0081] Furthermore, the present invention also includes the following embodiments:

[0082] 1. A composition containing vedolizumab with reduced levels of basic vedolizumab isoforms 1. A method of producing an article, comprising: providing a composition comprising vedolizumab at a pH greater than pH 6.5; incubating a composition comprising vedolizumab for more than 10 hours; This allows the administration of vedolizumab with reduced levels of basic vedolizumab isoforms. and forming a composition comprising:

[0083] 2. The method according to item 1, wherein the incubation is carried out at ambient temperature.

[0084] 3. The method according to item 1, wherein the incubation is carried out at 15 to 30°C.

[0085] 4. The method according to item 1, wherein the incubation is carried out at 20 to 25°C.

[0086] 5. The composition comprising vedolizumab is provided at a pH of about 6.5 to 8.5, Item 10. The method according to any one of items 1 to 7.

[0087] 6. The composition comprising vedolizumab is provided at a pH of about 7.0 to 8.0, Item 10. The method according to any one of items 1 to 7.

[0088] 7. The composition comprising vedolizumab is provided at a pH of about 7.0 to 7.5, Item 10. The method according to any one of items 1 to 7.

[0089] 8. The composition comprising vedolizumab has a pH of about 6.5, pH 6.6, pH 6.7, pH 6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8. 1, the preceding paragraphs are available in pH 8.2, pH 8.3, pH 8.4, or pH 8.5. 2. The method according to any one of the preceding claims.

[0090] 9. The composition comprising vedolizumab is incubated for a period of about 10 to 120 hours. , A method according to any one of the preceding items.

[0091] 10. The composition comprising vedolizumab is incubated for a period of about 12 to 120 hours. 5. The method according to any one of the preceding items,

[0092] 11. The composition comprising vedolizumab is incubated for a period of about 12 to 96 hours. , A method according to any one of the preceding items.

[0093] 12. The composition comprising vedolizumab is incubated for a period of about 12 to 72 hours. , A method according to any one of the preceding items.

[0094] 13. The composition comprising vedolizumab is incubated for a period of about 12 to 48 hours. , A method according to any one of the preceding items.

[0095] 14. The composition comprising vedolizumab is incubated for a period of at least 12 hours. 5. The method according to any one of the preceding items,

[0096] 15. The composition comprising vedolizumab is incubated for a period of about 24 to 120 hours. 5. The method according to any one of the preceding items,

[0097] 16. The composition comprising vedolizumab is incubated for a period of about 24 to 96 hours. , A method according to any one of the preceding items.

[0098] 17. The composition comprising vedolizumab is incubated for a period of about 24 to 72 hours. , A method according to any one of the preceding items.

[0099] 18. The composition comprising vedolizumab is incubated for a period of about 24 to 48 hours. , A method according to any one of the preceding items.

[0100] 19. The composition comprising vedolizumab is administered for about 12 hours, about 24 hours, about 36 hours, or about 4 hours. Incubate for about 8 hours, about 72 hours, about 96 hours, or about 120 hours, prior to Item 10. The method according to any one of items 1 to 7.

[0101] 20. The composition comprising vedolizumab is a mammalian cell culture expressing vedolizumab. 2. The method according to any one of the preceding items, derived from

[0102] 21. The mammalian cell culture is Chinese Hamster Ovary (CHO) cells. 21. The method according to item 20.

[0103] 22. The CHO cell culture is a CHO cell culture lacking dihydrofolate reductase (DHFR) expression. 22. The method of claim 21, comprising HO cells.

[0104] 23. The CHO cell culture is a CHO cell culture lacking glutamine synthetase (GS) expression. 22. The method of claim 21, comprising administering to said subject a therapeutically effective amount of

[0105] 24. Affinity Chromatography, Cation Exchange Chromatography, Anion Exchange Chromatography Ceramic hydroxyapatite (CHT) chromatography using one or more chromatographic separation steps selected from the group consisting of and purifying a composition comprising vedolizumab from mammalian host cell proteins (HCPs). 7. The method of any one of the preceding items, further comprising:

[0106] 25. The composition comprising vedolizumab is subjected to affinity chromatography containing protein A. 25. The method according to claim 24, wherein the product is purified using a fluororesin.

[0107] 26. The composition comprising vedolizumab is subjected to affinity chromatography prior to the incubation. 26. The method according to claim 25, wherein the product is purified using chromatography.

[0108] 27. The composition comprising vedolizumab is affinity-activated after the incubation. 26. The method according to claim 25, wherein the purification is carried out using chromatography.

[0109] 28. The composition comprising vedolizumab is purified using cation exchange chromatography. The method according to item 24, wherein the compound is prepared by the method according to item 24.

[0110] 29. The composition comprising vedolizumab is subjected to a cation exchanger prior to the incubation. 29. The method according to claim 28, wherein the purification is carried out using chromatography.

[0111] 30. The composition comprising vedolizumab is cation-exchanged after the incubation. 29. The method according to claim 28, wherein the purification is carried out using chromatography.

[0112] 31. The composition comprising vedolizumab is purified by anion exchange chromatography. 25. The method according to item 24, wherein the product is purified.

[0113] 32. The composition comprising vedolizumab is subjected to an anion exchange prior to the incubation. 32. The method according to claim 31, wherein the purification is carried out using chromatography.

[0114] 33. The composition comprising vedolizumab is an anion exchanger after the incubation. 32. The method according to claim 31, wherein the purification is carried out using chromatography.

[0115] 34. The composition comprising vedolizumab is purified using CHT chromatography. 25. The method according to item 24,

[0116] 35. The composition comprising vedolizumab is a CHT chromosome prior to the incubation. 35. The method according to claim 34, wherein the purified product is purified using HPLC.

[0117] 36. The composition comprising vedolizumab inhibits CHT chromosomes after the incubation. 35. The method according to claim 34, wherein the purified product is purified using HPLC.

[0118] 37. A composition comprising vedolizumab, comprising the method according to any one of items 1 to 36. The composition produced by

[0119] 38. The basic vedolizumab isoform species is present in the composition. 38. The composition according to item 37, comprising less than 10% of the mab species.

[0120] 39. Producing a composition comprising vedolizumab with reduced amounts of host cell proteins (HCPs) 1. A method for producing a pharmaceutical composition comprising the steps of: (a) A sample containing vedolizumab and HCP was diluted with an anionic and contacting the loading buffer with an exchange resin, wherein the conductivity of the loading buffer is less than or equal to HC The contacting step has a pH of 11 mS / cm or less so that P binds to the anion exchange resin. and; (b) collecting a flow-through material from the anion exchange resin; wherein the flow-through material comprises vedolizumab and a reduced amount of HCPs. and said collecting.

[0121] 40. The loading buffer has a conductivity of 9 mS / cm to 11 mS / cm. 39. The method according to claim 39.

[0122] 41. The loading buffer has a conductivity of 10 mS / cm or less. The method according to

[0123] 42. The loading buffer has a conductivity of 9 mS / cm or less. The method according to

[0124] 43. The loading buffer has a concentration of about 9 mS / cm, 9.5 mS / cm, 10 mS / cm 39. The method according to claim 39, having a conductivity of 10.5 mS / cm, 10.5 mS / cm, or 11 mS / cm. method.

[0125] 44. The HCP is a Chinese Hamster Ovary (CHO) cell protein. 43. The method according to any one of items 39 to 42.

[0126] 45. The HCP is transfected into CHO cells lacking dihydrofolate reductase (DHFR) expression. 45. The method according to item 44, derived from

[0127] 46. ​​The HCP is derived from CHO cells lacking glutamine synthetase (GS) expression. Item 45. The method according to item 44.

[0128] 47. The method according to claim 39, further comprising contacting the anion exchange resin with a wash buffer. 46. ​​The method according to any one of claims 46 to 47.

[0129] 48. The method according to item 47, wherein the wash buffer has a conductivity of less than 11 mS / cm. .

[0130] 49. Item 4, wherein the wash buffer has a conductivity of 9 mS / cm to 11 mS / cm. 7. The method according to claim 7.

[0131] 50. The method according to claim 47, wherein the washing buffer has the same conductivity as the loading buffer. method.

[0132] 51. The loading buffer contains sodium chloride and / or sodium phosphate 51. The method according to any one of Items 39 to 50.

[0133] 52. The washing buffer contains sodium chloride and / or sodium phosphate, 9. The method according to any one of claims 9 to 50.

[0134] 53. The anion exchange resin is formed as an anion exchange column or an anion exchange membrane. 53. The method according to any one of items 39 to 52, wherein the substrate is matted.

[0135] 54. Any of items 39 to 53, wherein the anion exchange resin contains a quaternary amine functional group. 2. The method according to claim 1.

[0136] 55. A sample containing vedolizumab and HCPs is subjected to one or more chromatographic separation steps. 55. The method according to any one of items 39 to 54, which is derived from a mammalian cell culture followed by a step of .

[0137] 56. One or more chromatographic separation steps are performed by affinity chromatography. cation exchange chromatography, and ceramic hydroxyapatite (CHT Item 55. The method according to item 55, further comprising one or more steps selected from the group consisting of: The method described.

[0138] 57. The amount of HCP in the eluate is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6. 5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5ppm or less, 4 ppm or less, 3.5ppm or less, 3ppm or less, 2.5ppm or less, or 2ppm or less 57. The method according to any one of Items 39 to 56,

[0139] 58. The amount of HCP in the flow-through material has a conductivity greater than 12 mS / cm. The same loading buffer was used to carry out the method, but the same sample was used to carry out the method. at least 50% reduction compared to the amount of HCPs in the flow-through material. 39 to 57. A method according to any one of claims 39 to 57.

[0140] 59. Ultrafiltration into a buffer containing one or more pharma- ceutically acceptable carriers or excipients; and / or to exchange the elution buffer by a process involving diafiltration. 59. Any one of items 39 to 58, further comprising treating the flow-through material. The method according to claim 5.

[0141] 60. A composition containing vedolizumab produced by the method according to any one of items 39 to 59. Composition.

[0142] 61. The amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, or 7 ppm or less. , 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5ppm or less below, 4ppm or less, 3.5ppm or less, 3ppm or less, 2.5ppm or less, or 2ppm or less Item 61. The composition according to item 60, wherein the molecular weight of the composition is less than or equal to m.

[0143] 62. Yield of vedolizumab recovered after elution from mixed-mode chromatography resin a method for increasing the amount of chromatographic activity of a mixed-mode chromatography resin by equilibrating the resin with an equilibration buffer; and equilibrating the solution containing vedolizumab and loading buffer in the mixed-mode culture. chromatographic resin, and vedolizumab was washing the mixed-mode chromatography resin with a wash buffer to allow binding to the resin; and washing and eluting the vedolizumab from the mixed-mode chromatography resin with an elution buffer. wherein said equilibration buffer, said loading buffer and / or said elution buffer are combined. wherein the pH of said wash buffer is 7.0 or less.

[0144] 63. The equilibration buffer, the loading buffer, and / or the washing buffer Item 63. The method according to item 62, wherein the pH is 6.0 to 7.0.

[0145] 64. The equilibration buffer, the loading buffer, and / or the washing buffer Item 63. The method according to item 62, wherein the pH is 6.5 to 7.0.

[0146] 65. The equilibration buffer, the loading buffer, and / or the washing buffer Item 63. The method according to item 62, wherein the pH is 6.6 to 6.8.

[0147] 66. The equilibration buffer, the loading buffer, and / or the washing buffer 66. The method according to any one of items 62 to 65, wherein the salt concentration is 30 mM to 70 mM.

[0148] 67. The equilibration buffer, the loading buffer, and / or the washing buffer Item 67. The method according to Item 66, wherein the salt concentration is 40 mM to 70 mM.

[0149] 68. The equilibration buffer, the loading buffer, and / or the washing buffer Item 67. The method according to Item 66, wherein the salt concentration is 50 mM to 65 mM.

[0150] 69. The equilibration buffer, the loading buffer, and / or the washing buffer Item 67. The method according to Item 66, wherein the salt concentration is 55 mM to 65 mM.

[0151] 70. The salt comprises sodium chloride and / or sodium phosphate, 10. The method according to any one of claims 9 to 10.

[0152] 71. The equilibration buffer, the loading buffer, and / or the washing buffer The sodium chloride concentration is 30 mM to 70 mM, Method of posting.

[0153] 72. The equilibration buffer, the loading buffer, and / or the washing buffer Item 72. The method according to Item 71, wherein the sodium chloride concentration is 40 mM to 70 mM.

[0154] 73. The equilibration buffer, the loading buffer, and / or the washing buffer Item 72. The method according to Item 71, wherein the sodium chloride concentration is 40 mM to 60 mM.

[0155] 74. The equilibration buffer, the loading buffer, and / or the washing buffer Item 72. The method according to Item 71, wherein the sodium chloride concentration is 45 mM to 55 mM.

[0156] 75. The equilibration buffer, the loading buffer, and / or the washing buffer 72. The method of claim 71, wherein the sodium chloride concentration is about 50 mM.

[0157] 76. The equilibration buffer, the loading buffer, and / or the washing buffer 76. The method according to any one of items 71 to 75, further comprising sodium phosphate.

[0158] 77. The equilibration buffer, the loading buffer, and / or the washing buffer 77. The method according to any one of items 62 to 76, wherein the pH is the same.

[0159] 78. The equilibration buffer, the loading buffer, and / or the washing buffer 78. The method according to any one of items 62 to 77, wherein the salt concentrations are the same.

[0160] 79. The equilibration buffer, the loading buffer, and / or the washing buffer 77. The method according to any one of items 62 to 76, wherein the buffer is the same.

[0161] 80. The mixed mode resin is a ceramic hydroxyapatite resin, 79. The method according to any one of claims 79 to 80. [Brief description of the drawings]

[0162] [Figure 1]1 shows the cation exchange (CEX)-high performance liquid chromatography (HPLC) profile of vedolizumab, with peaks representing acidic species, basic species, and the major isoforms of vedolizumab. [Diagram 2] FIG. 1 shows the elution profile of vedolizumab purified using standard ceramic hydroxyapatite (CHT) equilibration and wash buffers after loading the CHT column with 27 mg / ml protein or 35 mg / ml protein. [Diagram 3] FIG. 1 shows the elution profile of vedolizumab purified using standard CHT equilibration and wash buffer or low pH buffer after loading 38 mg / ml protein on the CHT column. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0163] Provided herein are methods for administering anti-α4β7 integrin antibodies, such as vedolizumab, to, for example, For example, in a method for purifying a liquid solution derived from a clarified harvest of a mammalian cell culture. The present invention relates to, inter alia, an anti-α4β7 integrin antibody, or an antigen-binding fragment thereof. Product-related substances (e.g., bases, etc.) present in purified preparations of and / or acidic isoform species) and / or process-related impurities (e.g., Chief cell proteins (HCPs), host cell nucleic acids, viruses, chromatographic materials, and Vedolizumab is a relatively Highly hydrophobic antibodies, especially when the antibodies are produced in large quantities with the level of purity required for therapeutic use In this case, purification is problematic.

[0164] I. Definition In order that the present invention may be more readily understood, certain terms are first defined.

[0165] The cell surface molecule "α4β7 integrin" or "α4β7" (used interchangeably throughout) It is a heterodimer of α4 chain (CD49D, ITGA4) and β7 chain (ITGB7). Human α4 integrin and β7 integrin genes are available from GenBank (Nationa l Center for Biotechnology Information,B ethesda, Md.) RefSeq accession number NM_00088, respectively. 5 and NM_000889) inhibit B and T lymphocytes, particularly memory CD4+ lymphocytes. Typical of many integrins, α4β7 is expressed by lymphocytes in both resting and activated states. Ligands for α4β7 may exist in either activated or activated states. adhesion molecule (VCAM), fibronectin and mucosal addressin (MAdCAM (e.g., M Antibodies that bind to α4β7 integrin are referred to herein as "anti- It is called the "α4β7 antibody."

[0166] As used herein, an antibody, or antigen-binding fragment thereof, is an antibody that binds to α4β7. However, α4β1 also has α E "Binding specificity to the α4β7 complex" that does not bind to B7 Vedolizumab is an example of an antibody that has binding specificity for the α4β7 complex.

[0167] The word "about" means that the value that follows is not an exact value and is within the bounds of + / - 5% of that value. It means that the value is the center point of the range. It is a relative value expressed as a percentage. In some cases, the word "about" means that the value following it is not an exact value and is within the range of + / - 5% of that value. This means that the upper limit of the range cannot exceed 100%. I can't come.

[0168] As used herein, the term "aggregate" or "aggregate(s)" refers to a compound having a structure of 2 An aggregate refers to an association of one or more antibodies or antibody fragments. For example, an aggregate may comprise antibodies and / or The antibody fragments were dimers, trimers, tetramers, or multimers larger than tetramers. The antibody aggregates may be soluble or insoluble. The association between them may be either covalent or noncovalent, regardless of the mechanism by which they associate. The association may be direct between the aggregated molecules or through other interactions that link them together. Examples of the latter include, but are not limited to, other Disulfide bonds with proteins, hydrophobic associations with lipids, charge associations with DNA, leaching protons The aggregates can be formed by affinity association with tein A or by mixed association with multiple components. Irreversible during protein expression in culture, downstream protein purification, or during storage The presence of aggregates in a solution can be determined, for example, by size exclusion chromatography (SE C) (e.g., SEC with UV detection, SEC with light scattering detection (SEC-LSD) ), field-flow fractionation, analytical ultracentrifugation (sedimentation velocity), or capillary centrifugation (CAP). Electrophoresis using sodium dodecyl sulfate (CE-SDS, reduced and non-reduced) The above can be determined.

[0169] As used herein, the term "antibody" refers to a nucleic acid molecule that is interconnected by disulfide bonds. Immunoglobulins consist of four polypeptide chains, two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (referred to herein as HCVR or VH The heavy chain constant region is composed of three domains: Each light chain is composed of a light chain variable region (referred to herein as a light chain variable region), ... and a light chain variable region (referred to herein as a light chain variable region). The light chain constant region is composed of a light chain constant region (LCVR or VL). The VH and VL regions are composed of one domain CL. The complementarity determining regions (CDs) are interspersed with more conserved regions called FRs. Each VH and VL can be further subdivided into regions of hypervariability called CDR. It is composed of R and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: The sequences are: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the antibody has a fragment crystallizable (Fc) region. The antibody is of the IgG1 isotype and has kappa light chains.

[0170] As used herein, the term "basic species" or "basic isoform species" refers to a The term "antibody" refers to a variant of an antibody or antigen-binding portion thereof, such as vedolizumab, which is The basic species of an antibody or antigen-binding portion thereof is characterized by a positive basic charge. ion-exchange high performance liquid chromatography (CEX-HPLC), CEX-mass spectrometry, or The antibody can be detected by a variety of methods known in the art, such as isoelectric focusing. Group species include, but are not limited to, charge variants, structural variants, and / or or fragmentation variants. In some embodiments, the antibody or or an antigen-binding portion thereof, the composition may include two or more types of basic isoform species. In some embodiments, the present invention can be used to identify chromatin-binding proteins based on differences in retention times during CEX-HPLC separation. For example, an antibody, such as vedolizumab, may be used to identify multiple basic isoform species. When a composition containing the above-mentioned amines was analyzed using CEX-HPLC, the following was obtained: one or more basic isoform peaks representing one or more basic isoform species of the antibody For example, in some embodiments, the basic isoform species may be aspartate. It is an antibody isoform in which the lignin residues are isomerized to succinimide. The pure substance or other impurities not related to the antibody or its antigen-binding portion are identified by primary sequence. , a "basic species" or "basic isoform species" of an antibody, or an antigen-binding portion thereof is not considered.

[0171] As used herein, the term "buffer" refers to a compound that is an acid-base conjugate. A buffer solution is a solution that resists changes in pH by the action of water. a processing step or a chromatographic step such as a chromatographic resin or membrane It is used to establish the biochemical conditions that mediate the regulation of the feed support.

[0172] The "CDRs" or "complementarity determining regions" are referred to as "framework regions" (FR). , are regions of hypervariability interspersed among more conserved regions. The term "antigen-binding fragment" or "antigen-binding portion" of an antibody includes Fab, Fa, b', F(ab')2, and Fv fragments, single chain antibodies, functional heavy chain antibodies (nanobody and at least one desired antibody that competes with the intact antibody for specific binding. Any portion of an antibody having specificity for an epitope of refers to an isolated portion of a complementarity determining region having sufficient framework sequences to bind to the Antigen-binding fragments can be produced by recombinant techniques or by enzymatic or chemical modification of antibodies. It can be produced by cleavage.

[0173] "Chromatographic support" as used herein refers to a material having a particular chemical composition or or a solid or porous matrix of specific three-dimensional structure, or specific chemical groups or The polymer is purified by affinity chromatography, gel filtration (size exclusion chromatography) or ion exchange chromatography. The term chromatographic support refers to a solid or porous matrix onto which a substance can be immobilized for the purpose of chromatography. Examples include, but are not limited to, a resin (e.g., agarose) or a membrane. As used herein, a "chromatography housing" refers to a chromatography housing. The term refers to a structure that contains a support. Examples of chromatography housings include columns or The container may be a cartridge or other container.

[0174] As used herein, the term "clarified harvest" refers to a product that is free of a protein of interest, e.g. For example, a liquid material containing an anti-α4β7 antibody, which is cultured in a cell culture, e.g., a fermentation bioreactor. from the material through one or more processing steps to remove solids such as cellular debris and particulate impurities derived from the material. It refers to the liquid material containing anti-α4β7 antibodies extracted after the removal of somatic particles. After cell culture, Typically, the harvest is purified to remove cells and other substances using separation techniques such as centrifugation and filtration. The initial clarification, particle removal step is used to remove the chromatographic This results in a "clarified harvest" that can be used in the roughy step (downstream processing). The oxidized harvest product is generally the starting material for downstream processing, such as the downstream processing steps described herein. It's quality.

[0175] As used herein, the terms "culture" and "cell culture" generally refer to a culture in which cells are Refers to the process of growing under controlled conditions, generally outside their natural environment (upstream). "Culturing" cells means culturing cells under conditions suitable for their survival and / or growth and / or proliferation. In certain embodiments, cell culture refers to contacting cells with a cell culture medium at a temperature of To generate and maintain a population of host cells capable of producing a recombinant protein, e.g., an anti-α4β7 antibody. The present invention provides methods and techniques for maintaining the integrity of proteins of interest, as well as methods and techniques for producing and harvesting proteins of interest. For example, once an expression vector is incorporated into a suitable host, e.g., a host cell in culture, For example, the host can be adapted to support the expression of the relevant nucleotide coding sequence and the production of the desired recombinant protein. A "cell culture" refers to a solution containing cells that is maintained under conditions suitable for collection and purification of the protein. It can also refer to the following.

[0176] As used herein, the term "downstream processing" refers to the process of producing a protein of interest, e.g. For example, it refers to one or more techniques used after upstream processing to purify an antibody. For example, downstream processing techniques include, for example, affinity chromatography, e.g., Protein A affinity chromatography, anion or cation exchange chromatography Chromatography, size exclusion chromatography, mixed chromatographies, etc. Hydrophobic interaction chromatography (HIC), or displacement chromatography This includes purification of the protein product using chromatography.

[0177] As used herein, the term "elution solution" or "eluate" refers to a solution containing a protein of interest. , e.g., to displace an antibody from a chromatographic support, e.g., a resin or membrane. In one embodiment, the elution solution is a formulated aqueous liquid that is capable of eluting the protein of interest, e.g. The antibody is bound to the elution solution rather than to the chromatographic support, e.g., a resin or membrane. have different biochemical properties than the equilibration and / or wash solutions, so as to prioritize .

[0178] As used herein, the term "equilibration solution" refers to a process step or a chromatography step. The feed support, e.g., is formulated to create the initial operating conditions of a chromatographic operation. The equilibration solution refers to the aqueous liquid that is used to load the protein of interest, e.g., an antibody. , e.g., preparing a solid phase, e.g., a chromatographic support, e.g., a resin or membrane Used for:

[0179] As used herein, a "flow-through operation" refers to, for example, a chromatography step. In relation to the impurities bind and remain bound to the chromatographic support, Refers to the process by which proteins elute during loading and washing.

[0180] The term "high molecular weight" or "HMW" refers to a molecule having a molecular weight greater than that of a monomeric antibody. In one embodiment, the HMW aggregate is about 147 kD The presence of high molecular weight aggregates can be determined by standard methods known in the art. The amount of the ion exchange reaction can be determined by methods such as size exclusion chromatography (SEC).

[0181] "Humanized" forms of non-human (e.g., rodent) antibodies are those that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are chimeric antibodies that contain the same structure as the recipient's antibodies. Residues from the variable regions are selected from mouse, rat, rabbit and / or human genomic DNA with the desired specificity, affinity, and potency. or by residues from a hypervariable region of a non-human species (donor antibody), such as a non-human primate. In some cases, human immunoglobulins (recipient antibodies) are replaced by human immunoglobulins (recipient antibodies). Framework region (FR) residues of the human antibody are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies have at least It comprises substantially all of at least one, but typically two, variable domains, and has hypervariable CDR loops. all or substantially all of the loops correspond to the hypervariable CDR loops of a non-human antibody, and all of the FR Or substantially all of the FRs are human antibody sequences. The humanized antibody optionally comprises an antibody At least a portion of an antibody constant region (Fc), typically a portion of an antibody constant region (Fc) of a human antibody. For further details, see, for example, Jones et al., ature 321:522-525(1986);Riechmann et al. ,Nature 332:323-329(1988); and Presta, Curr. See Op. Struct. Biol. 2:593-596 (1992).

[0182] "Impurities" as used herein with respect to impurities contained in the solution containing the antibody to be purified The term "process-related impurities" includes both process-related impurities and product-related impurities. The term "impurity" as used herein refers to a composition, e.g., a protein-containing It refers to the impurity or impurities present in the solution that are not derived from the protein itself. Examples: For example, process-related impurities include, but are not limited to, cell culture media components, host cells, Main cellular components (e.g., proteins (HCPs), host cell nucleic acids, or lipid-containing intracellular structures) or fragments thereof), viruses, trace metals or ions from buffer solutions, material processing vessels or Process-related impurities include leachable materials from the chromatographic support. may be formed during the preparation (upstream and / or downstream processing) of a protein, e.g., an antibody. As used herein, the term "host cell impurities" refers to host cell lines, cell culture Proteinaceous, nucleic acid, lipid, or other contaminants introduced by the liquid or cell culture The term "host cell proteins" refers to proteins that are produced by or through the host cell line, cell culture medium, or Impurities refer to proteinaceous by-products introduced by the cell culture or However, Chinese hamster ovary protein (CHOP), E. coli i protein, yeast protein, simian COS protein, or myeloma cell protein Substances (e.g., NS0 protein (murine plasmacytoma cells derived from BALB / c mice)) Host cell proteins include the proteins of interest produced in a host cell expression system. For example, CHO cells are used to produce recombinant antibodies or fragments thereof. When used, the term "host cell protein" refers to a recombinant antibody or fragment thereof. As used herein, "produced" includes proteins derived from CHO cells, other than the target protein. The term “product-related impurities” refers to impurities that are derived from the protein of interest, e.g., an antibody, itself. For example, product-related impurities include, but are not limited to, aggregation of the antibody of interest. misfolded species, oxidized or deamidated species, or low molecular weight flag Examples include:

[0183] As used herein, the term "recombinant antibody" refers to an antibody produced in a host cell, e.g., a mammalian cell. The gene(s) carried on the recombinant expression vector(s) introduced into a host cell In certain embodiments, the term "antibody" refers to an antibody produced as a result of transcription and translation of a recombinant protein. Proteins include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, and IgA. 1, an antibody of an isotype selected from the group consisting of IgA2, IgD, or IgE. In certain embodiments, the recombinant antibody is an IgG1.

[0184] The term "recombinant host cell" (used interchangeably herein with the term "host cell") As used herein, the term includes cells into which a recombinant expression vector has been introduced. It should be understood that this is intended to refer not only to the subject cell but to the progeny of such a cell. Certain modifications occur in subsequent generations, either due to mutations or environmental influences. Since certain genetic alterations may occur, such progeny may not, in fact, be identical to the parent cell, but Still falling within the scope of the term "host cell" as used herein. Unless otherwise specified, when the term "cell" is used, it refers to, for example, a host cell or mammalian cell. It is understood that a mammalian cell or mammalian host cell is intended to include a population of cells. sea ​​bream.

[0185] "Substantially purified" with respect to a desired protein refers to a purified product containing the protein. The sample is at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least At least 97.5%, at least 98%, at least 98.5%, or at least 99% Contains the desired recombinant protein and contains less than 3%, less than 2.5%, less than 2%, less than 1.5% impurities This means less than 1%, or less than 0.5%.

[0186] As used herein, the term "upstream" in the context of a protein, e.g., an antibody, preparation. The term "process" refers to the production and collection of a protein (e.g., an antibody) from a host cell. refers to activities that involve the production of a protein of interest, such as an antibody, during cell culture. ).

[0187] As used herein, the term "vector" refers to a nucleic acid capable of propagating another nucleic acid to which it is linked. The term refers to a nucleic acid molecule. The term includes vectors as self-replicating nucleic acid structures, as well as Certain vectors are operably linked to the host cell genome. Such vectors are referred to herein as "expression vectors." It is called.

[0188] As used herein, the term "wash" or "wash solution" refers to a solution that is Aqueous liquids formulated to displace unbound contaminants from chromatographic supports In some embodiments, the wash is loaded with a protein of interest, e.g., an antibody. After the incubation, and prior to elution of the protein of interest, e.g., an antibody, a solid support, e.g., a resin or In one embodiment, the wash solution has similar biochemical properties as the equilibration solution. .

[0189] II. Anti-α4β7 integrin antibodies The methods disclosed herein provide a highly purified composition comprising an anti-α4β7 integrin antibody. As will be apparent, the anti-α4β Any of the methods for producing integrin antibodies may be used individually or in combination. In an exemplary embodiment, the antibody is vedolizumab or vedolizumab. Vedolizumab is also sold under the trademark ENT. YVIO® (Takeda Pharmaceuticals, Inc.) Vedolizumab is made up of human IgG1 framework and constant regions, as well as Vedolizumab C is a humanized antibody that contains the antigen-binding CDRs derived from the mouse antibody Act-1. The DR, variable region, and mutated Fc region (mutated to eliminate Fc effector function) No. 7,147,851, which is incorporated herein by reference in its entirety. It is being done.

[0190] Vedolizumab specifically binds to α4β7 integrin, e.g., the α4β7 complex Vedolizumab is a humanized monoclonal antibody that binds to the α4β7 integrin and mucosal address receptors. Blocks the interaction with MAdCAM-1, which promotes inflammation Inhibits the migration of memory T lymphocytes across the endothelium into the gastrointestinal parenchyma. It is known that integrin α4β1 or αEβ7 binds to or inhibits their function. It also antagonizes the interaction between α4 integrin and vascular cell adhesion molecule-1 (VCAM-1). Don't resist.

[0191] α4β7 integrin is a distinct subset of memory T lymphocytes that preferentially traffic to the gastrointestinal tract. MAdCAM-1 is expressed primarily on the surface of intestinal epithelial cells and in intestinal lymphoid tissues. α4β7 integrin and MAdCA play an important role in T lymphocyte homing to the endothelium. The interaction with M-1 may be involved in the development of mucosal inflammation, such as the chronic inflammation characteristic of ulcerative colitis and Crohn's disease. Vedolizumab is an important treatment for inflammatory bowel disease, e.g. ulcerative colitis, pouchitis, e.g., chronic pouchitis, graft-versus-host disease, and It can be used to treat HIV.

[0192] The heavy chain variable region of vedolizumab is provided herein as SEQ ID NO: 1, and The vedolizumab light chain variable region is provided herein as SEQ ID NO:5. The mab comprises CDR1 of SEQ ID NO:2, CDR2 of SEQ ID NO:3, and CDR3 of SEQ ID NO:4. Vedolizumab comprises a heavy chain variable region comprising CDR1 of SEQ ID NO:6, CDR2 of SEQ ID NO:7, and a light chain variable region comprising the CDR3 of SEQ ID NO: 8. A heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10. Vedolizumab and the sequence of vedolizumab are also incorporated herein by reference in their entireties. No. 2014 / 0341885 and U.S. Pat. No. 6,341,925, which are expressly incorporated herein by reference. and U.S. Patent Application Publication No. 2014-0377251. The method can be performed using an antibody that includes a binding region, e.g., a CDR or variable region, as described above. You may go.

[0193] The method of the present invention involves the administration of an anti-α4β7 antibody, particularly vedolizumab or vedolizumab, in mammalian cells. The binding domain of riboma, i.e., CDR or variable domain, can be used to produce antibodies. It is useful.

[0194] Exemplary Strategies for Antibody Production In certain embodiments, the methods described herein include one or more of the steps described below. one or more additional steps to facilitate production and / or purification of vedolizumab, Long-term, high-yield production of recombinant proteins such as vedolizumab To this end, mammalian host cells are cultured to stably express an anti-α4β7 antibody (e.g., vedolizumab). Exemplary cell culture processes and methods for the production of monoclonal antibodies such as vedolizumab. For a discussion of the production of clonal antibodies, see Li et al. (2010) mAbs 2:5,4 66-477, and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685, the entire contents of which are incorporated herein by reference. (incorporated herein).

[0195] In certain embodiments, the primary recovery is performed using sequential steps of pH reduction, centrifugation, and filtration. to remove cells and cell debris (including HCPs) from the production bioreactor harvest. In certain embodiments, the present invention may proceed by providing a sample from said primary collection. The mixture was purified by affinity chromatography, anion exchange (AEX), cation exchange (CEX) and cyclohexane chromatography. (CEX), Hydrophobic Interaction Chromatography (HIC), Ceramic Hydroxyapatite Tight chromatography (CHT) and / or mixed mode (MM) purification steps In some embodiments, the order of steps includes subjecting the The antibody composition can be produced by adjusting the levels of aggregates, impurities, or isoforms. This can affect the resulting quality.

[0196] In an exemplary embodiment, a composition comprising vedolizumab is The order of the ion exchange, cation exchange, and anion exchange is chosen based on the type of ion exchange (e.g., protein A), mixed mode, and cation exchange. The product can be purified using a process including the steps of:

[0197] In another exemplary embodiment, a composition comprising vedolizumab is HPLC (e.g., Protein A), mixed mode, cation exchange, and anion exchange. It can be purified using a process including steps.

[0198] In another exemplary embodiment, a composition comprising vedolizumab is HPLC (e.g., Protein A), cation exchange, mixed mode, and anion exchange. It can be purified using a process including steps.

[0199] In another exemplary embodiment, a composition comprising vedolizumab is HPLC (e.g., Protein A), anion exchange, mixed mode, and cation exchange. It can be purified using a process including steps.

[0200] In another exemplary embodiment, a composition comprising vedolizumab is a mixed mode, affinity - Chromatography (e.g., Protein A), anion exchange, cation exchange, in that order It can be purified using a process including steps.

[0201] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), CHT chromatography, cation exchange, anion exchange The product can be purified using a process that includes the steps of ion exchange.

[0202] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), CHT chromatography, anion exchange, cation exchange The product can be purified using a process that includes the steps of ion exchange.

[0203] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), cation exchange, CHT chromatography, anion The product can be purified using a process that includes the steps of ion exchange.

[0204] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), hydrophobic interaction chromatography, cation exchange The product can be purified using a process comprising the steps of anion exchange, followed by cation exchange.

[0205] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), hydrophobic interaction chromatography, anion exchange The product can be purified using a process comprising the steps of cation exchange, cation exchange, and the like.

[0206] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), anion exchange, hydrophobic interaction chromatography The product can be purified using a process comprising the steps of cation exchange, cation exchange, and the like.

[0207] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography (e.g., Protein A), cation exchange, hydrophobic interaction chromatography The product can be purified using a process comprising the steps of anion exchange, followed by cation exchange.

[0208] In another exemplary embodiment, a composition comprising vedolizumab is chromatography (e.g., Protein A), cation exchange, anion exchange, hydrophobic interaction chromatography The product may be purified using a process that includes steps in the order of chromatography.

[0209] In another exemplary embodiment, a composition comprising vedolizumab is chromatography (e.g., Protein A), anion exchange, cation exchange, hydrophobic interaction chromatography The product may be purified using a process that includes steps in the order of chromatography.

[0210] In another exemplary embodiment, a composition comprising vedolizumab is Chromatography, affinity chromatography (e.g., Protein A), anion exchange The product can be purified using a process comprising the steps of cation exchange, cation exchange, and the like.

[0211] It should be understood that the purification steps are not necessarily immediately adjacent to each other. Various other process steps, such as filtering or viral reduction steps, can be performed using the charge profile. without disturbing the effect of the order of chromatography steps on the It may be inserted between the first and second steps.

[0212] To modulate the level of a basic isoform species present in a vedolizumab composition, As described herein, the incubation step may be performed prior to any of the aforementioned purification steps. Additionally or alternatively, the purification process may be incorporated into any of the methods described herein. The antibody may be adapted to minimize the period of exposure to low pH conditions, such as .

[0213] An anion can be used to regulate the levels of host cell proteins present in the vedolizumab composition. At any stage of the vedolizumab purification process using ion exchange chromatography, AEX may be carried out using low conductivity buffers as described herein.

[0214] In order to control the yield of vedolizumab in a purification process that includes CHT, The CHT conditions described were compared using ceramic hydroxyapatite chromatography. It may be used at any stage of the dolizumab purification process.

[0215] Certain embodiments of the invention include a further purification step: Ion Exchange Chromatography Examples of additional purification steps that can be performed before, during, or after the purification procedure include ethanol precipitation. , isoelectric focusing, reversed-phase HPLC, chromatography on silica, heparin sepharose Chromatography on 100% ethanol, further anion exchange chromatography and / or or further cation exchange chromatography, chromatofocusing, SDS-PA GE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis , dialysis, and affinity chromatography (e.g., protein G, antibodies, specific using a substrate, ligand or antigen as a capture reagent.

[0216] In certain embodiments, the unbound flow-through and wash fractions may be further fractionated to separate the target Combinations of fractions that provide product purity may be pooled.

[0217] In certain embodiments, the protein concentration is adjusted to provide a good correlation between the antibody product and product-related materials. Different partitioning behavior can be achieved, which can result in further improvements in purity and / or yield. In an embodiment, loading is performed at different protein concentrations during the loading procedure, The product quality / yield of any particular purification step may be improved.

[0218] In certain embodiments, to improve the separation efficiency and / or yield of a particular purification step Additionally, the column temperature may be changed independently.

[0219] In certain embodiments, the loading and washing solution matrix is As can be achieved, the resins may be different while achieving similar "resin interaction" behavior. or a mixture of chemicals. For example, but not limited to, loading The solution and the wash solution may differ in ionic strength or pH, while the wash solution may differ in pH. The functionality remains substantially similar with respect to washout of the product achieved during the step. In certain embodiments, additives such as amino acids, sugars, PEG, etc., may be added during the loading step. Add to the wash or wash steps to adjust separation behavior to achieve better separation efficiency and / or yield. possible.

[0220] In certain embodiments, the loading & washing steps improve the quality of the target product and / or The level of column output or collected pool, or both, to achieve a yield Product-related impurity / substance levels in-line, at-line, or off-line In certain embodiments, the loading concentration can be controlled by line measurements. and / or into the solution or other solutions to achieve the necessary partitioning to improve yield. The concentration can be dynamically controlled by in-line or batch or continuous dilution.

[0221] Certain embodiments of the present invention use ultrafiltration and diafiltration steps to: The protein of interest, e.g., an antibody product, is further concentrated and formulated. , Microfiltration and Ultrafiltration: Pr principles and applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996); and Ultrafiltration Handbook, Munir Cheryan(Technomic Publishing,1986;ISBN N One of the filtration processes is described in detail in the paper "Phase filtration". armaceutical Process Filtration Catalog "Millipore Catalog" (Bedford, Massachusetts), pp. 177-202. ss., 1995 / 96). Ultrafiltration is generally performed using a pore size of This is considered to mean filtration using a filter with a diameter of less than 0.1 μm. Small pore size filters are used to retain proteins such as antibodies on the membrane surface The volume of the sample can be reduced by allowing the sample solution to permeate the pores of the filter membrane while do.

[0222] Diafiltration is the process of removing salts, sugars, and non-aqueous solvents using a membrane filter. Remove and exchange, separate from bound species, remove low molecular weight species, and / or remove ions and The method involves the rapid change of the pH and / or pH environment. The most efficient method is to add solvent to the solution being diafiltered at approximately the same rate. This allows the microspecies to be washed out of the solution in a consistent manner and retained. In a particular embodiment of the present invention, the diafiltrate is used to effectively purify the target protein. The purification step may optionally be performed prior to chromatography or other purification steps. To exchange various solutions used in connection with the invention and to remove impurities from protein preparations. Used to remove things.

[0223] Ultrafiltration / diafiltration (UF / DF) of vedolizumab involves the use of equipment and membranes, For example, PELLICON® cassettes (MilliporeSigma, Bu rlington MA), for example polyethersulfone or regenerated cellulose, e.g. For example, ULTRACEL® or BIOMAX® membranes are available. In one embodiment, the UF is a microporous polyethylene with a molecular weight cutoff of 30 kDa. This is carried out using a cellulose membrane cast onto a cellulose membrane.

[0224] III. Preparation of Vedolizumab Compositions Containing Altered Levels of Basic Isoform Species Preparations of vedolizumab from mammalian host cells typically contain major (or In addition to the main vedolizumab isoform, there are also small amounts of acidic and / or The acidic and basic isoform species include, for example, cationic isoform species. and cation exchange high pressure liquid chromatography (CEX-HPLC), known in the art. The acidic and basic vedolizumab isoforms can be quantified by the CEX tree. The major isoforms can be separated based on their different retention times in lipids. As shown in Figure 1, acidic vedolizumab isoform species present in liquid preparations of vedolizumab has a shorter retention time than the major isoform, and the basic vedolizumab isoform species The vedolizumab preparations show a longer retention time compared to the major isoform. There is slight variation in the retention times of multiple acidic species, which result in elution from the CEX resin at different retention times. and / or multiple basic species. Multiple basic peaks may be due to retention on the CEX resin. The major isoform peaks are referred to herein in relation to time, followed by the CEX resin. The first basic isoform peak to elute from the elution column is referred to herein as "basic peak 1." A second basic isoform elutes from the CEX resin following the major isoform peak. The main isoform peak is referred to herein as "basic peak 2" and is the major isoform peak. The third basic isoform peak eluting from the CEX resin following this peak is referred to herein as " It is called "basic peak 3", etc.

[0225] Percentage of antibodies present as major isoforms in pharmaceutical antibody preparations While maximizing the percentage of basic and / or acidic isoforms In one aspect, the present invention relates to a composition comprising vedolizumab. Methods for modulating the percentage of basic vedolizumab isoforms are provided. The method revealed the surprising finding that the distribution of vedolizumab isoforms can be modulated by changes in pH. The basic isoform of vedolizumab is particularly sensitive to pH changes. As described herein, this pH-dependent regulation of isoform distribution This is at least in part due to the change in the level of basic peak 2 and the associated It is driven by variations in the levels of the major isoforms.

[0226] While not wishing to be bound by theory, it is believed that the discoveries provided herein may be at least in part due to Based on these results, at least two basic isoform species were identified in some vedolizumab preparations. First, it is believed that there exists a basic peak 1 eluting from the CEX resin. This is due to the presence of a lysine residue at the carboxy terminus of the IgG heavy chain. Elution from the CEX resin as "basic peak 2" indicates that one or more asparagines of the antibody This is due to the isomerization of the carboxylic acid residue to form a succinimide intermediate. In , one or more aspartic acid residues undergo isomerization to form a succinimide intermediate The glycine or serine residue adjacent to the aspartic acid is the “n+1 position” (calcium). (near the carboxy terminus, one amino acid away) isomerization of the aspartic acid residue to succinimide Substituting succinyltransferase (Succinimidyl) for aspartic acid at residue 102 of SEQ ID NO:1 may be advantageous for the synthesis of ribozymes. The identification of this variant of vedolizumab containing an imide confirms that this basic isoform In some embodiments, vedolizumab preparations are provided with reduced, minimized, or In some embodiments, the basic isoform The variants can be, for example, fractionated (e.g., on a CEX resin) from a preparation containing vedolizumab. Also, the fraction of vedolizumab eluting from the CEX resin as “basic peak 2” was Also identifiable as a compound having a succinyl group instead of aspartic acid at residue 102 of SEQ ID NO:1. and removing (or failing to collect) those fractions that contain the aniline imide. In some embodiments, the basic isoform variant (as described herein) may be removed. In literature it is called "succinimide variant" or alternatively "basic isoform peak 2." or "BP2" variants) regulate the pH exposure of antibodies. This can be minimized during the production and purification of vedolizumab. Compositions with reduced levels of basic isoform variants are This may include a corresponding increase in the relative proportions of isoforms. In this embodiment, the composition having a reduced level of the basic isoform variant is There may be increased efficacy compared to compositions containing elevated levels of the isoform variant.

[0227] In one embodiment, provided herein is a method for treating a pulmonary edema in which the level of a basic isoform species is reduced. The present invention relates to a method for purifying vedolizumab obtained by ultrafiltration / diafiltration. Cell cultures are harvested in pharma- ceutically acceptable carriers by filtration (UF / DF). The total time from initial recovery of vedolizumab from the harvest to the time vedolizumab is formulated. At least 60%, at least 65%, at least 70%, at least 75% of the At least 80%, at least 85%, at least 90%, at least 95%, at least At least 97%, at least 98%, or at least 99% of the time, the pH is greater than or equal to 5.5, pH 5. 6 or above, pH5.7 or above, pH5.8 or above, pH5.9 or above, pH6.0 or above, pH6. 1 or higher, pH 6.2 or higher, pH 6.3 or higher, pH 6.4 or higher, or pH 6.5 or higher. Vedolizumab is maintained at pH 5.5 or higher and pH 5.6 for most of the purification process. Above, pH5.7 or above, pH5.8 or above, pH5.9 or above, pH6.0 or above, pH6. 1 or higher, pH 6.2 or higher, pH 6.3 or higher, pH 6.4 or higher, or pH 6.5 or higher. By retaining the basic bendolizumab isoforms, the basic bendolizumab isoforms were subjected to an equivalent purification process. In this process, the concentration of glycerol in the blood can be reduced by ultrafiltration / diafiltration. The vedolizumab is first recovered from the cell culture harvest in a pharma- ceutically acceptable carrier, A significant portion of the total time it takes for vedolizumab to be formulated, e.g., greater than 5%, 10%, Between more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, or more than 40%, The lizumab has a pH of less than pH 5.5 to 6.5. For example, 1000L scale, 2000L scale, 3000L scale, 4000L scale The bed derived from the harvest of cell cultures produced at the L-scale or 5000 L-scale The preparation of tafamidis is carried out on a commercial manufacturing scale (e.g., at least 30 00L scale).

[0228] Thus, in one aspect, the present invention provides a method for the preparation of a low-order α4β7 antibody or an antigen-binding portion thereof. The present invention provides a method for producing a composition comprising: (i) an anti-α4β7 antibody or an antigen thereof; Harvesting of clarified cell culture obtained from culture of recombinant host cells expressing the binding moiety (ii) administering to the patient an anti-α4β7 antibody or an antigen-binding portion thereof, to a patient in need of treatment with a cell culture. and purifying the antibody from a harvest, wherein the antibody is purified at a pH of 3.5 or less (e.g., pH 2.5 to 3.5, less than pH 3.0, or less than pH 3.5) for 20 minutes, 30 minutes, or 45 minutes Within, within 1 hour, within 3 hours, within 5 hours, within 7 hours, within 10 hours, or 12 o'clock Within a period of time, the anti-α4β7 antibody or antigen-binding portion thereof exhibits a basal The levels of the sex isoform species were decreased (as determined by CEX) in this control. or a composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof produced by the same method, The antibody may be used at a pH of 3.5 or less (e.g., pH 2.5 to 3.5, pH less than 3.0, or (pH less than 3.5) for long periods of time, i.e., more than 20 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, Exposure for more than 3 hours, more than 5 hours, more than 7 hours, more than 10 hours, or more than 12 hours. In an embodiment, the low basic species composition comprises a lower level of BP2 compared to a control. In some embodiments, the antibody or antigen-binding portion thereof comprises the amino acid sequence set forth in SEQ ID NO:1. A heavy chain variable region comprising a set of sequences, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the antibody or antigen-binding portion thereof comprises vedolizumab, or an antigen-binding portion thereof. In some embodiments, the anti-α4β7 antibody or The step of purifying the antigen-binding portion may include protein A chromatography, anion exchange chromatography, or the like. Chromatography, Cation Exchange Chromatography, Mixed Mode Chromatography, hydrophobic interaction chromatography, and combinations thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or antigen-binding portion thereof is In another embodiment, the host cell is a DHFR-CHO cell. In some embodiments, the composition of low basic species is less than 16%, less than 15%, less than 14%. Less than 13%, Less than 12%, Less than 11%, Less than 10%, or Less than 9% basic isoforms In some embodiments, the composition of the low based species comprises less than 4%, less than 3% Contains less than, less than 2%, or less than 1% basic isoform peak 2.

[0229] In another aspect, the present invention provides a low basic species composition of an anti-α4β7 antibody or antigen-binding portion thereof. The present invention provides a method for producing an anti-α4β7 antibody, or an antigen-binding portion thereof, comprising: (i) administering to said patient an anti-α4β7 antibody or an antigen-binding portion thereof; and providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing (ii) isolating an anti-α4β7 antibody or antigen-binding portion thereof from the cell culture harvest; wherein the antibody is at a pH of 4.0 or less (e.g., pH 3.6 to 4.0). , within 20 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 3 hours, within 5 hours, 10 o'clock Within 1 hour, 12 hours, 15 hours, 18 hours, or 24 hours of exposure. The anti-α4β7 antibody or antigen-binding portion thereof inhibits the basic isoform species as compared to a control. The levels were decreased (as determined by CEX) and the control was made in the same manner. a composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof, the antibody having a β-glutamylase activity of 4.0 or more; Long periods at lower pH levels (e.g., pH 3.6 to 4.0), i.e., more than 20 minutes, more than 30 minutes, 4 Over 5 minutes, Over 1 hour, Over 3 hours, Over 5 hours, Over 10 hours, Over 12 hours, Over 15 hours, 18:00 In some embodiments, the low basic species composition is exposed to the solvent for more than 24 hours. In some embodiments, the antibody or antibody thereof comprises a lower level of BP2 as compared to a control. The antigen-binding portion of the present invention comprises a heavy chain variable region comprising the set of amino acid sequences set forth in SEQ ID NO:1, and In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5. The antibody or antigen-binding portion thereof is vedolizumab, or an antigen-binding portion thereof. In some embodiments, the step of purifying the anti-α4β7 antibody, or antigen-binding portion thereof, comprises purifying the antibody by Protein A chromatography, anion exchange chromatography, cation exchange chromatography topography, mixed mode chromatography, hydrophobic interaction chromatography, and and one or more of these combinations. or an antigen-binding portion thereof. In some embodiments, the host cell is a DHFR-CHO cell. The composition is less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%. In some embodiments, the nucleotide sequence includes less than 10%, or less than 9% of the basic isoform species. In the present invention, a low basicity composition is one that has less than 4%, less than 3%, less than 2%, or less than 1% basicity. Contains isoform peak 2.

[0230] In another aspect, the present invention provides a low basic species composition of an anti-α4β7 antibody or antigen-binding portion thereof. The present invention provides a method for producing an anti-α4β7 antibody, or an antigen-binding portion thereof, comprising: (i) administering to said patient an anti-α4β7 antibody or an antigen-binding portion thereof; and providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing (ii) isolating an anti-α4β7 antibody or antigen-binding portion thereof from the cell culture harvest; wherein the antibody is prepared at a pH of 4.5 or less (e.g., pH 4.1 to 4.5). ) within 3 hours, within 5 hours, within 10 hours, within 12 hours, within 18 hours, within 24 hours Within 36 hours, within 48 hours, within 72 hours, within 96 hours, exposed to this anti-α The 4β7 antibody or an antigen-binding portion thereof reduces the level of basic isoform species compared to a control. The effect of the antibody was reduced (as determined by CEX) and this control was compared with that of the antibody produced in the same manner. A composition comprising an α4β7 antibody or an antigen-binding portion thereof, the antibody having a pM of 4.5 or less. H (e.g., pH 4.1-4.5) for a long period of time, i.e., more than 3 hours, more than 5 hours, or more than 10 hours More than 12 hours, more than 18 hours, more than 24 hours, more than 36 hours, more than 48 hours, more than 72 hours, and In some embodiments, the low basic species composition is exposed to a long period of time of 96 hours. In some embodiments, the antibody or its anti- The original binding portion comprises a heavy chain variable region comprising the set of amino acid sequences set forth in SEQ ID NO:1, and the sequence In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in claim 5. or an antigen-binding portion thereof is vedolizumab, or an antigen-binding portion thereof. In embodiments, the step of purifying the anti-α4β7 antibody, or antigen-binding portion thereof, comprises purifying a protein A-chromatography, anion exchange chromatography, cation exchange chromatography Chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and In some embodiments, the anti-α4β7 antibody or In another embodiment, the host cell expressing the antigen-binding portion is a GS-CHO cell. In some embodiments, the host cell is a DHFR-CHO cell. Composition: Less than 16%, Less than 15%, Less than 14%, Less than 13%, Less than 12%, Less than 11%, In some embodiments, the nucleic acid sequence comprises less than 10%, or less than 9%, of a basic isoform species. The low basic species composition may contain less than 4%, less than 3%, less than 2%, or less than 1% basic isoforms. Includes 2 of the 2nd peaks.

[0231] In another aspect, the present invention provides a low basic species composition of an anti-α4β7 antibody or antigen-binding portion thereof. The present invention provides a method for producing an anti-α4β7 antibody, or an antigen-binding portion thereof, comprising: (i) administering to said patient an anti-α4β7 antibody or an antigen-binding portion thereof; and providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing (ii) isolating an anti-α4β7 antibody or antigen-binding portion thereof from the cell culture harvest; wherein the antibody is prepared at a pH of 5.0 or less (e.g., pH 4.6 to 5.0). ) within 3 hours, within 5 hours, within 10 hours, within 12 hours, within 18 hours, within 24 hours Within 36 hours, within 48 hours, within 72 hours, or within 96 hours of exposure. The anti-α4β7 antibody or antigen-binding portion thereof inhibits the basic isoform species as compared to a control. The levels were decreased (as determined by CEX) and the control was made in the same manner. a composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof, the antibody having a β-glutamylase activity of 5.0 or more; Long periods at lower pH levels (e.g., pH 4.6 to 5.0), i.e., more than 3 hours, more than 5 hours, or 1 More than 0 hours, more than 12 hours, more than 18 hours, more than 24 hours, more than 36 hours, more than 48 hours, more than 72 hours In some embodiments, the low basic species composition is exposed to a control for 10 to 20 hours. In some embodiments, the antibody or its anti- The original binding portion comprises a heavy chain variable region comprising the set of amino acid sequences set forth in SEQ ID NO:1, and the sequence In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in claim 5. or an antigen-binding portion thereof is vedolizumab, or an antigen-binding portion thereof. In embodiments, the step of purifying the anti-α4β7 antibody, or antigen-binding portion thereof, comprises purifying a protein A-chromatography, anion exchange chromatography, cation exchange chromatography Chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and In some embodiments, the anti-α4β7 antibody or In another embodiment, the host cell expressing the antigen-binding portion is a GS-CHO cell. In some embodiments, the host cell is a DHFR-CHO cell. Composition: Less than 16%, Less than 15%, Less than 14%, Less than 13%, Less than 12%, Less than 11%, In some embodiments, the nucleic acid sequence comprises less than 10%, or less than 9%, of a basic isoform species. The low basicity species composition has less than 4%, less than 3%, less than 2%, or less than 1% basic isomer. Includes Soform Peak 2.

[0232] In another aspect, the present invention provides a low basic species composition of an anti-α4β7 antibody or antigen-binding portion thereof. The present invention provides a method for producing an anti-α4β7 antibody, or an antigen-binding portion thereof, comprising: (i) administering to said patient an anti-α4β7 antibody or an antigen-binding portion thereof; and providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing (ii) isolating an anti-α4β7 antibody or antigen-binding portion thereof from the cell culture harvest; wherein the antibody is prepared at a pH of 5.5 or less (e.g., pH 5.1 to 5.5). ) within 3 hours, within 5 hours, within 10 hours, within 12 hours, within 18 hours, within 24 hours Within 36 hours, within 48 hours, within 72 hours, or within 96 hours of exposure. The anti-α4β7 antibody or antigen-binding portion thereof inhibits the basic isoform species as compared to a control. The levels were decreased (as determined by CEX) and the control was made in the same manner. a composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof, the antibody having a β- Long periods at lower pH levels (e.g., pH 5.1 to 5.5), i.e., more than 3 hours, more than 5 hours, or more than 1 More than 0 hours, more than 12 hours, more than 18 hours, more than 24 hours, more than 36 hours, more than 48 hours, more than 72 hours In some embodiments, the low basic species composition is exposed to a control for 10 to 20 hours. In some embodiments, the antibody or its anti- The original binding portion comprises a heavy chain variable region comprising the set of amino acid sequences set forth in SEQ ID NO:1, and the sequence In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in claim 5. or an antigen-binding portion thereof is vedolizumab, or an antigen-binding portion thereof. In embodiments, the step of purifying the anti-α4β7 antibody, or antigen-binding portion thereof, comprises purifying a protein A-chromatography, anion exchange chromatography, cation exchange chromatography Chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and In some embodiments, the anti-α4β7 antibody or In another embodiment, the host cell expressing the antigen-binding portion is a GS-CHO cell. In some embodiments, the host cell is a DHFR-CHO cell. Composition: Less than 16%, Less than 15%, Less than 14%, Less than 13%, Less than 12%, Less than 11%, In some embodiments, the nucleic acid sequence comprises less than 10%, or less than 9%, of a basic isoform species. The low basicity species composition has less than 4%, less than 3%, less than 2%, or less than 1% basic isomer. Includes Soform Peak 2.

[0233] In some embodiments, the method is carried out at, for example, a 1000 L scale, a 2000 L scale, 3000L scale, 4000L scale, or 5000L scale (e.g. Vedolizumab derived from the harvest of cell cultures produced at a scale of at least 3000 L This is carried out on a commercial manufacturing scale using the preparation of the present invention.

[0234] In some embodiments, the methods described herein provide a composition, e.g., vedolizumab. The present invention is used to regulate the level of a basic vedolizumab isoform in a liquid composition comprising In some embodiments, the method can be used to detect low levels of basic vedolizumab. Vedolizumab compositions having seeds may be prepared.

[0235] In one aspect, the present invention provides a method for reducing the levels of basic vedolizumab isoform species. Incubating the composition at a pH above pH 6.5 for a sufficient period of time can result in the formation of β-lactamase. Methods for reducing levels of basic vedolizumab isoform species in compositions containing vedolizumab - Patents.com In one embodiment, the method is carried out at ambient temperature, e.g., 20-25°C. In another embodiment, the method is carried out at 2-8°C.

[0236] In another aspect, the present invention provides a method for the treatment of vedolizumab comprising administering to a patient a therapeutically effective amount of vedolizumab having reduced levels of the basic vedolizumab isoform species. A method for producing a composition comprising dolizumab is provided. The method comprises administering to the patient a pH of 6.5 or greater. providing a composition comprising vedolizumab in a basic vedolizumab solution; Incubate for a time sufficient to reduce the levels of limumab isoforms. Contains vedolizumab with reduced levels of basic vedolizumab isoforms The method may include forming a composition.

[0237] Efficiently reducing the levels of basic vedolizumab isoform species in vedolizumab compositions To this end, the incubation is preferably carried out at a pH of at least pH 6.5.

[0238] In an exemplary embodiment, the pH of the vedolizumab composition is about pH 6.5 to 9.0. For example, the pH of the vedolizumab composition may be about pH 6.5 to 9.0, pH 6.5 to 8.5, The pH range may be H6.5-8.0, pH6.5-7.5, or pH6.5-7.0. Alternatively, the pH of the vedolizumab composition is about pH 7.0 to 9.0, pH 7.5 to 9.0, The pH range may be from 8.0 to 9.0, or from 8.5 to 9.0. The pH of the vedolizumab composition is about 6.5 to 7.5, for example, pH 6.6 to 7.3, pH6.6~7.5, pH6.7~7.5, pH6.8~7.5, pH6.9~7.5, pH7.0~7.5, pH7.1~7.5, pH7.2~7.5, pH7.3~7.5, Alternatively, the pH may be in the range of 7.4 to 7.5. The pH range is approximately pH 6.5-7.5, pH 6.5-7.4, pH 6.5-7.3, and pH 6.5. ~7.2, pH6.5~7.1, pH6.5~7.0, pH6.5~6.9, pH6.5 The pH range may be from 6.5 to 6.8, 6.5 to 6.75, or 6.5 to 6.6. In an embodiment, the pH of the vedolizumab composition may range from about 7.0 to 7.5. In an embodiment, the pH of the vedolizumab composition may range from about 7.5 to 8.0. In embodiments, the pH of the vedolizumab composition may range from about 8.0 to 8.5. In certain embodiments, the pH of the vedolizumab composition is about pH 6.5, pH 6.6, pH 6.7 , pH6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7 .4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, p H8.1, pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7 , pH 8.8, pH 8.9, or pH 9.0.

[0239] Compositions containing vedolizumab at the above pH, for example at a pH of 6.5 or higher, Sufficient to reduce the percentage of basic vedolizumab isoform species in the composition. In an exemplary embodiment, the incubation is for 20 minutes or more. Above, 30 minutes or more, 45 minutes or more, 1 hour or more, 1.5 hours or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 1 8 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 96 hours or more, 120 hours or more For example, the treatment may be performed for a period of 144 hours or more, or 168 hours or more. So, incubation is about 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more. In some embodiments, the ink is applied for a period of 5 days or more, 6 days or more, or 7 days or more. Incubation is about 20 minutes to about 1 hour, about 20 minutes to about 1 hour, about 20 minutes to about 2 hours, about 2 0 minutes to about 3 hours, about 1 hour to about 3 hours, about 1 hour to about 5 hours, or about 5 hours to about 8 hours In some embodiments, the incubation may be performed for about 8 hours to about 168 hours. In some embodiments, the incubation may be performed for 1 hour (7 days) or more. The duration is about 8 to 168 hours, for example, about 8 to 144 hours (6 days), about 8 to 120 hours (5 days), ), about 8 to 96 hours (4 days), about 8 to 72 hours (3 days), about 8 to 48 hours (2 days), about 8 ~36 hours, approx. 8~24 hours (1 day), approx. 8~18 hours, approx. 8~12 hours, approx. 15~36 In another embodiment, the incubation is performed for about 8 to 10 hours. About 10 hours to about 168 hours or more, for example, about 10 to 168 hours, about 12 to 168 hours, about 18~168 hours, approx. 24~168 hours, approx. 36~168 hours, approx. 48~168 hours , about 72 to 168 hours, about 96 to 168 hours, about 120 to 168 hours, or about 144 to In some embodiments, the incubation may be for about 0.5 to 168 hours. 7 days, for example, about 0.5 to 5 days, about 0.5 to 4 days, about 0.5 to 3 days, about 0.5 to 2 days, In some embodiments, the incubation may be performed for about 0.5 to 1 day. The incubation may be performed for about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days. Incubation at H6.5 or higher can be for periods of 1-2 days, 1-3 days, or 1-5 days. In another exemplary embodiment, the incubation at pH 6.5 or higher is performed for Total purification time, e.g., starting from providing a harvest of clarified cell culture and culminating in the production of purified antibody. This occurs for more than 25% of the time, ending with the body's UF / DF.

[0240] In other embodiments, the incubation step comprises incubating the basic vedolizumab isoform in the composition. The percentage of the game is 1% or more, e.g. 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, 6% or more, 7% or more, 8 % or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 1 5% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more , 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% In an exemplary embodiment, the method comprises: Incubation determines the percentage of basic vedolizumab isoform species in the composition. In another embodiment, the incubation is performed for a period of time sufficient to reduce the amount of The percentage of basic vedolizumab isoforms in the composition is between 1% and 10%. In another embodiment, the incubation is performed for a period of time sufficient to reduce the composition by %. Reduce the percentage of basic vedolizumab isoforms in the sample by 2% to 10% In another embodiment, the incubation is performed for a period of time sufficient to A period sufficient to reduce the percentage of vedolizumab isoforms by 5% to 10% In other embodiments, the incubation is performed for a period of time after the basic vedolizumab in the composition is This is performed for a period of time sufficient to reduce the percentage of isoform species by 2%-20%. In other embodiments, the incubation step comprises incubating the basic vedolizumab isoform in the composition. This is done for a period of time sufficient to reduce the percentage of germline species by 5%-20%.

[0241] In other embodiments, the incubation is with an anti-α4β7 antibody (e.g., vedolizumab). and for a period of time sufficient to produce a low basic species composition comprising less than 16% Less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, or 9 In some embodiments, the method comprises the step of: The level of basic isoform peak 2 is less than 4%, less than 3%, less than 2%, or less than 1%. Incubating the composition at a pH greater than 6.3 for a period of time sufficient to achieve In some embodiments, the method comprises administering a composition comprising vedolizumab at a pH of 6. pH higher than 3 (e.g., pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6 .8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, p H7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8.1 , pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7, pH8 0.8, pH 8.9, or pH 9.0); incubate with anti-α4β7 antibody (e.g. For example, vedolizumab) is added to the composition for 20 minutes or more, for example, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more , 10 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 24 hours or more, 48 hours 72 hours or more, 96 hours or more, 120 hours or more, 144 hours or more, or 168 hours This includes incubating for at least 2 hours.

[0242] During incubation, in some embodiments, the pH of the vedolizumab composition is adjusted to pH During incubation, in another embodiment, the vedolizumab antibody may be maintained at 6.3 or higher. The pH of the composition may be maintained at or above pH 6.5. The composition is maintained at approximately the same pH during the incubation period.

[0243] The incubation may be carried out at any suitable temperature. The incubation may be carried out at a temperature ranging from about 0 to 40° C. In another embodiment, the incubation The incubation may be carried out at a temperature ranging from about 1 to 37° C. In some embodiments, the incubation The reaction is carried out at a temperature in the range of about 0-4° C. or in the range of 4-8° C. In the present case, the incubation is carried out at ambient temperature. For example, the incubation is carried out The incubation may be carried out at a temperature ranging from about 20 to 25° C. In another embodiment, the incubation is In some embodiments, the incubation is carried out at about 37° C. In some embodiments, the incubation is carried out at a temperature ranging from about 5 to 100° C. In some embodiments, the incubation is carried out at a temperature in the range of about 18° C. The incubation is carried out at a temperature ranging from 15 to 30° C. In some embodiments, the incubation The incubation is carried out at a temperature ranging from about 33 to 37° C. In an exemplary embodiment, the incubation The options are 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 ℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃ It is carried out at °C.

[0244] The above method optionally includes the additional step of adjusting the pH of the vedolizumab composition. For example, the percentage of basic vedolizumab species in a composition comprising vedolizumab may be If it is desired to reduce the amount of phosphate ... For example, the composition comprising vedolizumab may include a step of increasing H to less than 6.5. If the pH is 6.5 or more, the method includes increasing the pH of the composition to a pH of 6.5 or more. Conversely, the percentage of basic vedolizumab species in a composition comprising vedolizumab may be If it is desired to increase the pH of the composition, the method may further comprise increasing the pH of the composition prior to incubation. For example, the composition comprising vedolizumab may include a step of lowering the pH of the composition to 6.5 or less. If the composition is in the range of 0.1 to 1.5, the method may further include reducing the pH of the composition to a pH below 6.5. good.

[0245] The composition comprising vedolizumab may be a liquid solution. The composition comprising dolizumab can be used to produce vedolizumab or an antigen-binding portion thereof. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell, e.g. For example, CHO cells lacking dihydrofolate reductase (DHFR) expression, or glutamine These are CHO cells lacking GS synthetase (GS) expression.

[0246] The aforementioned methodology is applicable to small- or large-scale purification of vedolizumab following isolation of the antibody from cell culture. In certain embodiments, the vedolizumab extract from a host cell culture may be incorporated into a microorganism-derived extract process. Primary recovery of the enzyme, e.g., bioreactor harvest, may involve centrifugation and filtration steps. The methods described herein may include steps before or after centrifugation and / or before or after filtration. The vedolizumab composition is then subjected to a corresponding purification step to remove bases in the composition. After primary recovery, process-related impurities and / or or downstream processing steps that may be used to purify vedolizumab from product-related impurities. Examples of such methods include, but are not limited to, affinity chromatography (Protein A chromatography), depth filtration, cation exchange (CEX), anion exchange (AEX ), mixed mode chromatography (MM), ceramic hydroxyapatite chromatography Chromatography (CHT), Hydrophobic Interaction Chromatography (HIC), Ultrafiltration, and The methods described herein may include downstream processes such as filtration, diafiltration, and / or diafiltration. to the vedolizumab composition before or after the step to obtain a composition at a corresponding step of purification. The levels of basic isoform species in the blood may be reduced.

[0247] For example, the vedolizumab composition may be subjected to affinity chromatography prior to or after affinity chromatography. The cells may be incubated as described herein. In one embodiment, the cells may be incubated in a manner similar to that described herein. The method includes dissolving a vedolizumab composition, e.g., an affinity chromatography load material. or the pH of the affinity chromatography eluate should be adjusted to pH 6.5 or higher. This may include adjusting the

[0248] In another embodiment, the vedolizumab composition is administered either before or after depth filtration using a method described herein. In one embodiment, the method is performed at a pH of 6.5 or higher. The method may include adjusting the pH of the vedolizumab composition before or after depth filtration. .

[0249] In another embodiment, the vedolizumab composition is, before or after cation exchange (CEX), In one embodiment, the method comprises the steps of: The pH of the dolizumab composition, e.g., the CEX load material, or the CEX eluate, may be adjusted to pH 6. This may include adjusting the pH to 6.5 or greater.

[0250] In another embodiment, the vedolizumab composition is, prior to or after anion exchange (AEX): In one embodiment, the method comprises: , vedolizumab composition, e.g., AEX load material, or AEX flow-through This may include adjusting the pH to a pH of 6.5 or greater.

[0251] In another embodiment, the vedolizumab composition is It may be incubated before or after as described herein. The method includes administering to the patient a vedolizumab composition, e.g., a MM load material, or a MM eluate. to a pH of 6.5 or greater.

[0252] In another embodiment, the vedolizumab composition is a ceramic hydroxyapatite chromatograph. Incubation as described herein was performed before or after CT imaging. In one embodiment, the method includes administering to the subject a vedolizumab composition, e.g., a CHT-loaded mate. This may include adjusting the pH of the real or CHT eluate to a pH of 6.5 or higher. good.

[0253] In another embodiment, the vedolizumab composition is purified by hydrophobic interaction chromatography (HI C) may be incubated as described herein before or after. In this method, a vedolizumab composition, e.g., a HIC load material, or a HI C may include adjusting the pH of the eluate to a pH of 6.5 or greater.

[0254] In another embodiment, the vedolizumab composition is subjected to ultrafiltration and / or diafiltration. Incubation as described herein was performed before or after UF / DF. In one embodiment, the method further comprises adjusting the pH of the vedolizumab composition before or after UF / DF. may include a subsequent pH adjustment to pH 6.5 or greater.

[0255] IV. Vedolizumab Compositions Containing Reduced Basic Isoform Species In some embodiments, the present invention provides a method for treating low levels of basic vedolizumab isoform species. In some embodiments, the basic isoform Compositions having reduced levels of a species can be used in accordance with the methods provided herein (e.g., Section III). In some embodiments, the basic aryl group can be obtained by the method of the present invention. Compositions having reduced levels of an isoform species can be used in accordance with the methods provided herein (e.g., See Section III. Provided herein is a composition comprising vedolizumab, wherein the basic isoform in the composition is Bedli at pH greater than 6.5 for a period of time sufficient to reduce the level of foam species. The method includes, inter alia, incubating a composition comprising izumab. Following incubation, the vedolizumab-containing composition is added to, e.g., To reduce the levels of process-derived impurities and / or product-derived impurities in the composition. The product may then be optionally subjected to further purification steps designed to

[0256] In another aspect, provided herein is a method for treating cancer comprising administering to the patient a therapeutically effective amount of vedolizumab or an antigen-binding portion thereof. The method includes, for example, providing an antibody that is exposed to low pH conditions during a purification process. This composition is obtained by limiting the period of exposure.

[0257] In some embodiments, provided herein is a method for treating vedolizumab, or an antigen thereof. A composition comprising a binding moiety, the composition comprising, inter alia: (i) vedolizumab; or an antigen-binding portion thereof. (ii) administering vedolizumab, or an antigen-binding portion thereof, to a cell. and purifying the antibody or antigen-binding portion thereof from a harvest of the cell culture. pH below 3.5 (e.g., pH 2.5 to 3.5, pH less than 3.0, or pH less than 3.5) 20 minutes or less, 30 minutes or less, 45 minutes or less, 1 hour or less, 3 hours or less, 5 hours or less, Within 7 hours, 10 hours, or 12 hours, the anti-α4β7 antibody or The antigen-binding portion exhibits reduced levels of basic isoform species compared to a control. (as determined by CEX), this control was either an anti-α4β7 antibody or is a composition comprising an antigen-binding portion thereof, the antibody being capable of binding to a pH below 3.5 (e.g., pH 2.5 to 3.5, pH less than 3.0, or pH less than 3.5) for a long period of time, i.e., 2 More than 0 minutes, More than 30 minutes, More than 45 minutes, More than 1 hour, More than 3 hours, More than 5 hours, More than 7 hours, More than 10 hours, or exposing the composition to the liquid for more than 12 hours.

[0258] In some embodiments, provided herein is a method for treating vedolizumab, or an antigen thereof. A composition comprising a binding moiety, the composition comprising, inter alia: (i) vedolizumab; or an antigen-binding portion thereof. (ii) administering vedolizumab, or an antigen-binding portion thereof, to a cell. and purifying the antibody or antigen-binding portion thereof from a harvest of the cell culture. At a pH of 4.0 or less (e.g., pH 3.6 to 4.0), within 20 minutes, within 30 minutes, or within 45 minutes Within, within 1 hour, within 3 hours, within 5 hours, within 10 hours, within 12 hours, over 15 hours Within 18 hours or 24 hours after exposure, the anti-α4β7 antibody or its antigen binding The combined area showed a decrease in the levels of basic isoform species compared to the control (CEX). The control was an anti-α4β7 antibody or its antigen produced in the same manner. A composition comprising a binding moiety, the antibody being capable of binding to a substance at a pH of 4.0 or less (e.g., pH 3.6 to 4. .0) for a long period of time, i.e., more than 20 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, more than 3 hours, more than 5 hours More than 10 hours, more than 12 hours, more than 15 hours, more than 18 hours, or more than 24 hours of exposure and

[0259] In another aspect, provided herein is a method for the treatment of cancer, comprising administering to the patient a therapeutically effective amount of vedolizumab, or an antigen-binding portion thereof. A composition comprising, inter alia: (i) vedolizumab, or an anti-cancer agent thereof; Harvesting of clarified cell culture obtained from culture of recombinant host cells expressing the original binding moiety. (ii) incubating a cell culture with vedolizumab, or an antigen-binding portion thereof, and purifying said antibody or antigen-binding portion thereof from said harvested product, said antibody or antigen-binding portion having a molecular weight of 4.5 or less. (e.g., pH 4.1 to 4.5) within 3 hours, within 5 hours, within 10 hours, or within 1 Within 2 hours, Within 18 hours, Within 24 hours, Within 36 hours, Within 48 hours, Within 72 hours or within 96 hours, and the anti-α4β7 antibody or antigen-binding portion thereof is The levels of basic isoform species were decreased compared to those of α-terminal β-terminal β-terminal The control contains an anti-α4β7 antibody or an antigen-binding portion thereof produced in the same manner. The composition is characterized in that the antibody can be maintained at a pH of 4.5 or less (e.g., pH 4.1 to 4.5) for a long period of time. , i.e., more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, Those who are exposed for more than 36 hours, more than 48 hours, more than 72 hours, or more than 96 hours The composition is obtainable by the method.

[0260] In another aspect, provided herein is a method for the treatment of cancer, comprising administering to the patient a therapeutically effective amount of vedolizumab, or an antigen-binding portion thereof. A composition comprising, inter alia: (i) vedolizumab, or an anti-cancer agent thereof; Harvesting of clarified cell culture obtained from culture of recombinant host cells expressing the original binding moiety. (ii) incubating a cell culture with vedolizumab, or an antigen-binding portion thereof, and purifying said antibody or antigen-binding portion thereof from said harvested product, said antibody or antigen-binding portion having a molecular weight of 5.0 or less. (e.g., pH 4.6 to 5.0) within 3 hours, within 5 hours, within 10 hours, or within 1 Within 2 hours, Within 18 hours, Within 24 hours, Within 36 hours, Within 48 hours, Within 72 hours or within 96 hours, and the anti-α4β7 antibody or antigen-binding portion thereof is The levels of basic isoform species were decreased compared to those of α-terminal β-terminal β-terminal The control contains an anti-α4β7 antibody or an antigen-binding portion thereof produced in the same manner. The composition is characterized in that the antibody can be maintained at a pH of 5.0 or less (e.g., pH 4.6 to 5.0) for a long period of time. , i.e., more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, Those who are exposed for more than 36 hours, more than 48 hours, more than 72 hours, or more than 96 hours The composition is obtainable by the method.

[0261] In another aspect, provided herein is a method for the treatment of cancer, comprising administering to the patient a therapeutically effective amount of vedolizumab, or an antigen-binding portion thereof. A composition comprising, inter alia: (i) vedolizumab, or an anti-cancer agent thereof; Harvesting of clarified cell culture obtained from culture of recombinant host cells expressing the original binding moiety. (ii) incubating a cell culture with vedolizumab, or an antigen-binding portion thereof, and purifying said antibody or antigen-binding portion thereof from said harvested product, said antibody or antigen-binding portion being less than or equal to 5.5. (e.g., pH 5.1 to 5.5) within 3 hours, within 5 hours, within 10 hours, or within 1 Within 2 hours, Within 18 hours, Within 24 hours, Within 36 hours, Within 48 hours, Within 72 hours or within 96 hours, and the anti-α4β7 antibody or antigen-binding portion thereof is The levels of basic isoform species were decreased compared to those of α-terminal β-terminal β-terminal The control contains an anti-α4β7 antibody or an antigen-binding portion thereof produced in the same manner. The composition is characterized in that the antibody can be maintained at a pH of 5.5 or less (e.g., pH 5.1 to 5.5) for a long period of time. , i.e., more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, Those who are exposed for more than 36 hours, more than 48 hours, more than 72 hours, or more than 96 hours The composition is obtainable by the method.

[0262] In some embodiments of the foregoing aspects, the antibody or antigen-binding portion thereof is selected from the group consisting of SEQ ID NO:1. and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5. It contains the chain variable region.

[0263] In some embodiments of the foregoing aspects, the anti-α4β7 antibody, or antigen-binding portion thereof, is purified. The steps for preparation are Protein A chromatography, anion exchange chromatography, and , cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography and combinations thereof.

[0264] In some embodiments of the foregoing aspects, the present invention relates to a method for the expression of an anti-α4β7 antibody or an antigen-binding portion thereof. In another embodiment, the host cell is a GS-CHO cell. CHO cells.

[0265] In some embodiments of the aforementioned aspects, the composition has less than 16%, less than 15%, less than 14%, %, 13%, 12%, 11%, 10%, or 9% basic acid Includes isoform species.

[0266] In some embodiments of the above aspects, the composition comprises less than 4%, less than 3%, less than 2% , or containing less than 1% basic isoform peak 2.

[0267] In some embodiments, provided herein is a composition comprising a basic species of vedolizumab. The vedolizumab composition contains 15% or less of the vedolizumab isoforms in the composition. For example, in some embodiments, provided herein is about 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% Basic isoforms at levels of ≤4%, ≤3%, ≤2%, or ≤1% A vedolizumab composition comprising seeds.

[0268] In some embodiments, the level of a basic isoform species in a vedolizumab composition is , about 1%~15%, 1%~14%, 1%~13%, 1%~12%, 1%~11%, 1%~ 10%, 1%~9%, 1%~8%, 1%~7%, 1%~6%, 1%~5%, 1%~4%, In other embodiments, the base in the vedolizumab composition is 1% to 3%, or 1% to 2%. The levels of the sex isoform species were approximately 2%-11%, 3%-11%, 4%-11%, 5%- 11%, 6%-11%, 7%-11%, 8%-11%, 9%-11%, or 10%-1 In another embodiment, the level of basic isoform species in the vedolizumab composition is The range is approximately 1%-10%, 2%-10%, 3%-10%, 4%-10%, 5%-10%, 6%-10%, % to 10%, 7% to 10%, 8% to 10%, or 9% to 10%. The level of basic isoform species in vedolizumab compositions is about 1% to 9%, 2% to 9%, 3%-9%, 4%-9%, 5%-9%, 6%-9%, 7%-9%, or 8%-9 In other embodiments, the level of a basic isoform species in a vedolizumab composition is is approximately 1%-8%, 2%-8%, 3%-8%, 4%-8%, 5%-8%, 6%-8%, or In another embodiment, the basic isoform in the vedolizumab composition is 0.05 to 0.5%. The levels of spores range from approximately 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, or In another embodiment, the basic isoform in the vedolizumab composition is 0.05 to 0.5%. The levels of gram species are approximately 1%-6%, 2%-6%, 3%-6%, 4%-6%, or 5%-6%. In other embodiments, the level of a basic isoform species in a vedolizumab composition is is about 1% to 5%, 2% to 5%, 3% to 5%, or 4% to 5%. In some embodiments, the level of a basic isoform species in the vedolizumab composition is about 5% or less. .

[0269] In some embodiments, the percentage of basic isoform species in a vedolizumab composition The stage may include incubation at a pH above 6.5, as described herein. Furthermore, the pattern of basic isoform species in vedolizumab compositions produced by the same method was -Percentage of about 1% or more, e.g., 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, 6% or more, 7% or more, 8 % or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 1 5% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more , 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or reduced by 100% or more.

[0270] The percentage of basic isoform species in a composition containing vedolizumab is CEX- It may be determined by any suitable method, including but not limited to HPLC. In embodiments, provided herein is an anti-α4β7 antibody or antigen-binding portion thereof (e.g., For example, a low base species composition comprising vedolizumab, the composition comprising less than 16%, 1 Less than 5%, Less than 14%, Less than 13%, Less than 12%, Less than 11%, Less than 10%, or 9% The total basic isoform species of an anti-α4β7 antibody, or antigen-binding portion thereof, The basic isoform species of α4β7 is the major isoform of anti-α4β7 antibodies or their antigen-binding portions. form, has a net positive charge as determined by CEX, and this anti-α4β7 antibody or an antigen binding portion thereof, In some embodiments, the composition comprises a first basic isoform The peak (BP1) and the second basic isoform peak (BP2). In such an embodiment, the composition comprises less than 2% BP2, less than 1.5% BP2, 1 In some embodiments, the composition contains less than 0.7% BP2. The composition is 1.5%~2.5% BP2, 1.2%~2.2%, 1%~2% BP2, 1% ~1.8% BP2, 1%~1.6% BP2, 1%~1.5% BP2, 0.8%~1 .8% BP2, 0.8%~1.6% BP2, 0.8%~1.4% BP2, 0.8% ~1.2% BP2, 0.8%~1% BP2, 0.7%~1.7% BP2, 0.7% ~1.5% BP2, 0.7%~1.3% BP2, 0.7%~1% BP2, 0.6% ~1.6% BP2, 0.6%~1.4% BP2, 0.6%~1.2% BP2, 0. 6%~1% BP2, 0.6%~0.8% BP2, 0.5%~1.5% BP2, 0. 5%-1.3% BP2, 0.5%-1% BP2, or 0.5%-0.8% BP2 Includes.

[0271] In some embodiments, the ratio of BP1 to BP2 in the composition is at least 3 (e.g., At least 5, at least 6, at least 7, at least 8, at least 9, or at least In some embodiments, the ratio of BP1 to BP2 is 2 to 12, 2 to 10, 10, 2 vs 8, 2 vs 6, 2 vs 4, 3 vs 12, 3 vs 11, 3 vs 9, 3 vs 6, 3 vs 5, 3 vs 4 , 4 vs 12, 4 vs 11, 4 vs 10, 4 vs 8, 4 vs 6, 4 vs 5, 5 vs 12, 5 vs 11, 5 vs 10, 5 vs 9, 5 vs 8, 5 vs 6, 6 vs 12, 6 vs 11, 6 vs 10, 6 vs 8, 6 vs 7, 7 vs 12, 7 vs 11, 7 vs 10, 7 vs 9, 7 vs 8, 8 vs 12, 8 vs 11, 8 vs 10, 8 vs 9, 9 vs 12, 9 vs 11, or 9 vs 10.

[0272] In some embodiments, the composition of low basic species is less than 8% (e.g., less than 7%, 6% or less than 5% of the total basic isoform species of the anti-α4β7 antibody (e.g., In some embodiments, the low basic species composition comprises 4% to 8%, 4% ~7.5%, 4%~7%, 4%~6.5%, 4%~6%, 4%~5.5%, 4%~5%, 5%~8%, 5%~7.5%, 5%~7%, 5%~6.5%, 5%~6%, 6%~8%, The total basic isoform species of anti-α4β7 antibodies, 6%–7.5% or 6%–7% ( For example, vedolizumab).

[0273] In some embodiments, the composition comprises an anti-α4β7 antibody, or an antigen-binding portion thereof. (e.g., vedolizumab), and a pharma- ceutical composition comprising a pharma- ceutical carrier or excipient. Thus, in some embodiments, provided herein are , a low basic species of an anti-α4β7 antibody, or an antigen-binding portion thereof (e.g., vedolizumab), and and a pharma- ceutical composition comprising a pharma- ceutical carrier. The antibody formulation remains liquid. In one embodiment, the antibody formulation may be a dried antibody formulation that has been freeze-dried. The lyophilized antibody formulation is available in 150 mg, 180 mg, 240 mg, 300 mg, and 360 mg doses. The drug is provided in a single-dose vial containing 450 mg or 600 mg of anti-α4β7 antibody. In another embodiment, the anti-α4β7 antibody, e.g. For example, vedolizumab may be stored in a container, e.g., until it is administered to a subject in need thereof. , a stable liquid pharmaceutical stored in a vial, syringe or cartridge at about 2-8°C. In some embodiments, the syringe or cartridge contains 54 mg, A single dose of 108 mg, 160 mg, or 216 mg of antibody may be provided. In embodiments, a reconstituted lyophilized formulation or a stable liquid pharmaceutical composition of an anti-α4β7 antibody The substances include, but are not limited to, amino acids (e.g., arginine, histidine, and / or or histidine monohydrochloride), sugars (e.g., sucrose), surfactants (e.g., polysol bate 80), and / or buffer (e.g., citrate, phosphate, etc.) In one embodiment, a reconstituted lyophilized preparation of an anti-α4β7 antibody may be used. The agent or stable liquid pharmaceutical composition may contain L-arginine, L-histidine, L-histidine monohydrochloride, sucrose, and / or polysorbate 80. The reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an α4β7 antibody is formulated with citrate. , arginine, histidine, and / or polysorbate 80. Additional formulations and uses are described, for example, in U.S. Pat. No. 9,764,033 and U.S. Pat. No. 040,855, the entire contents of each of the foregoing patents being incorporated herein by reference. Incorporated by.

[0274] V. Preparation of Vedolizumab Compositions Containing Low Levels of Host Cell Proteins In some embodiments, the present invention provides a vegetative model comprising a vegetative cell having a reduced amount of host cell protein. The present invention provides a method for producing a composition comprising izumab, the method comprising the steps of: Using an AEX buffer with reduced conductivity (e.g., loading buffer) The levels of host cell proteins (HCPs) were significantly increased compared to vedolizumab compositions produced under The present invention is based on the surprising discovery that vedolizumab compositions having reduced creatine phosphate esterase (ATP) levels can be obtained.

[0275] Thus, in one aspect, the present invention provides a vector having reduced amounts of host cell proteins (HCPs). A method for producing a composition comprising dolizumab is provided, the method comprising: contacting a sample containing the compound with an anion exchange resin in the presence of a loading buffer; , where the loading buffer has reduced conductivity compared to standard buffer conditions. and collecting the flow-through material from the anion exchange resin, and the flow-through contains vedolizumab and a reduced amount of HCP. In an embodiment, AEX is used in a method of isolating vedolizumab without a separate elution step, where vedolizumab does not bind to the AEX resin. The sample is run in flow-through mode, with the sample collected in the flow-through material.

[0276] Standard operating range of buffer conductivity for anion exchange (AEX) (e.g., anion exchange Q membrane adsorption The average value of the ion exchange rate (through the ion exchange device) is about 11 to 15 mS / cm (average 13.6 mS / cm). Thus, in some embodiments, standard AEX buffer conditions are about 11 mS / cm or higher. , about 12 mS / cm or more, about 13 mS / cm or more, about 14 mS / cm or more, or about 15 In some embodiments, the loading buffer has a conductivity of at least mS / cm. The conductivity of standard buffer solutions is about 11 mS / cm to about 12 mS / cm, and about 11 mS / cm to about 13mS / cm, about 11mS / cm to about 14mS / cm, or about 11mS / cm to about 1 5 mS / cm. In some embodiments, the conductivity of a standard buffer solution is about 14 mS / cm. cm ~ approx. 15mS / cm, approx. 13mS / cm ~ approx. 15mS / cm, approx. 12mS / cm ~ approx. 15 mS / cm, or from about 11 mS / cm to about 15 mS / cm. In this state, the conductivity of standard buffer solutions is about 11 mS / cm, about 12 mS / cm, and about 13 mS / cm. cm, about 14 mS / cm, or about 15 mS / cm.

[0277] The reduced conductivity AEX buffer (e.g., AEX buffer) used in the methods described herein The loading buffer has a reduced conductivity compared to the standard AEX buffer conditions. For example, in certain embodiments, the reduced conductivity loading buffer has a conductivity of about 15 mS / cm or less. Bottom, approx. 14mS / cm or less, approx. 13mS / cm or less, approx. 12mS / cm or less, approx. 11mS / cm or less, approximately 10mS / cm or less, approximately 9mS / cm or less, approximately 8mS / cm or less, approximately 7m S / cm or less, approximately 6mS / cm or less, approximately 5mS / cm or less, approximately 4mS / cm or less, approximately 3m In some embodiments, the conductive material has a conductivity of about 2 mS / cm or less. The reduced conductivity loading buffer has a conductivity of about 11 mS / cm or less.

[0278] In certain embodiments, the reduced conductivity loading buffer has a conductivity of about 1 mS / cm to about 1 1mS / cm, approx. 2mS / cm ~ approx. 11mS / cm, approx. 3mS / cm ~ approx. 11mS / cm , approx. 4mS / cm ~ approx. 11mS / cm, approx. 5mS / cm ~ approx. 11mS / cm, approx. 6mS / cm ~ approx. 11mS / cm, approx. 7mS / cm ~ approx. 11mS / cm, approx. 8mS / cm ~ approx. 11 mS / cm, about 9mS / cm to about 11mS / cm, or about 10mS / cm to about 11mS / cm (including any one or more of the ranges above). In the form, the loading buffer with reduced conductivity is about 11 mS / cm to about 12 mS / cm. m, about 11 mS / cm to about 13 mS / cm, about 11 mS / cm to about 14 mS / cm, is about 11 mS / cm to about 14.5 mS / cm (including any one or more of the ranges above) In certain embodiments, the reduced conductivity loading buffer has a conductivity of about 1mS / cm ~ approx. 2mS / cm, approx. 1mS / cm ~ approx. 3mS / cm, approx. 1mS / cm ~ approx. 4mS / cm, approx. 1mS / cm ~ approx. 5mS / cm, approx. 1mS / cm ~ approx. 6mS / cm, approx. 1mS / cm ~ approx. 7mS / cm, approx. 1mS / cm ~ approx. 8mS / cm, approx. 1mS / cm ~ approx. 9mS / cm, about 1mS / cm to about 10mS / cm, or about 1mS / cm to about 11mS / cm (including any one or more of the ranges above).

[0279] In certain embodiments, the reduced conductivity loading buffer has a conductivity of about 1 mS / cm, about 1 .5mS / cm, approx. 2mS / cm, approx. 2.5mS / cm, approx. 3mS / cm, approx. 3.5mS / cm, approx. 4mS / cm, approx. 4.5mS / cm, approx. 5mS / cm, approx. 5.5mS / cm, Approx. 6mS / cm, Approx. 6.5mS / cm, Approx. 7mS / cm, Approx. 7.5mS / cm, Approx. 8mS / cm, approx. 8.5mS / cm, 9mS / cm, 9.5mS / cm, 10mS / cm, 10 .5mS / cm, 11mS / cm, 11.5mS / cm, 12mS / cm, 12.5mS / cm, 13mS / cm, 13.5mS / cm, 14mS / cm, 14.5mS / cm, Or has a conductivity of 15 mS / cm.

[0280] In some embodiments, the method further comprises applying a loading buffer comprising vedolizumab. Following the step of 200-200, the method may further comprise applying a wash buffer to the AEX resin. In one embodiment, the elution buffer has the same conductivity as the loading buffer. The wash buffer has an increased conductivity compared to the loading buffer. The wash buffer has a reduced conductivity compared to the loading buffer.

[0281] In some embodiments, the loading buffer contains sodium chloride and / or phosphate In an exemplary embodiment, the loading buffer contains 40-70 mM Na Cl (e.g., 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, or 7 In some embodiments, the loading buffer contains 55-60 mM NaCl. Additionally or alternatively, the loading buffer contains sodium phosphate. In an exemplary embodiment, the loading buffer may contain 20-50 mM Sodium phosphate (e.g., 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, In some embodiments, the loader comprises 50 mM sodium phosphate. The incubation buffer contains 35 to 45 mM sodium phosphate. The loading buffer has a pH of 6.5 or more, for example, pH 6.5 to 8.5, pH 7.0 In some embodiments, the loading buffer is pH 6.8 to 7.4. The liquid is approximately 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, 8.0, 8.1, 8.2, 8.3, The pH is 8.4 or 8.5.

[0282] In some embodiments, provided herein are low conductivity ion exchangers in flow-through mode. Contacting the AEX resin equilibrated in a buffer with a solution containing an antibody, and A method for purifying an α4β7 antibody, such as vedolizumab, comprising collecting eluate material. In one embodiment, the low-conductivity solution is a mixture of NaCl and a buffer solution having a pH of 6.5 to 8.5. In some embodiments, the low conductivity solution comprises a mixture of 5-15 mS / cm, 5- It has a conductivity of 11 mS / cm, 7 to 10 mS / cm or about 10 mS / cm.

[0283] In some embodiments, the anion exchange resin is formatted as an anion exchange membrane. In some embodiments, the anion exchange resin is an anion exchange chromatograph. In some embodiments, the column is formatted as an anion exchange column. contains quaternary amine functional groups. Exemplary AEX resins include, but are not limited to, , Mustang Q(Pall Corporation,Port Washing ton, NY), Sartobind Q (Sartorius GmbH, Goett Other exemplary AEX resins include, for example, , Eshmuno Q resin(EMD Millipore, Burlingto n, MA) and Nuvia Q resin (Bio-Rad, Hercules, CA) ) are mentioned.

[0284] HCP refers to the host cells used to produce vedolizumab or its antigen-binding portion. For example, in some embodiments, vedolizumab may be derived from Chinese hamburger. The HCP is a CHO cell protein. In an embodiment, the HCP is expressed in CHO cells lacking dihydrofolate reductase (DHFR) expression. In certain embodiments, the HCP lacks glutamine synthetase (GS) expression. Derived from CHO cells.

[0285] In certain embodiments, the amount of HCPs in the flow-through is about 8 ppm or less, about 7.5 ppm or less. m or less, about 7ppm or less, about 6.5ppm or less, about 6ppm or less, about 5.5ppm or less, About 5ppm or less, about 4.5ppm or less, about 4ppm or less, about 3.5ppm or less, about 3pp m or less, about 2.5 ppm or less, about 2 ppm or less, about 1.5 ppm or less, or about 1 ppm The following is the result.

[0286] In some embodiments, the amount of HCPs in the flow-through is between 1 ppm and 8 ppm, 2 ppm pm~8ppm, 3ppm~8ppm, 4ppm~8ppm, 5ppm~8ppm, 6p pm to 8 ppm, or 7 ppm to 8 ppm (including any one or more of the above ranges). In some embodiments, the amount of HCPs in the flow-through is between 1 ppm and 2 ppm, 1 ppm m~3ppm, 1ppm~4ppm, 1ppm~5ppm, 1ppm~6ppm, 1pp 5 ppm to 7 ppm, or 1 ppm to 8 ppm (including any one or more of the above ranges). In some embodiments, the amount of HCPs in the flow-through is between 1 ppm and 3 ppm, ~5ppm, or 5ppm~7ppm.

[0287] In certain embodiments, the amount of HCPs in the flow-through is determined by a standard conductivity (e.g., 11 to 1 The same sample was used with a loading buffer having a conductivity of 5 mS / cm or more. The amount of HCPs in the flow-through material generated when the method was performed was At least about 0.5%, at least about 1%, at least about 2%, at least about 5%, At least about 10%, at least about 15%, at least about 20%, at least about 25%, At least about 30%, at least about 35%, at least about 40%, at least about 45%, At least about 50%, at least about 55%, at least about 60%, at least about 65%, At least about 70%, at least about 75%, at least about 80%, at least about 85%, At least about 90%, at least about 95%, or at least about 98% or more than about 98%, It has been reduced.

[0288] In certain embodiments, the amount of HCPs in the flow-through is about 0.5% to about 50%, about 1% ~50%, ~2%~50%, ~5%~50%, ~10%~50%, ~15%~ 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, or about 45% to about 50%. The amount of HCP in the flow-through is determined by the standard conductivity (e.g., about 11-15 mS / cm). The results are shown in Table 1. About 50% to about 55%, about 50% to about 60%, about 50%~65%, 50%~70%, 50%~75%, 50%~80%, 50% to about 85%, about 50% to about 90%, about 50% to about 95%, or about 50% to about 98% % reduction.

[0289] In certain embodiments, the amount of HCPs in the flow-through is about 1% to about 10%, about 10% to about 10%. About 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% or more About 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 9 In certain embodiments, the amount of HCPs in the flow-through is reduced by between about 0% and about 98%. Repeat the same procedure using a loading buffer with a conductivity of approximately 11-15 mS / cm. for the amount of HCP in the flow-through generated when running this method using , about 0.5% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% ~15%, ~20%, ~25%, ~30%, ~30% ~35%, ~40%, ~40%~45%, ~50%, ~50% ~55%, ~60%, ~60%~65%, ~70%, ~70% ~75%, ~80%, ~80%~85%, ~90%, ~90% by about 95%, or by about 95% to about 98%.

[0290] The sample containing vedolizumab and HCPs can optionally be purified by, for example, affinity chromatography. Chromatography, cation exchange chromatography, hydrophobic interaction chromatography, Chromatographic and mixed-mode chromatography After one or more additional purification steps, such as chromatography, chromatography, or a combination thereof, the mammalian Additionally, vedolizumab-containing The level of HCPs in samples containing the IgG can be determined using, for example, affinity chromatography, cation exchange chromatography, or other methods. Chromatography, Hydrophobic interaction chromatography, Ceramic hydroxyapatite CHT chromatography and / or mixed mode chromatography Optionally, the affinity of the amine is further reduced by one or more additional purification steps, including a combination of obtain.

[0291] Thus, the methods described herein, in some embodiments, include vedolizumab. The method includes one or more additional purification steps to further reduce the level of HCPs in the sample. In one embodiment, the present invention uses the AEX method described herein and further comprises: and reducing the level of HCPs in a composition comprising vedolizumab, comprising one or more additional purification steps. One or more additional purification steps may be performed in the AEX method described herein. In some embodiments, one or more additional purification steps may be performed before or after. In an exemplary embodiment, one or more additional chromatographic separations are included. The purification step can be performed using affinity chromatography (e.g., protein A chromatography). chromatograph), cation exchange chromatography, hydrophobic interaction chromatography , ceramic hydroxyapatite (CHT) chromatography, or mixed-mode chromatography chromatography, or a combination thereof.

[0292] In an exemplary embodiment, the present invention provides a method for reducing the levels of HCPs in a composition comprising vedolizumab. The present invention provides a method for reducing the level of vedolizumab in a subject, the method comprising providing a composition comprising vedolizumab and an HCP. and affinity chromatography, mixed mode chromatography, and / or Purify vedolizumab from HCP by performing cation exchange chromatography and, further comprising carrying out the AEX method described herein to obtain Vedriz from the HCP. In one embodiment, the load of the AEX method described herein is The material contains a cation exchange eluate. Using the method described above, the elution peaks are 11–15 mS / c. In the composition resulting from carrying out the same method, an anion exchange buffer having a conductivity of at least m is used. A composition containing vedolizumab with reduced levels of HCP compared to the levels of HCP present It can generate things.

[0293] In another exemplary embodiment, the present invention provides a method for determining the level of HCPs in a composition comprising vedolizumab. The present invention provides a method for reducing the amount of vedolizumab in a composition comprising vedolizumab and an HCP. and affinity chromatography, cation exchange chromatography and / or hydroxyapatite chromatography (e.g., ceramic hydroxyapatite The HCP was isolated from the vegetative state by performing hydrophilic hydrochloride (HCH) chromatography. The mab can be purified and further purified by the AEX method described herein to obtain HCP and further purifying vedolizumab from the A described herein. The load material for the EX method includes hydroxyapatite chromatography eluate. Using the method described above, use an anion exchange buffer with a conductivity of 11-15 mS / cm or higher. and compared to the level of HCPs present in a composition resulting from the practice of the same method. Compositions containing vedolizumab with reduced levels of

[0294] Vedolizumab content and / or host cell protein content were determined by HCP ELISA, Chromatography (e.g., SEC), analytical ultracentrifugation, light scattering (DLS or MA LLS), mass spectrometry (e.g., MALDI-TOF MS), or nanoparticle tracking analysis Nanoscale measurement systems such as NanoSight Ltd, Wiltshire, UK) The present invention may be measured by any method known in the art, including, but not limited to, obtain.

[0295] In some embodiments, the method further comprises administering to the subject the composition a medicament comprising one or more pharma- ceutically acceptable carriers or excipients. by a process involving ultrafiltration and / or diafiltration into a buffer containing the agent. The method further comprises treating the AEX flow-through material to exchange the buffer.

[0296] VI. Vedolizumab Compositions with Reduced Host Cell Proteins In some embodiments, the present invention provides a method for the preparation of a medicament for the treatment of a vascular endothelial disorder comprising administering to a subject a subject a subject in need thereof a therapeutically effective amount of a medicament for the treatment of a vascular endothelial disorder. In some embodiments, the compositions are reduced in host cell proteins. The products are produced by the methods provided herein (see, e.g., Section V). Thus, in one aspect, provided herein is a composition comprising vedolizumab. wherein the composition comprises a sample comprising vedolizumab and HCPs in a loading buffer. contacting the anion exchange resin in the presence of a loading buffer; The solution has a reduced conductivity compared to standard buffer conditions and is free of anion exchange resins. collecting flow-through material from the sample, said flow-through being vedolizumab and containing reduced amounts of HCPs. The flow-through material can then be optionally treated to convert the elution buffer to one or more pharma- ceutical acceptable salts. The drug is then exchanged into a buffer containing a carrier or excipient that is acceptable for the drug, thereby reducing the amount of HCP. This buffer exchange step can be carried out, for example, by ultrafiltration and / or dialysis. This can be done using standard techniques, including filtration.

[0297] HCP refers to the host cells used to produce vedolizumab or its antigen-binding portion. For example, in some embodiments, vedolizumab may be derived from Chinese hamburger. The HCP is a CHO cell protein. In an embodiment, the HCP is expressed in CHO cells lacking dihydrofolate reductase (DHFR) expression. In certain embodiments, the HCP lacks glutamine synthetase (GS) expression. Derived from CHO cells.

[0298] In certain embodiments, the amount of HCP in the composition is about 8 ppm or less, about 7.5 ppm or less. Below, about 7ppm or less, about 6.5ppm or less, about 6ppm or less, about 5.5ppm or less, about 5ppm or less, approximately 4.5ppm or less, approximately 4ppm or less, approximately 3.5ppm or less, approximately 3ppm less than about 2.5ppm, less than about 2ppm, less than about 1.5ppm, or less than about 1ppm Below.

[0299] In some embodiments, the amount of HCPs in the composition is from 1 ppm to 8 ppm, 2 ppm to 5 ppm, m~8ppm, 3ppm~8ppm, 4ppm~8ppm, 5ppm~8ppm, 6pp 5 ppm to 8 ppm, or 7 ppm to 8 ppm (including any one or more of the above ranges). In some embodiments, the amount of HCPs in the composition is from 1 ppm to 2 ppm, from 1 ppm to 3ppm, 1ppm~4ppm, 1ppm~5ppm, 1ppm~6ppm, 1ppm~ 7 ppm, or 1 ppm to 8 ppm (including any one or more of the above ranges). In some embodiments, the amount of HCP in the composition is from 1 ppm to 3 ppm, from 3 ppm to 5 ppm. pm, or 5 ppm to 7 ppm.

[0300] In certain embodiments, the amount of HCP in the composition is greater than or equal to a standard conductivity (e.g., 11 to 15 The same samples were analyzed using a loading buffer with a conductivity of 0.1 mS / cm or higher. At least about 0.5%, less than about 0.5%, relative to the amount of HCP in the composition produced by the method. At least about 1%, at least about 2%, at least about 5%, at least about 10%, at least At least about 15%, at least about 20%, at least about 25%, at least about 30%, At least about 35%, at least about 40%, at least about 45%, at least about 50%, At least about 55%, at least about 60%, at least about 65%, at least about 70%, At least about 75%, at least about 80%, at least about 85%, at least about 90%, Both are reduced by about 95%, or at least about 98% or greater than about 98%.

[0301] In certain embodiments, the amount of HCP in the composition is from about 0.5% to about 50%, from about 1% to about About 50%, about 2% to about 50%, about 5% to about 50%, about 10% to about 50%, about 15% to about 5 0%, approx. 20% to approx. 50%, approx. 25% to approx. 50%, approx. 30% to approx. 50%, approx. 35% to approx. In certain embodiments, the concentration of the ionizing radiation is reduced by 0%, by about 40% to about 50%, or by about 45% to about 50%. The amount of HCP in the flow-through material is determined by the standard conductivity (e.g., about 11-15 mS / The same samples were run using a loading buffer with a conductivity of 0.1% or higher (conductivity 0.1% or higher). About 50% to about 55%, about ... About 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to About 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, or about 5 Reduced by 0% to approximately 98%.

[0302] In certain embodiments, the amount of HCP in the composition is from about 1% to about 10%, from about 10% to about 20%. %, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60 %, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to In certain embodiments, the amount of HCPs in the flow-through material is reduced by about 98%. Loading buffer with standard conductivity (e.g., conductivity of about 11-15 mS / cm or higher) The amount of HCP in the composition produced by the method performed using the same sample with About 0.5% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, About 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, About 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, About 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, About 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, It is reduced by about 90% to about 95%, or about 95% to about 98%.

[0303] Vedolizumab content and / or host cell protein content were determined by HCP ELISA, Chromatography (e.g., SEC), analytical ultracentrifugation, light scattering (DLS or MA LLS), mass spectrometry (e.g., MALDI-TOF MS), or nanoparticle tracking analysis Nanoscale measurement systems such as NanoSight Ltd, Wiltshire, UK) The present invention may be measured by any method known in the art, including, but not limited to, obtain).

[0304] VII. Preparation of Vedolizumab using Mixed-Mode Chromatography Described herein is the use of vectors after elution from a mixed-mode chromatography resin. This buffer maximizes the yield of dolizumab. Protein loss is reduced by the high loading concentration. Conditions for testing (e.g., at least 14g / L, 15g / L, 17g / L, 10g / L, 25g / L, 30g / L, 35g / L or more) This can occur during the wash steps of mixed-mode purification using hydroxyapatite resin. Increase the loading capacity of ceramic resins, e.g., ceramic hydroxyapatite resins. To improve the resulting yield of vedolizumab, The column washing buffer and wash buffer can be optimized to reduce antibody loss when washing the column. Importantly, the methods described herein allow for greater loading concentrations on mixed mode resins. without altering the quality of the product (e.g., percent aggregates) in the final eluate. It also increases yields by 2-3% compared to standard mixed-mode buffer.

[0305] Thus, in one aspect, the present invention provides a method for recovering a mixed-mode chromatography resin after elution. A method for increasing the yield of vedolizumab obtained by mixed mode chromatography. Equilibrating the resin with an equilibration buffer; a solution comprising vedolizumab and a loading buffer. The eluate was loaded onto a mixed-mode chromatography resin to detect vedolizumab in the mixed-mode chromatographic Chromatography resin and mixed-mode chromatography with wash buffer. - Wash the resin and remove the beads from the mixed-mode chromatography resin with elution buffer. and eluting the izumab, wherein the equilibration buffer, loading buffer and / or wherein the pH of the wash buffer is 7.0 or less. Standard buffers for the assay may be at a higher pH, i.e., a pH of 7.2 or higher. In some embodiments, an equilibration buffer having a pH of 7.0 or less is used. In some embodiments, a loading buffer having a pH of 7.0 or less is used. In some embodiments, a wash buffer having a pH of 7.0 or less is used.

[0306] To prevent loss of vedolizumab during the wash steps of mixed-mode purification, A buffer pH in the range of 0.1 to 100 may be used. For example, in some embodiments, the equilibration buffer The pH of the solution, loading buffer, and / or washing buffer is less than 7.0, less than 6.9, Less than 6.8, Less than 6.7, Less than 6.6, Less than 6.5, Less than 6.4, Less than 6.3, Less than 6.2 Full, less than 6.1, less than 6.0, less than 5.9, less than 5.8, less than 5.7, less than 5.6, or For example, the buffer may have a pH of about 7.0, 6.9, 6.8, 6.7, 6. 6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5. In some embodiments, the buffer may have a pH of about 7.0 to 5. In another embodiment, the buffer has a pH in the range of about 7.0 to 6.0. In another embodiment, the buffer has a pH in the range of about 7.0 to 6.5. In one embodiment, the buffer has a pH in the range of about 6.8 to 5.8. In one embodiment, the buffer has a pH in the range of about 6.8 to 6.0. In another embodiment, the buffer has a pH in the range of about 6.6 to 6.8. The pH is

[0307] Additionally or alternatively, mixed mode chromatography for the purification of vedolizumab Buffers used for equilibrating, loading, and / or washing the resin should have a total salt concentration of less than 70 mM. For example, the buffer may have a concentration of less than 70 mM, less than 65 mM, less than 60 mM, less than 55 mM, Less than M, less than 50 mM, less than 45 mM, less than 40 mM, less than 35 mM, or less than 30 mM In some embodiments, the buffer may have a salt concentration of about 70 mM, about 65 mM, or less. M, about 60 mM, about 55 mM, about 50 mM, about 45 mM, about 40 mM, about 35 mM, In another embodiment, the buffer has a salt concentration in the range of 30 to 70 mM. In some embodiments, the buffer has a salt concentration in the range of 40 to 65 mM. In some embodiments, the buffer has a salt concentration in the range of 40 to 65 mM. In some embodiments, the buffer has a salt concentration in the range of 50 to 60 mM. In some embodiments, the buffer has a salt concentration in the range of 40 to 50 mM. In some embodiments, the buffer has a salt concentration in the range of 45 to 55 mM. Equilibration, loading, and purification of CHT chromatography resin for purification of vedolizumab The salts present in the buffers used for washing and / or cleaning are sodium chloride and / or phosphate. Contains sodium.

[0308] In some embodiments, mixed mode chromatography for the purification of vedolizumab Buffers used for equilibrating, loading, and / or washing the resin should contain less than 70 mM For example, the buffer may have a sodium chloride concentration of less than 70 mM, less than 65 mM, Full, Less than 60mM, Less than 55mM, Less than 50mM, Less than 45mM, Less than 40mM, 35m In some embodiments, the sodium chloride concentration is less than 30 mM, or less than 30 mM. In the form of about 70 mM, about 65 mM, about 60 mM, about 55 mM, about 50 mM M, about 45 mM, about 40 mM, about 35 mM, or about 30 mM sodium chloride concentration In another embodiment, the buffer has a sodium chloride concentration in the range of 30 to 70 mM. In some embodiments, the buffer has a concentration in the range of 40-65 mM sodium chloride. In some embodiments, the buffer has a sodium concentration in the range of 40 to 65 mM. In some embodiments, the buffer has a sodium chloride concentration of 50 to 60 mM In some embodiments, the buffer has a sodium chloride concentration in the range of 40 to 50%. In some embodiments, the buffer has a sodium chloride concentration in the range of 50 mM. In some embodiments, the sodium chloride concentration ranges from 45 to 55 mM. The sodium chloride buffer may further include sodium phosphate. In one embodiment, the sodium chloride buffer is 1 to 30 mM sodium phosphate, for example, 5 mM sodium phosphate. ~20mM sodium phosphate, 10~20mM sodium phosphate, etc. In an exemplary embodiment, the sodium chloride buffer may further comprise 1 mM, 2 mM, 3 mM mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20m The solution may contain sodium phosphate at a concentration of 10 mM, 25 mM, or 30 mM.

[0309] The above buffers are used for equilibration of the mixed-mode chromatography resin during the purification of vedolizumab. In an exemplary embodiment, a mixed mode tree may be used for loading and / or washing. The resin may be a ceramic hydroxyapatite resin.

[0310] In some embodiments, two of the equilibration, loading, and wash buffers are the same. In some embodiments, the equilibration, loading, and wash buffers have the same pH. The two have the same salt concentration. The two of the wash buffers have the same pH and the same salt concentration. In some experiments, the equilibration, loading, and washing buffers all have the same pH. In an embodiment, the equilibration, loading, and washing buffers all have the same salt concentration. In some embodiments, the equilibration, loading, and wash buffers all have the same pH and have the same salt concentration.

[0311] In some embodiments, the mixed-mode chromatography resin is loaded and then recovered. The method to increase the yield of vedolizumab is to run the column at a pH below 7 and between pH 5.5 and 6.9. or pH 6.5-6.8 and a salt concentration of, for example, 40-60 mM or 45-55 mM The method involves elution with a pH of less than 7, pH 5.5-6.9 or may further comprise washing the column at pH 6.5 to 6.8.

[0312] The buffers and methods described herein may include any suitable buffer for equilibration, loading, and / or washing steps. The group is not suitable for use in buffers with a pH greater than 7.0 and / or salt concentrations greater than 70 mM and / or When run using sodium chloride concentrations greater than 70 mM, the mixture yields The yield of vedolizumab eluted from the multiple mode chromatography column can be improved. In some embodiments, the yield is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, Improved by 8%, 9%, 10%, 12%, 15%, 18%, 20% or more. In the form, the yield is improved by more than 2% compared to buffers having a pH greater than 7.0. In some embodiments, the yield is greater than or equal to 100% relative to a buffer having a pH greater than 7.0. In some embodiments, the yield is improved by more than 3% at a pH of greater than 7.0. In some embodiments, the yield is improved by more than 4% compared to a buffer having This is an improvement of over 5% compared to buffers having a pH greater than 0.0.

[0313] VIII. Methods for assessing the purity of compositions containing anti-α4β7 antibodies The purity of a composition comprising an antibody, e.g., vedolizumab, can be determined by any method, including those described herein. The evaluation may be performed by any suitable method, including but not limited to.

[0314] (a) Evaluation of basic isoform species The present invention relates to a method for the detection of basic isoforms in compositions comprising vedolizumab or an antigen-binding portion thereof. Methods for modulating, e.g., reducing, levels of rheumatoid arthritis species and methods for the treatment of rheumatoid arthritis in compositions comprising vedolizumab The present invention provides methods for regulating, e.g., increasing, the levels of a desired isoform. The relative amounts of basic vedolizumab isoform species present in the mab composition and the predominant vedolizumab The relative amounts of tafamidis isoforms were determined using positive and negative control as described in detail in the Examples section. The CEX method can be used to measure the overall Fractionation of antibody species according to surface charge. After dilution to low ionic strength using mobile phase, the test The sample is then loaded onto a CEX column, e.g., in a suitable buffer, e.g., 10 mM sodium phosphate, pH 6.6 Equilibrated Dionex Pro-Pac™ WCX-10 column (The rmo Fisher Scientific, Waltham, MA (USA) etc. The antibody may be eluted using a sodium chloride gradient in the same buffer. The elution of proteins can be monitored at 280 nm and peaks can be identified as acidic, basic, or The acidic peaks are assigned to the major isoform categories. The basic peak elutes from the column at a shorter retention time than the main isoform peak. The isoforms are eluted from the column at a longer retention time than the chromatin peak. The sum of the percentages of the active and basic species is reported. The retention times of the samples are compared to the retention times of a reference standard to determine suitability. In some cases, CEX-HPLC methods are used. For example, vedolizumab and its acidic and / or and a composition comprising a basic species, using cation exchange chromatography as described above, The eluted peaks can then be analyzed and separated using HPLC. HPLC was performed using an Agilent 1200 HPLC system (Agilent, San Jose, CA). Quantification may be performed using a chromatographic method (Ta Clara, CA). Based on area percent. CEX-HPLC profile of exemplary vedolizumab preparations The file is shown in Figure 1.

[0315] In one embodiment, the CEX assay method comprises diluting the test sample to low ionic strength, The injection was performed on a CEX column equilibrated with 2 mM sodium phosphate, pH 6.6. The column was eluted with a NaCl gradient in the buffer and the peaks were monitored at 280 nm. and assigning peaks as acidic, major, or basic, where The acidic peak elutes first with the shortest retention time, the main peak elutes second, and the basic peak The peak elutes with the longest retention time, the peak area is quantified, and the amount is the sum of all peak areas. Calculated as a percentage.

[0316] (b) Assessment of host cell protein levels The present invention relates to a method for preventing and treating residual host cell proteins in a composition comprising vedolizumab or an antigen-binding portion thereof. In some embodiments, the present invention provides a method for modulating, e.g., decreasing, the level of a protein. The amount of host cell protein present in the vedolizumab composition can be determined using standard techniques. , can be measured using an enzyme-linked immunosorbent assay (ELISA). CHO HCP EL from chnologies(Southport,NC(USA)) There are many commercially available ELISA kits designed for this purpose, such as the ISA Kit 3G. Host cell proteins in the test sample were detected by immunoblotting using immobilized polyclonal anti-CHOH The captured protein can then be detected by a suitable detection agent, e.g. For example, it can be detected using a horseradish peroxidase-labeled version of the same antibody. In an exemplary embodiment, the amount of trapped peroxidase is directly proportional to the concentration of CHO HCPs. The amount of peroxidase was determined using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB The HCP concentration can be measured colorimetrically at 450 nm using the The antibody preparation may be determined by comparison with a CHO HCP standard curve, such as that contained in In another embodiment, the protein level is reported as a percentage of the total level of protein in the HC P is a rabbit-rabbit ("RaR") Polyclonal anti-CHOHCP antibodies can be determined using the vedolizumab It was produced by immunizing rabbits with the harvest produced in null cells under similar production conditions. The antibody was affinity purified. Proteins were captured using an immobilized polyclonal anti-CHO HCP antibody and then incubated with the same antibody. followed by horseradish peroxidase-conjugated streptavidin. The amount of trapped peroxidase is directly proportional to the concentration of CHO HCP. The amount of peroxidase was determined using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB The HCP concentration can be measured colorimetrically at 450 nm using the Percentage of total protein determined by comparison to a standard curve of CHO HCP In another embodiment, the HCP is a rabbit-goat hybrid as exemplified herein. Polyclonal antibodies can be determined using the Rabbit-Goat ("RaGo") method. The anti-CHO HCP antibody is produced in null cells using a manufacturing process similar to that of vedolizumab. The antibody pools were generated by immunizing rabbits and goats with the harvested material. The host cell proteins in the MLN0002 test samples were affinity purified using immobilized The HCPs were captured using a polyclonal rabbit anti-CHO HCP antibody and then a goat anti-CH Affinity purified antibodies and donkey anti-goat IgG reagent (labeled with horseradish peroxidase) The concentration of CHO HCP is directly proportional to the concentration of the The amount of captured peroxidase was determined by the peroxidase substrate 3,3',5,5'-tetrahydroperoxidase. Measured colorimetrically at 450 nm using methylbenzidine (TMB). Concentrations were determined by comparison with a CHO HCP standard curve included in the test kit and were Reported as concentration (ng / mg) for limumab.

[0317] CHO HCP antigens suitable for use in connection with the various embodiments provided herein The assay uses a polyclonal anti-CHO HCP antibody to capture HCPs. which contains the peroxidase substrate 3,3',5,5'-tetramethylbenzidine ( Polyclonal antibody that converts trimethylsilyl methyl ... Detected after binding to a horseradish peroxidase-labeled version of the CHO HCP antibody In one embodiment, the present invention is suitable for use in connection with the various embodiments provided herein. The HCP ELISA described above uses immobilized polyclonal anti-CHO HCP antibodies, preferably CHO ygnus Technologies (Southport, NC (USA)) Capture HCPs using the antibodies provided in the HCP ELISA Kit 3G This is a LISA method, whereby the captured proteins are amplified by horseradish peroxidase with the same antibody. The amount of peroxidase detected and captured by the peroxidase-labeled version was 3, Colorimetric analysis at 450 nm using 3',5,5'-tetramethylbenzidine (TMB) and subsequently comparing to a CHO HCP standard curve to determine the HCP concentration.

[0318] (c) Size variant evaluation In certain embodiments, chromatography images generated using the techniques described herein are The levels of aggregates, monomers, and fragments in the sample are analyzed. In various embodiments, size exclusion chromatography (SEC) is used to identify antibodies or The antigen-binding portion thereof, e.g., a monomer present in the population of vedolizumab, a high molecular weight (HMW The SEC method can determine the relative levels of H, H2O, H3O4, H4O, H5O, H6O, H7O, H8O, H9O, H10O, H11O, H2O, H3O, H4O, H5O, H6O, H7O, H8O, H9O, H10O, H11O, H12O, H13O, H2O, H2O, H14O, H2O, H15 Provides size-based separation of antibody monomers from MW species and LMW degradation products. Samples and reference standards are analyzed using commercially available SEC columns using appropriate buffers. For example, in some embodiments, SEC analysis is performed using a G3000 SWxl column ( Tosoh Bioscience, King of Prussia, PA (USA) ), or two G3000 SWxl columns connected in tandem and an isocrate Protein synthesis can be carried out using a solid phosphate-sodium chloride buffer system (pH 6.8). The elution of the protein species is monitored at 280 nm. The main peak (monomer) and the total peak The area is evaluated to determine the purity. The purity (%) of the sample (calculated as % monomer), H The % MW aggregates and / or % LMW degradation products are reported.

[0319] In one embodiment, SEC analysis was performed using two G3000 SWxl columns connected in tandem. The sample was injected into the column and the elution of protein species was monitored at 280 nm. The main peak (monomer) and total peak areas are measured. It is carried out in a salt-sodium chloride buffer system (pH 6.8).

[0320] IX. Pharmaceutical Compositions and Uses Thereof Compositions comprising vedolizumab provided herein, e.g., the basic isoform species Compositions Comprising Reduced Levels of Vedolizumab and / or Host Cell Proteins The compositions comprising vedolizumab with reduced activity can be incorporated into pharmaceutical formulations for therapeutic use. The pharmaceutical formulation containing vedolizumab may be prepared by any suitable method.

[0321] In one aspect, the pharmaceutical formulation comprising the composition described herein is a lyophilized pharmaceutical formulation. In one embodiment, the lyophilized formulation is stored as a single dose in a single container, e.g., a vial. The container, e.g., vial, may be refrigerated, e.g., at about 2-8° C., or may be stored at room temperature. For example, the composition may be administered to a subject in need thereof at about 20° C. to 35° C., about 25° C., or about 30° C. The vials are stored in vials, e.g., 10, 20, or 50 cc vials. The container, e.g., vial, may contain about 90-115 mg, about 95-105 mg, or less. at least about 100 mg, at least about 135 to 160 mg, at least about 145 to 155 mg, at least about 15 0mg, about 180-220mg, about 190-210mg, about 195-205mg, less About 200mg, about 280mg to 320mg, about 290mg to 310mg, at least About 300 mg, about 380-420 mg, about 390-410 mg, at least about 400 mg , about 580-620 mg, about 590-610 mg, or at least about 600 mg of anti-α In one embodiment, the vial contains about 200 mg of an anti-α4β7 antibody. The vials may contain, for example, about 100 mg, about 108 mg, about 150 mg, about 200 mg , about 300 mg, about 400 mg, or about 600 mg of anti-α4β7 antibody, e.g., The antibody may contain sufficient anti-α4β7 antibody, e.g., vedolizumab, to permit manufacture. , the vial contains about 15%, about 12%, about 10%, or about 8% more anti-α4 than the dose. It may include a β7 antibody.

[0322] In some embodiments, the compositions described herein are prepared in a liquid form, such as sterile water, for administration. The reconstituted pharmaceutical preparation is formulated as a dry, lyophilized pharmaceutical preparation that can be reconstituted with Administration of the formulation may be by parenteral injection by one of the routes described above. Intravenous injections are administered in a sterile Isotonic saline, buffered saline, e.g., phosphate buffered saline or Ringer's (lactate or dextran This may be achieved by injection, followed by further dilution with tallow (sugar) solution.

[0323] In some embodiments, the compositions described herein are in a liquid form suitable for subcutaneous administration to humans. In some embodiments, the anti-α4β7 antibody is formulated as a pharmaceutical preparation. Thus, for example, about 2, 3, or 4 weeks after initiation of treatment or after the third subsequent dose. For example, about 54 mg, 108 mg, or about 165 mg or about 216 mg per dose. It is administered in doses.

[0324] In another embodiment, a composition described herein comprising an anti-α4β7 antibody, e.g., vedolizumab. The drug may be stored in a container, e.g., a vial, syringe, etc., until it is administered to a subject in need thereof. The pharmaceutical composition is a stable liquid pharmaceutical composition stored in a container or cartridge at about 2-8°C. In some embodiments, the stable liquid pharmaceutical composition of an anti-α4β7 antibody comprises about 0% to 5.0% %, 0%-2%, ≦2%, ≦1%, ≦0.6% or ≦0.5% aggregates. The container or cartridge may be a 1 mL or 2 mL container (e.g., 160 mL). g / mL dose) or, for example, a higher dose (at least 320 mg or 40 In the case of a volume of 0 mg or more, it may be 2 ml or more. The syringe or cartridge may be At least about 20 mg, at least about 50 mg, at least about 70 mg, at least about 80 mg g, at least about 100 mg, at least about 108 mg, at least about 120 mg, at least about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 400 mg g, or at least about 500 mg of anti-α4β7 antibody. The container, e.g., syringe or cartridge, may contain about 20 to 120 mg, about 40 mg to 70 mg, about 45-65 mg, about 50-57 mg, or about 54 mg of an anti-α4β7 antibody; For example, a syringe or The cartridges are approximately 90-120 mg, approximately 95-115 mg, approximately 100-112 mg, or manufactured to deliver about 108 mg of an anti-α4β7 antibody, e.g., vedolizumab. In another embodiment, the syringe or cartridge contains about 140 to 250 mg, about 1 50~200mg, approx. 160~170mg, approx. 160~250mg, approx. 175mg~21 0 mg, or about 160 mg, about 165 mg, about 180 mg, or about 200 mg of anti-α4 It can be manufactured to deliver a β7 antibody, such as vedolizumab.

[0325] Containers that can be used to store and freeze the purified compositions described herein include polyethylene terephthalate (PE)-based polymeric ... These include recarbonate bottles (for IV preparations) or PETG bottles (for subcutaneous preparations) After dispensing the formulation into bottles, freezing may occur (e.g., below -60 degrees Celsius). ).

[0326] The pharmaceutical composition may be any vedolizumab composition provided herein, e.g., a basic amide. Compositions comprising vedolizumab with reduced levels of isoform species, and / or host cell tachyarrhythmase Vedolizumab having reduced levels of protein and a pharma- ceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical composition may comprise a composition having a pH of 6.0 to 6.5. 7.0, for example, pH 6.0-6.2, pH 6.0-6.4, pH 6.0-6 .6, pH6.0~6.8, pH6.1~6.3, pH6.1~6.5, pH6.1~6 .7, pH6.1~6.9, pH6.2~6.4, pH6.2~6.6, pH6.2~6 .8, pH6.2~7.0, pH6.3~6.5, pH6.3~6.7, pH6.3~6 .9, pH6.4~6.6, pH6.4~6.8, pH6.4~7.0, pH6.5~6 .7, pH6.5~6.9, pH6.6~pH6.8, pH6.6~7.0, pH6.7 In some embodiments, the pharmaceutical composition has a pH of 6.8 to 7.0. , 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7 , about 6.8, about 6.9, or about 7.0.

[0327] The pharmaceutical composition may be further supplemented with amino acids or sugars. In some cases, the pharmaceutical composition further comprises an amino acid such as arginine or histidine. In some embodiments, the pharmaceutical composition further comprises a sugar, such as sucrose or trehalose. In some embodiments, the pharmaceutical compositions of anti-α4β7 antibodies provided herein include arginine, histidine, and / or polysorbate 80. In the present specification, the pharmaceutical composition of the anti-α4β7 antibody is a compound containing citrate, arginine, and arginine. , histidine, and / or polysorbate 80.

[0328] Compositions comprising vedolizumab provided herein, e.g., the basic isoform species Compositions Comprising Reduced Levels of Vedolizumab and / or Host Cell Proteins The composition comprising vedolizumab with reduced activity against integrin α4 in vitro or in vivo It can be used in methods to inhibit the activity of β7.

[0329] In one aspect, the compositions described herein are effective for treating a human disease or disorder. and administering to the subject a composition comprising an anti-α4β7 antibody in an amount of 0.1 to 100 mg / kg, The human subject may be an adult (e.g., 18 years of age or older), an adolescent, or The human subject may be a person aged 65 or older.

[0330] In one embodiment, the compositions described herein include an immunomodulatory agent, a TNF-alpha antagonist, Lack of adequate response, loss of response to treatment with nifedipine, ... It is used to treat subjects who may have been intolerant to treatment. Receiving treatment with at least one corticosteroid (e.g., prednisone); For example, patients with inadequate response to corticosteroids for the treatment of inflammatory bowel disease may have Patients may be resistant or dependent on corticosteroids. Response was defined as prednisone 30 mg orally daily for 2 weeks or intravenously for 1 week. Patients must have had at least one 4-week induction regimen containing a dose equivalent to Refers to signs and symptoms of persistent active disease despite resistance to corticosteroids. Loss of response was defined as the initiation of corticosteroid therapy equivalent to 10 mg of oral prednisone daily. Corticosteroid intolerance means two failed attempts to taper below the recommended dose. Cushing's syndrome, osteopenia / osteoporosis, hyperglycemia, insomnia and / or infections This includes medical history.

[0331] Immunomodulators include, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. An inadequate response to an immunomodulatory agent is defined as at least one 8-week consecutive regimen or oral azathioprine (≥ 1.5 mg / kg), 6-mercaptopurine ( 0.75 mg / kg or more), or methotrexate (12.5 mg / week or more) Refers to signs and symptoms of persistently active disease despite a history of disease. These include, but are not limited to, nausea / vomiting, abdominal pain, pancreatitis, abnormal LFTs, and lymphopenia. These include rheumatoid arthritis, TPMT gene mutations and / or infection.

[0332] A TNFα antagonist is, for example, an agent that inhibits the biological activity of TNFα and is preferably For example, TNFα, e.g., monoclonal antibodies, e.g., REMICADE (Inflammatory CIMZIA (certolizumab pegol), HUMIRA (adalimumab), S Circulating receptor fusion drugs such as IMPONI (golimumab) or ENBREL (etanercept) It is a factor that binds to the fusion protein. infliximab 5 mg / kg (IV) for two doses at least 2 weeks apart; mumumab 80 mg subcutaneously once, followed by 40 mg once at least 2 weeks apart; or certolizumab pegol, 400 mg subcutaneously, given twice at least 2 weeks apart. Persistently active despite a history of at least one 4-week induction regimen Refers to the signs and symptoms of the disease. Loss of response to TNF-α antagonists refers to the loss of response to previous It refers to the recurrence of symptoms during maintenance medication following clinical benefit. These include, but are not limited to, infusion-related reactions, demyelination, congestive heart failure, and / or infection. Examples include:

[0333] As used herein for a subject with ulcerative colitis, loss of maintenance of remission refers to at least A Mayo score of 3 and a Modified Barron score of at least 2 This refers to an increase in the Baron Score.

[0334] In one embodiment, diseases that may be treated accordingly include, but are not limited to: Inflammatory bowel disease (IBD), e.g., ulcerative colitis, Crohn's disease, ileitis, celiac disease, Non-tropical sprue, enteropathy associated with seronegative arthropathy, microscopic colitis or collagen ileal colitis, eosinophilic gastroenteritis, or pouchitis following proctocolectomy, and ileoanal In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Additional diseases that may be treated include, for example, primary sclerosing cholangitis (PSC) and colitis. ), and graft-versus-host disease (GVHD).

[0335] Ulcerative colitis is defined as moderate to severe active ulcerative colitis (e.g., submucosal ulcers) The score may be 2 or 3, and the Mayo score may be 6 to 12. Treatment is moderate to severe. Induction and maintenance of clinical response, clinical remission in patients suffering from moderately active ulcerative colitis Treatment may also result in induction and maintenance of corticosteroid therapy by the patient, or mucosal healing. This may result in the reduction, elimination, or reduction and elimination of corticosteroid use (e.g., remission without luteosteroids).

[0336] Crohn's disease may be moderately to severely active (e.g., Crohn's disease The CDAI score is 220-450. Treatment is for moderate to severe active In patients suffering from Rohn's disease, achieving a clinical response or achieving clinical remission Treatment may also involve reducing, eliminating, or decreasing corticosteroid use by the patient. In some cases, this may result in elimination (e.g., corticosteroid-free remission).

[0337] Pancreatitis and insulin-dependent diabetes mellitus can be treated using the compositions of the present invention. Other diseases. MAdCAM (e.g., MAdCAM-1) is involved in the development of NOD (non-obese diabetic retinopathy). ) Several blood vessels in the exocrine pancreas of mice, as well as from BALB / c and SJL mice. MAdCAM (e.g., MAdCAM-1) has been reported to be expressed by It has been reported that expression of α-amyloid β is induced in the endothelium of inflamed islets in the pancreas of NOD mice. MAdCAMs (e.g., MAdCAM-1) are expressed on NOD islet endothelium during the early stages of insulitis. It was the major addressin expressed by the ., J. Clin. Invest., 92: 2509-2515 (1993)). NO. Treatment of D mice with anti-MAdCAM or anti-beta7 antibodies prevented the onset of diabetes. (Yang et al., Diabetes, 46:1542-1547 (1997 In addition, accumulation of lymphocytes expressing α4β7 was observed in the pancreatic islets, and MAdCAM- 1 was associated with the binding of lymphoma cells to blood vessels from inflamed pancreatic islets via α4β7 (Ha nninen, A., et al., J. Clin. Invest., 92: 2509 -2515 (1993)) or mantle cell lymphoma in the gastrointestinal tract (Geissmann et al., Am. J. Pathol., 153:1701-1705 (1998) ) is related.

[0338] Examples of inflammatory diseases associated with mucosal tissue that can be treated using the compositions of the present invention include: Examples of these include cholecystitis and cholangitis (Adams and Eksteen Nature Rev iews 6:244-251(2006)Grant et al.,Hepatol ogy 33:1065-1072(2001)), for example, primary sclerosing cholangitis, e.g. Behçet's disease of the intestine, or pericholecititis (tissue surrounding the bile duct and liver), and graft-versus-host disease. Main disease (e.g., in the gastrointestinal tract (e.g., after bone marrow transplantation) (Petrovic et al. .Blood 103:1542-1547(2004)). As can be seen, inflammation often extends beyond the mucosal surface and thus may contribute to the development of sarcoidosis. chronic inflammatory diseases such as gastritis, chronic gastritis, e.g. autoimmune gastritis (Katakai et al. l., Int. Immunol., 14:167-175(2002)) and other idiopathic The condition may be amenable to treatment.

[0339] The present invention also relates to a method for inhibiting leukocyte infiltration of mucosal tissue. (e.g., α4β7 positive tumors such as lymphomas). Other examples of inflammatory diseases associated with mucosal tissue that can be treated using the formulation include mastitis ( mammary gland) and irritable bowel syndrome.

[0340] Pathogenesis exploits the interaction of MAdCAM (e.g., MAdCAM-1) with α4β7 The disease or pathogen can be treated with an anti-α4β7 antibody in a formulation described herein. Examples of such diseases include immunodeficiency disorders such as those caused by the human immunodeficiency virus. Harmful effects include (see, for example, WO2008140602).

[0341] The compositions of the invention comprise an effective amount of α4β7 integrin that inhibits binding to its ligand. In the case of treatment, an effective amount is an amount that exerts the desired therapeutic (including preventive) effect. sufficient to achieve (e.g., α4β7 integrin-mediated binding and / or signaling and reducing or preventing leukocyte adhesion and infiltration and / or associated cell proliferation. an amount sufficient to inhibit the response; Effective titers sufficient to maintain saturation, i.e., neutralization, of the agonist are essential for clinical response in inflammatory bowel disease. An effective amount of an anti-α4β7 antibody may induce the progression of ulcerative colitis or Crohn's disease. The formulations of the invention may be administered in unit doses or in multiple doses. The dosage may be determined by methods known in the art, e.g. For example, the dosage may depend on the individual's age, sensitivity, tolerance, and overall health. These include topical routes such as intranasal or inhaled or transdermal administration, via feeding tubes or suppositories. Enteral routes, such as intravenous, intramuscular, subcutaneous, intraarterial, intraperitoneal, or intravitreal administration, In one embodiment, the total dose is 165 mg. In one embodiment, the total dose is 108 mg. In another embodiment, the total dose is 216 mg. In an embodiment, the total dose is 300 mg.

[0342] In some embodiments, the method is for treating a disease described herein, e.g., UC or Crohn's disease. The dosing regimen for treating rheumatoid arthritis has two phases, an induction phase and a maintenance phase. The antibody or antigen-binding fragment thereof is a compound that binds to an antibody or antigen-binding fragment thereof. for specific purposes, such as inducing immune tolerance to inflammatory bowel disease, or to induce a clinical response The present invention provides a method for rapidly providing an effective amount of an antibody or an antigen-binding fragment thereof suitable for improving symptoms. When a patient is first treated with an anti-α4β7 antibody, the treatment When treated long after anti-α4β7 antibody therapy, for example, more than 3 months after anti-α4β7 antibody therapy, More than a month, more than six months, more than nine months, more than a year, more than 18 months or when treated after more than 2 years or when symptoms of inflammatory bowel disease recur, e.g. In cases where relapse from remission occurs, induction treatment during the maintenance phase of anti-α4β7 antibody therapy may be effective. In some embodiments, the induction phase regimen is maintained during the maintenance regimen. A mean trough serum concentration that is higher than the mean steady-state trough serum concentration expected to be achieved, e.g., Brings about the previous concentration.

[0343] In the maintenance phase, the antibody or antigen-binding fragment thereof maintains a stable level of the antibody or antigen-binding fragment thereof. in a manner that continues the response achieved by induction therapy with an antigen-binding fragment of A maintenance regimen may prevent the recurrence of symptoms or recurrence of inflammatory bowel disease. The treatment may be, for example, a simple medication regimen or may require only rare triages for treatment. In some embodiments, the maintenance regimen may provide convenience to the patient, such as being a For example, in the formulations described herein, low doses, infrequent administration, self-administration, and the aforementioned The use of an anti-α4β7 antibody or This may involve administration of an antigen-binding fragment.

[0344] In one embodiment, for example, during the induction phase of treatment, the dosing regimen is administered to a human patient to reduce inflammation. and administering to the patient an effective amount of an anti-α4β7 antibody or anti-α4β7 antibody in a formulation as described herein for inducing remission of chronic bowel disease. or an antigen-binding fragment thereof. The duration of the induction period is about 4 weeks, about 5 weeks, about 6 weeks, In some embodiments, the induction regimen may be for about 1 week, about 7 weeks, or about 8 weeks of treatment. The present study involves the use of high doses of anti-α4β7 antibodies or antigen-binding fragments thereof, frequent administration, and high A strategy selected from the group consisting of a combination of high doses and frequent administration is described, for example, in the present specification. The induction dose may be one or more doses, e.g., two or more During the induction period, the dose may be once daily, every other day, or once a week. May be administered twice a day, once a week, once every 10 days, once every 2 weeks, or once every 3 weeks. In some embodiments, the induction dose is administered within the first two weeks of treatment with the anti-α4β7 antibody. In one embodiment, the induction dose is administered once at the start of treatment (day 0) and once at the end of treatment. In another embodiment, the induction period is six weeks in duration. In another embodiment, the induction phase is six weeks in duration, with multiple induction doses administered during the first two weeks. is administered. In some embodiments, for example, when initiating treatment of a patient with severe inflammatory bowel disease, In some cases (e.g., in patients who have failed anti-TNFα therapy), the induction phase may be mild or moderate. In some embodiments, the patient has a longer period of time to be treated than the patient with the disease. The induction period for patients with the disease is at least 6 weeks, at least 8 weeks, or at least 10 weeks. In one embodiment, the period may be at least 12 weeks, or at least 14 weeks. For patients with severe disease, the induction dosing regimen is at week 0 (start of treatment), Patients with severe disease may receive treatment at week 0 (treatment week 2), and at week 6. may include administration at week 1, week 2, week 6, and week 10. .

[0345] This administration may be once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, It may be administered once every 8 weeks or once every 10 weeks. Higher or more frequent doses, e.g. For example, every other day, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks if active disease is present. To induce remission of a disease or to treat new patients, e.g., anti-α4β7 antibodies may be useful for inducing remission to rheumatoid arthritis. Once every 5 weeks, once every 6 weeks, once every 8 weeks, or once every 10 weeks may be administered without prior notice. In one embodiment, the method may be useful in preventative therapy, for example, to maintain remission in patients with chronic disease. The treatment regimen consisted of: on day 0, at about week 2, at about week 6, and then every week or two. In certain embodiments, the treatment regimen includes a treatment on day 0, about week 2, about week 6, and thereafter. In another embodiment, the induction treatment regimen is for a total of six treatments. This is a treatment given every other day.

[0346] In some embodiments, durable clinical remission, e.g., 6 months or 1 year after initiation of treatment. At least two, at least three, at least four visits by the caring physician within a period of Sustained clinical remission throughout the course of treatment can be achieved with optimized dosing regimens.

[0347] In some embodiments, a sustained clinical response, e.g., at least 6 months after initiation of treatment. Clinical responses lasting at least 9 months and at least 1 year were achieved with an effective dosing regimen. It can be done.

[0348] The present disclosure is further described in the following examples. The examples provided are for illustrative purposes only. and are not to be construed as limiting the scope or content of the present disclosure in any manner. . EXAMPLES

[0349] The following example describes an exemplary process for purifying vedolizumab from CHO cell culture. This section describes the various steps in the process.

[0350] Example 1. Control of Vedolizumab Charge Isoforms Vedolizumab exists in three charged isoforms: acidic, basic, and positive. Chromatographic analysis was performed using ion exchange (CEX)-HPLC to identify acidic, major, and basic species. Based on the relative areas of the lamellae, the isoform distribution of vedolizumab can be quantified. An exemplary CEX-HPLC profile showing the three vedolizumab species is shown in FIG.

[0351] Using CEX-HPLC, the charge isoform distribution of vedolizumab was characterized using various As summarized in Table 1, the manufacturing process of vedolizumab was Retention within the process affects the distribution of charged isoform species, while basic isoforms are more abundant under retention conditions. were most affected by the matter. [Table 1]

[0352] These results suggest that the ratio of different vedolizumab isoform species varies with antibody under certain conditions. It has been shown that the basic isoforms can be predictably regulated by retaining The ome species tends to increase during the holding period at pH below about 6.5. The isoform species decrease during the hold period at pH above about 6.5. These changes are The results were observed for vedolizumab produced at both pilot (Table 2) and manufacturing scale (Table 3). It is suspected. [Table 2] [Table 3]

[0353] To evaluate the charge isoform distribution of vedolizumab derived from GS-CHO cells Further experiments were performed. The ratio of the major acidic and basic species of vedolizumab was determined at various pH values. H (i.e., pH 4.7, 5.1, 5.3, 5.7, 5.9, 6.1, 6.5, or The results were evaluated after storage at 5°C or room temperature for 0 to 7 days. Enhanced stability was observed at pH levels above pH 5.9. Following capture and elution of Protein A, The typical pH range for neutralization (pH 4.9 to 5.2) is associated with increased formation of basic species. When vedolizumab was stored at pH 5.9 or 6.1, storage at pH below 5.9 The increase in basic species was slower than that observed during the incubation. As shown, the increase in basic species at pH levels above 6.5 is halted or reversed. Yes. [Table 4] [Table 5] [Table 6]

Table 7

Table 8

Table 9

Table 10

Table 11

[0354] This data shows that the levels of the major isoforms and basic isoform species in the vedolizumab preparation can be regulated by pH. In particular, the basic isoform species increase upon exposure of the antibody to low pH (<pH5.9), and concomitantly, the major isoform decreases. The level of the basic isoform species increases more rapidly and to a greater extent upon exposure to a decrease in pH. Furthermore, this trend is reversible upon exposure to an increase in pH, for example, >pH6.5.

[0355] Example 2. Reduction of the basic isoform of vedolizumab To further evaluate the effect of pH on the formation of the basic isoform of vedolizumab, the antibody was exposed to high pH conditions (200 mM Tris, pH9), and the isoform distribution of vedolizumab was quantified using cation exchange (CE X)-HPLC. For each condition, the relative to the chromatogram peaks corresponding to the acidic, major, and basic isoforms The relative amount of each vedolizumab isoform was quantified by determining the area.

[0356] As shown in Table 12, three peaks corresponding to the basic species of vedolizumab were detected at pH 6.3 ( control) (i.e., "basic peak 1," "basic peak 2," and "basic peak As shown in Table 12, when the pH was increased, a significant decrease in basic peak 2 was observed. It was noticed. [Table 12] The material eluting from the CEX resin with a retention time characteristic of basic peak 2 was collected and analyzed by enzyme analysis. After digestion, the product was analyzed by mass spectrometry (MS). The β-lactam was present in the basic peak 2 CEX fraction of the control preparation, but was not observed after exposure to pH 9. It was not present in the preparations analyzed later. Analysis of the primary amino acid sequence of vedolizumab , a residue that favors isomerization of aspartic acid to succinimide, i.e., at the n+1 position [ka] Aspartic acid in the CDR-H3 of vedolizumab located near the glycine or serine in This finding explains the increased basic species of vedolizumab observed at low pH. The large increase in the β-aspartic acid residues may be due to isomerization of the aspartic acid residue to succinimide. Maintaining the antibody at or near neutral pH slows the formation of basic peak 2. The isomerization reaction can be prevented or even prevented by increasing the pH (>pH 6.9). The succinimide can be converted back to aspartic acid (or isoaspartic acid) by conversion of the succinimide back to aspartic acid (or isoaspartic acid).

[0357] To further evaluate the effect of pH on basic peak 2, the antibody was incubated at various pH conditions. After exposure to conditions (pH 6.5, pH 7, pH 8, pH 8.5, or pH 9), CEX- The relative peak areas were determined by HPLC. As shown in Table 13, basic peak 2 was It was highly pH sensitive, decreasing with increasing pH, consistent with the results reported in Example 1. is doing. [Table 13]

[0358] Next, the levels of the vedolizumab isoform corresponding to basic peak 2 were analyzed using two different Vedolizumab preparations derived from CHO cell lines (DHFR-CHO and GS-CHO) As shown in Table 14, the DHFR-CHO cell line and the GS-CHO cell line were evaluated based on the following: A decrease in basic peak 2 was observed in vedolizumab preparations derived from both [Table 14]

[0359] Example 3. Effect of anion exchanger loading conductivity on host cell protein clearance sound Reducing the amount of host cell protein contaminants in a therapeutic protein composition is generally To generate vedolizumab compositions having reduced host cell protein content, as desired. We investigated a manufacturing method for the above.

[0360] Standard operating range of buffer conductivity for anion exchange (AEX) (e.g., anion exchange Q membrane adsorption The average ion current (through the ion exchanger) is about 11 to 15 mS / cm (average 13.6 mS / cm). To evaluate impurity clearance beyond the sub-operating range, A The composition obtained using EX conditions was tested for impurity clearance. Two independent starting preparations of clarified harvest were tested (Harvest 1 and Harvest 2). The electrical conductivity can be set to standard conductivity (approx. 13.6 mS / cm) or low conductivity (approx. 11 mS After adjusting to 100% HCP / cm, all samples were subjected to AEX purification. The level is the difference between the low conductivity AEX load material and the standard conductivity AEX load material. It decreased in Al. [Table 15]

[0361] Next, the AEX capacity (flow-through mode) was evaluated under standard operating conditions (i.e., 13–1 Tested using loading buffers with a range of conductivity lower than 5 mS / cm Table 16 shows the loading conditions, conductivity, host cell protein content, and acidity. The study summarized the percentage of CHO host cell protein isoforms. As shown in Table 16, the amount of host cell protein was , decreased with decreasing conductivity of the AEX loading buffer. [Table 16]

[0362] Next, the effect of reduced AEX conductivity on HCP clearance was examined using two different HCP The assay was performed using ELISA. The first method used rabbit primary and anti-rabbit secondary antibodies. was used in the HCP ELISA (RaRa). (RaGo) was used as a rabbit primary antibody and a goat secondary antibody. Tables 17 and 18 show the results. These two HCP ELISA methods after using standard or low conductivity AEX loads, respectively. Both methods reduce the conductivity of the AEX load material. These results indicate that HCP clearance is improved by administering [Table 17] [Table 18]

[0363] The amount of HCP in a recombinant protein preparation can vary. HCP clearance from preparations of dolizumab was significantly increased by reducing conductivity during purification using AEX. Regardless of the HCP concentration in the starting material, the low conductivity A significant reduction in HCPs was achieved in the AEX condition compared to the standard high conductivity AEX condition. It was.

[0364] Example 4. Effect of equilibration and washing conditions on the loading capacity of mixed-mode resin To minimize the loss of vedolizumab during purification, different purification conditions were investigated. We identified a step that reduces the yield of antibody product. Protein loss occurs during the wash steps of CHT purification. For example, Figure 2 shows that the standard wash and equilibration buffer (75 mM N aCl, 10 mM sodium phosphate, pH 7.2) at 27 mg / ml or 35 Comparison of the elution profile of vedolizumab from the CHT column after loading at 100 mg / ml. As shown in Figure 2, during the wash step under high loading conditions, The protein began to slowly deplete from the column (35 mg / ml).

[0365] The loading capacity of vedolizumab on the CHT column was increased to prevent loss during the CHT wash step. To improve vedolizumab yield by reducing the amount of protein consumed, an alternative C HT equilibration, loading, and wash buffers were evaluated. The loading amount (g / l) and the resulting yield achieved were calculated using standard CHT buffer at high pH ( 75 mM NaCl, 10 mM sodium phosphate, pH 7.2) for CHT column In comparison to the procedure, a low pH buffer (50 mM NaCl, 10 After running the CHT column using 10 mM sodium phosphate, pH 6.7, the results were as shown in Figure 3. As shown in Fig. 1, during the wash step using standard CHT buffer conditions (dashed line), significant When low pH buffer (solid line) was used, the protein loss was Furthermore, as summarized in Table 19, the equilibration and washing steps Using low pH buffers in the HPLC column allows for higher loadings on the CHT column. This allows for the elution of the standard without altering the quality of the product (e.g., percent aggregates) in the final eluate. Yields 2-3% higher were obtained compared to those achieved using standard CHT buffer. [Table 19]

[0366] Equivalent Although the present invention has been described with reference to its detailed description, the foregoing description is intended to limit the scope of the present invention. It is intended to be illustrative, not limiting, and is intended to be illustrative, not limiting, as defined in the appended claims. It should be understood that other aspects, advantages, and modifications are within the scope of the following claims.

[0367] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not limiting. Unless otherwise defined, all techniques and methods used herein are All scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. can be used, suitable methods and materials are described herein.

[0368] [Table 20]

Claims

1. 1. A method of producing a composition comprising vedolizumab, comprising: providing a composition comprising vedolizumab at a pH greater than pH 6.5; incubating said composition comprising vedolizumab for at least 20 minutes to 10 hours; ； thereby producing a composition comprising vedolizumab; and The method comprising:

2. The method comprises administering vedolizumab having reduced levels of basic vedolizumab isoform species.

16. A method for producing a composition comprising a basic vedolizumab isoform species, 13. The method of claim 1, producing a composition comprising reduced levels of vedolizumab.

3. The method comprises the steps of: < 16%, < 15%, < 14%, < 13%, < 12%, < 11% or < Producing a composition comprising vedolizumab having 10% basic vedolizumab isoform species The method according to claim 1 or 2,

4. The incubation is carried out during the purification of vedolizumab, and the incubation is a) prior to ultrafiltration / diafiltration (UF / DF) of the antibody, or (b) during pharmacy 4. The method according to claim 1, wherein the antibody is formulated in a physiologically acceptable buffer. The method described.

5. The method according to any one of claims 1 to 4, wherein the incubation is carried out at ambient temperature. method.

6. The incubation is carried out at 15 to 30° C. or The method according to any one of claims 1 to 4, wherein the step of heating is carried out at 20 to 25°C.

7. The method of claim 1, wherein the composition comprising vedolizumab is provided at a pH of about 6.5 to 8.

5. The method according to any one of claims 1 to 5

8. The method of claim 1, wherein the composition comprising vedolizumab is provided at a pH of about 7.0 to 8.

0.

13. The method according to any one of claims 1 to 12.

9. The method of claim 1, wherein the composition comprising vedolizumab is provided at a pH of about 7.0 to 7.

5.

13. The method according to any one of claims 1 to 12.

10. The composition comprising vedolizumab has a pH of about 6.5, pH 6.6, pH 6.7, pH 6. 8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH 7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8.1, The prior claims are provided at a pH of pH 8.2, pH 8.3, pH 8.4, or pH 8.

5. Item 10. The method according to any one of items 1 to 5.

11. The composition comprising vedolizumab is incubated for a period of about 10 to 120 hours.

13. A method according to any one of claims 1 to 12.

12. The composition comprising vedolizumab is incubated for a period of about 12 to 120 hours.

13. A method according to any one of claims 1 to 12.

13. The composition comprising vedolizumab is incubated for a period of about 12 to 96 hours.

10. The method according to claim 1 .

14. The composition comprising vedolizumab is incubated for a period of about 12 to 72 hours.

10. The method according to claim 1 .

15. The composition comprising vedolizumab is incubated for a period of about 12 to 48 hours.

10. The method according to claim 1 .

16. The composition comprising vedolizumab is incubated for a period of at least 12 hours.

13. A method according to any one of claims 1 to 12.

17. The composition comprising vedolizumab is incubated for a period of about 24 to 120 hours.

13. A method according to any one of claims 1 to 12.

18. The composition comprising vedolizumab is incubated for a period of about 24 to 96 hours.

10. The method according to claim 1 .

19. The composition comprising vedolizumab is incubated for a period of about 24 to 72 hours.

10. The method according to claim 1 .

20. The composition comprising vedolizumab is incubated for a period of about 24 to 48 hours.

10. The method according to claim 1 .

21. The composition comprising vedolizumab is , about 72 hours, about 96 hours, or about 120 hours. Item 10. The method according to any one of items 1 to 5.

22. The humanized anti-α4β7 antibody or antigen-binding portion thereof is isolated from the clarified cell culture harvest. A method for purifying a soluble cellulose derivative from a soluble cellulose derivative, comprising the steps of: (i) a culture of a recombinant host cell expressing the anti-α4β7 antibody or an antigen-binding portion thereof; providing a harvest of the clarified cell culture obtained from the (ii) purifying the anti-α4β7 antibody or antigen-binding portion thereof from the cell culture harvest. wherein the antibody is exposed to a pH of 4.0 or less for no more than 24 hours. and purifying said wherein the anti-α4β7 antibody or antigen-binding portion thereof has 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. How to write.

23. The anti-α4β7 antibody, or antigen-binding portion thereof, inhibits basic isoforms compared to a control. The levels of .ALPHA. species (as determined by CEX) were reduced, and the control was a composition comprising the anti-α4β7 antibody, or an antigen-binding portion thereof, produced by The antibody may be incubated at a pH below 4.0 (e.g., pH 3.6-4.0) for a longer period of time, i.e.

23. The method of claim 22, wherein the exposure is for more than 24 hours.

24. The anti-α4β7 antibody or antigen-binding portion thereof is vedolizumab or an antigen-binding portion thereof The method according to claim 22 or 23.

25. The composition comprising vedolizumab or an antigen-binding portion thereof comprises a first basic isoform a first basic isoform peak (BP1) and a second basic isoform peak (BP2), The present invention provides a composition comprising vedolizumab or an antigen-binding portion thereof having reduced levels of BP2.

13. A method according to any one of the preceding claims,

26. The method comprises vedolizumab or an antigen-binding portion thereof, 26. The method of claim 25, which produces a composition having less than 1%, or less than 0.7% BP2. method.

27. The composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

13. A method according to any one of the preceding claims,

28. 28. The method of claim 27, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. The method described above.

29. The CHO cell culture is a CHO cell culture lacking dihydrofolate reductase (DHFR) expression.

30. The method of claim 28, comprising administering to said subject a therapeutically effective amount of

30. The CHO cell culture comprises CHO cells lacking glutamine synthetase (GS) expression. The method of claim 28.

31. Affinity chromatography, cation exchange chromatography, anion exchange Chromatography and Ceramic Hydroxyapatite (CHT) Chromatography The mammalian animal is then subjected to one or more chromatographic separation steps selected from the group consisting of: Purifying said composition comprising vedolizumab from mammalian host cell proteins (HCPs).

2. The method of any one of the preceding claims, further comprising:

32. The composition comprising vedolizumab is subjected to affinity chromatography using Protein A.

32. The method of claim 31 , wherein the product is purified using a glycerol resin.

33. The composition comprising vedolizumab is subjected to affinity chromatography prior to the incubation.

33. The method of claim 32, wherein the product is purified using chromatography.

34. The composition comprising vedolizumab is subjected to affinity chromatography after the incubation.

33. The method of claim 32, wherein the product is purified using chromatography.

35. The composition comprising vedolizumab is purified using cation exchange chromatography. The method of claim 31 .

36. The composition comprising vedolizumab is treated with a cation exchange chromium salt prior to the incubation.

36. The method of claim 35, wherein the purified product is purified using microcentrifugation.

37. The composition comprising vedolizumab is, after the incubation, 36. The method of claim 35, wherein the purified product is purified using microcentrifugation.

38. The composition comprising vedolizumab is purified using anion exchange chromatography. The method of claim 31 .

39. The composition comprising vedolizumab is treated with an anion exchange chromium salt prior to the incubation.

39. The method of claim 38, wherein the product is purified using column chromatography.

40. The composition comprising vedolizumab is subjected to anion exchange chromium after the incubation.

39. The method of claim 38, wherein the product is purified using column chromatography.

41. The composition comprising vedolizumab is purified using CHT chromatography. The method according to claim 31.

42. The composition comprising vedolizumab is subjected to CHT chromatography prior to the incubation. The method of claim 41, wherein the product is purified using a fermentation process.

43. The composition comprising vedolizumab is subjected to CHT chromatography after the incubation. The method of claim 41, wherein the product is purified using a fermentation process.

44. The composition according to any one of claims 1 to 43, comprising incorporating the composition into a pharmaceutical formulation. method.

45. 45. The method of claim 44, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

46. 46. ​​The lyophilized pharmaceutical formulation of claim 45, wherein the lyophilized pharmaceutical formulation is a dry lyophilized pharmaceutical formulation. The method described above.

47. Reconstituting the dry lyophilized pharmaceutical formulation with a liquid so that it is suitable for administration.

47. The method of claim 46, further comprising the steps of:

48. 45. The method of claim 44, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

49. The composition is produced by the method of any one of claims 1 to 48. A composition comprising limumab.

50. A composition comprising vedolizumab, the composition being prepared by the method according to any one of claims 1 to 48. The composition is obtainable by

51. The basic vedolizumab isoform species is the vedolizumab isoform present in the composition. Less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or 51. The composition of claim 49 or 50, comprising less than 10%.

52. 1. A low basic species composition comprising an anti-α4β7 antibody, the composition comprising: less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the total basic isoform species, said basic isoform species being anti-α4 It has a net positive charge compared to the major isoform of the β7 antibody, and Peaks that elute slower from cation exchange (CEX) resins than peaks corresponding to foams The anti-α4β7 antibody can be quantified by determining the relative area of ​​the and a light chain variable region comprising SEQ ID NO:

2.

53. The composition comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2).

53. The composition of claim 52, comprising Foam Peak (BP2).

54. 54. The composition of claim 53, wherein the composition contains less than 2% BP2.

55. 54. The composition of claim 53, wherein the composition comprises less than 1.5% BP2.

56. 54. The composition of claim 53, wherein the composition contains less than 1% BP2.

57. 54. The composition of claim 53, wherein the composition comprises less than 0.7% BP2.

58. 54. The composition of claim 53, wherein the ratio of BP1 to BP2 is at least 3.

59. 54. The composition of claim 53, wherein the ratio of BP1 to BP2 is at least 5.

60. 54. The composition of claim 53, wherein the ratio of BP1 to BP2 is at least 7.

61. 54. The composition of claim 53, wherein the ratio of BP1 to BP2 is at least 10.

62. A composition according to any one of claims 52 to 61, and a pharma- ceutically acceptable carrier or A pharmaceutical composition comprising an excipient.

63. 1. A method of producing a composition comprising vedolizumab, comprising: (a) A sample containing vedolizumab and host cell proteins (HCPs) was added to a loading buffer. contacting the anion exchange resin with the anion exchange resin in the presence of a loading buffer solution, The conductivity of the buffer solution is 11 mS / cm or less so that HCP binds to the anion exchange resin. said contacting; (b) collecting the flow-through material from the anion exchange resin. 、 wherein the eluate comprises vedolizumab.

64. The method produces a composition comprising vedolizumab having a reduced amount of HCP. wherein the flow-through material comprises vedolizumab and a reduced amount of HCP.

64. The method of claim 63.

65. The loading buffer has a conductivity of 9 mS / cm to 11 mS / cm. Item 65. The method according to item 63 or 64.

66. 63. The method of claim 62, wherein the loading buffer has a conductivity of 10 mS / cm or less.

64. The method according to claim 64.

67. 63. The method of claim 62, wherein the loading buffer has a conductivity of 9 mS / cm or less.

64. The method according to claim 64.

68. The loading buffer is about 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10 65. The method according to claim 63 or 64, having a conductivity of 0.5 mS / cm or 11 mS / cm. The method described.

69. 7. The method of claim 6, wherein the HCP is a Chinese Hamster Ovary (CHO) cell protein.

69. The method according to any one of claims 3 to 68.

70. The HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression.

70. The method of claim 69 .

71. The HCP is derived from CHO cells lacking glutamine synthetase (GS) expression. The method of claim 69.

72. 72. The method of claim 63, further comprising contacting the anion exchange resin with a wash buffer.

2. The method according to any one of the preceding claims.

73. 73. The method of claim 72, wherein the wash buffer has a conductivity of less than 11 mS / cm.

74. 73. The method of claim 72, wherein the wash buffer has a conductivity of 9 mS / cm to 11 mS / cm. The method described.

75. 73. The method of claim 72, wherein the wash buffer has the same conductivity as the loading buffer. 。

76. The loading buffer comprises sodium chloride and / or sodium phosphate. Item 63 to 75. The method according to any one of items 63 to 75.

77. The washing buffer comprises sodium chloride and / or sodium phosphate.

75. The method according to any one of claims 75 to 75.

78. The anion exchange resin is formatted as an anion exchange column or an anion exchange membrane. The method according to any one of claims 63 to 77, wherein

79. 79. Any one of claims 63 to 78, wherein the anion exchange resin comprises a quaternary amine functional group. The method according to

80. The sample containing vedolizumab and HCP is subjected to one or more chromatographic separation steps. The method according to any one of claims 63 to 79, which is derived from a mammalian cell culture after cloning. 。

81. The one or more chromatographic separation steps may be performed by affinity chromatography. -, cation exchange chromatography, and ceramic hydroxyapatite (CHT) 81. The method of claim 80, further comprising one or more steps selected from the group consisting of: chromatography. The method described.

82. The amount of HCP in the flow-through material is 8 ppm or less, 7.5 ppm or less, 7 ppm or less pm or less, 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5 ppm or less, 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, The method according to any one of claims 63 to 81, wherein the concentration of 100% or less of the cation exchange resin is 2 ppm or less.

83. The amount of HCP in the flow-through material is determined by a low conductivity of greater than 12 mS / cm. The fragments produced when the method is performed using the same sample with a filling buffer are 63. The amount of HCP in the waste material is reduced by at least 50%.

83. The method according to any one of claims 1 to 82.

84. Ultrafiltration into a buffer containing one or more pharma- ceutically acceptable carriers or excipients and / or The filtration step is carried out to exchange the elution buffer by a process including diafiltration.

84. The method of claim 63, further comprising treating the drop-through material. The method described.

85. The method according to any one of claims 63 to 84, comprising incorporating the composition into a pharmaceutical formulation. How to.

86. 86. The method of claim 85, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

87. 87. The lyophilized pharmaceutical formulation of claim 86, wherein the lyophilized pharmaceutical formulation is a dry lyophilized pharmaceutical formulation. The method according to

88. Reconstituting the dry lyophilized pharmaceutical formulation with a liquid so that it is suitable for administration.

88. The method of claim 87, further comprising the steps of:

89. 86. The method of claim 85, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

90. 89 or 48, wherein the liquid pharmaceutical formulation is suitable for subcutaneous administration to humans. method.

91. A composition comprising vedolizumab produced by the method of any one of claims 63 to 90. 。

92. The composition is a bed obtainable by the method according to any one of claims 63 to 90. A composition comprising limumab.

93. the amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, 7 ppm or less; 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5ppm or less , 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, or 2 ppm 93. The composition of claim 91 or 92, wherein:

94. Increasing the yield of vedolizumab recovered after elution from a mixed-mode chromatography resin The method includes equilibrating the mixed-mode chromatography resin with an equilibration buffer. and subjecting a solution containing vedolizumab and a loading buffer to the mixed-mode chromatography. chromatographic resin, and vedolizumab was loaded onto the mixed-mode chromatography resin. allowing the resin to bind; and washing the mixed-mode chromatography resin with a wash buffer. and removing vedolizumab from said mixed-mode chromatography resin with an elution buffer. wherein said equilibration buffer, said loading buffer and / or The method, wherein the pH of the wash buffer is 7.0 or less.

95. The pH of the equilibration buffer, the loading buffer, and / or the washing buffer is 6.

95. The method of claim 94, wherein the .0 to 7.

0.

96. The pH of the equilibration buffer, the loading buffer, and / or the washing buffer is 6.

95. The method of claim 94, wherein the .5 to 7.

0.

97. The pH of the equilibration buffer, the loading buffer, and / or the washing buffer is 6.

95. The method of claim 94, wherein the pH is 6 to 6.

8.

98. The salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is The method according to any one of claims 94 to 97, wherein the concentration is between 30 mM and 70 mM.

99. The salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 99. The method of claim 98, wherein the concentration is between 40 mM and 70 mM.

100. The salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 99. The method of claim 98, wherein the concentration is between 50 mM and 65 mM.

101. The salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is 99. The method of claim 98, wherein the concentration is between 55 mM and 65 mM.

102. Claims 98 to 101, wherein the salt comprises sodium chloride and / or sodium phosphate.

2. The method according to any one of the preceding claims.

103. The sodium chloride of the equilibration buffer, the loading buffer, and / or the wash buffer The method according to any one of claims 94 to 97, wherein the sodium concentration is between 30 mM and 70 mM. 。

104. The sodium chloride of the equilibration buffer, the loading buffer, and / or the wash buffer 104. The method of claim 103, wherein the sodium concentration is between 40 mM and 70 mM.

105. The sodium chloride of the equilibration buffer, the loading buffer, and / or the wash buffer 104. The method of claim 103, wherein the sodium concentration is between 40 mM and 60 mM.

106. The sodium chloride of the equilibration buffer, the loading buffer, and / or the wash buffer 104. The method of claim 103, wherein the sodium concentration is between 45 mM and 55 mM.

107. The sodium chloride of the equilibration buffer, the loading buffer, and / or the wash buffer 104. The method of claim 103, wherein the sodium concentration is about 50 mM.

108. The equilibration buffer, the loading buffer, and / or the washing buffer may further The method of any one of claims 103 to 107, comprising sodium phosphate.

109. The pH of the equilibration buffer, the loading buffer, and / or the washing buffer is the same. The method according to any one of claims 94 to 108, wherein

110. The salt concentration of the equilibration buffer, the loading buffer, and / or the washing buffer is The method of any one of claims 94 to 109, wherein the

111. The equilibration buffer, the loading buffer, and / or the washing buffer are the same buffer. The method according to any one of claims 94 to 108, wherein the liquid is a liquid.

112. Claims 94 to 11, wherein the mixed mode resin is a ceramic hydroxyapatite resin.

1. The method according to any one of claims 1 to 9.

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