Methods of producing anti-alpha4beta7 antibodies

Abstract

Provided herein are methods for purifying an anti-alpha4beta7 integrin antibody, such as a vildozumab, from a liquid solution, e.g., from a mammalian cell culture clarified harvest. The present invention relates, inter alia, to purification methods for controlling the amount of product-related substances and / or process-related impurities present in a purified formulation of an anti-alpha4beta7 integrin antibody or antigen-binding fragment thereof (e.g., vildozumab). Compositions comprising an anti-alpha4beta7 antibody and their use to treat disorders are also provided.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of June 10, 2020, application number 202080049572.2, and invention name “Method for producing anti-α4β7 antibodies”. Technical Field

[0002] The present invention relates to a method for purifying an anti-α4β7 antibody or a fragment thereof.

[0003] Related Applications

[0004] This application claims priority to U.S. Provisional Application No. 62 / 859,494, filed on June 10, 2019. The entire contents of the foregoing application are incorporated herein by reference.

[0005] Sequence Listing

[0006] This application contains a sequence listing submitted electronically in ASCII format and hereby incorporated by reference in its entirety. The ASCII copy was created on June 5, 2020, named T103022_1090WO_SL.txt, and is 10,009 bytes in size. Background Art

[0007] Large-scale, economical purification of proteins is an increasingly important problem in the biotechnology industry. Typically, biopharmaceuticals are produced by cell culture using prokaryotic (e.g., bacteria) or eukaryotic (e.g., mammalian or fungal) cell lines that have been engineered to produce a large amount of therapeutic proteins of interest. Because the cell lines used are living organisms, they must be supplied with complex cell culture media (sometimes supplied by animal serum preparations) containing sugars, amino acids, and growth factors. It is a difficult challenge to separate the desired recombinant therapeutic protein from process-related impurities (including, for example, cell culture media components, host cell proteins (HCPs), host nucleic acids, and / or chromatographic materials) and product-related impurities (such as aggregates, misfolded species, or fragments of the protein of interest) to a purity sufficient to be used as a human therapeutic agent.

[0008] Product-related and process-related impurities, including aggregates, have the potential to interfere with the purification process, affect the protein during storage, and / or may be the cause of adverse reactions when the antibody is administered to a subject (Shukla et al., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 848(1), 28-39).

[0009] Therefore, there remains a need in the art for improved methods of purifying therapeutic proteins (eg, antibodies) while effectively removing impurities, improving protein recovery, and maintaining therapeutic needs. Summary of the invention

[0010] The present invention is based at least in part on the development of a process for producing an anti-α4β7 antibody or an antigen-binding portion thereof. In some embodiments, the anti-α4β7 antibody or an antigen-binding portion thereof is a humanized anti-α4β7 antibody or an antigen-binding portion thereof. In any of the following aspects and embodiments, the humanized anti-α4β7 antibody or an antigen-binding portion thereof may comprise a heavy chain variable region comprising CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4, and / or a light chain variable region comprising CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8. In some embodiments, the humanized anti-α4β7 antibody or an antigen-binding portion thereof comprises a heavy chain variable region comprising SEQ ID NO: 1 and / or a light chain variable region comprising SEQ ID NO: 2. In some embodiments, the humanized anti-α4β7 antibody or an antigen-binding portion thereof comprises a heavy chain comprising SEQ ID NO: 9 and / or a light chain comprising SEQ ID NO: 10. In exemplary embodiments, the anti-α4β7 antibody is vedolizumab, or an antigen-binding portion thereof.

[0011] Therefore, in one aspect, the present invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof, the method comprising: providing a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof having a pH greater than 6.5; and incubating the composition comprising the humanized anti-α4β7 antibody or an antigen-binding portion thereof for a period of at least 20 minutes to 10 hours; thereby producing a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof.

[0012] In another aspect, the present invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, having reduced levels of basic isoform species, the method comprising: providing a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, having a pH greater than 6.5; and incubating the composition comprising the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, for a period of time sufficient to reduce the level of basic isoform species of the anti-α4β7 antibody, or an antigen-binding portion thereof, in the composition; thereby producing a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, having reduced levels of basic isoform species.

[0013] In some embodiments of the aforementioned aspects, the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising 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 light chain variable region comprising CDR1 set forth in SEQ ID NO:6, CDR2 set forth in SEQ ID NO:7, and CDR3 set forth in SEQ ID NO:8. In some embodiments, the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising SEQ ID NO:1 and / or a light chain variable region comprising SEQ ID NO:2. In some embodiments, the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain comprising SEQ ID NO:9 and / or a light chain comprising SEQ ID NO:10. In exemplary embodiments, the anti-α4β7 antibody is vedolizumab or an antigen-binding portion thereof.

[0014] In some embodiments, the methods produce a composition comprising a humanized anti-α4β7 antibody, or antigen-binding portion thereof, having <16%, <15%, <14%, <13%, <12%, <11%, or <10% basic isoform species.

[0015] In some embodiments, the incubation is performed during purification of the humanized anti-α4β7 antibody or antigen-binding portion thereof, and wherein the incubation is performed prior to (a) ultrafiltration / diafiltration (UF / DF) of the antibody or (b) prior to formulating the antibody in a pharmaceutically acceptable buffer.

[0016] In some embodiments, the incubation is performed at ambient temperature. In some embodiments, the incubation is performed at 15°C-30°C. In some embodiments, the incubation is performed at 20°C-25°C.

[0017] In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen binding portion thereof is provided at a pH of about 6.5-8.5. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen binding portion thereof is provided at a pH of about 7.0-8.0. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen binding portion thereof is provided at a pH of about 7.0-7.5. In other embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen binding portion thereof is provided at a pH of about 6.6-7.3. In some embodiments, the composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, is provided at a pH of about pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, or pH 8.5.

[0018] In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 10 to 120 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 120 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 96 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 72 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 12 to 48 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of at least 12 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of at least 15 to 36 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 24 to 120 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 24 to 96 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 24 to 72 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 24 to 48 hours. In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is incubated for a period of about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, or about 120 hours.

[0019] In another aspect, provided herein is a method for purifying a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, from a clarified cell culture harvest, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing the anti-α4β7 antibody, or an antigen-binding portion thereof, and (ii) purifying the anti-α4β7 antibody, or an antigen-binding portion thereof, from the cell culture harvest, wherein the antibody is exposed to a pH of 4.0 or below for no more than 24 hours, wherein the anti-α4β7 antibody, or an antigen-binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:5.

[0020] In some embodiments, the anti-α4β7 antibody, or antigen-binding portion thereof, has reduced levels of basic isotype species (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.0 or below (e.g., pH 3.6 to 4.0) for a longer period of time, i.e., greater than 24 hours.

[0021] In some embodiments, the anti-α4β7 antibody, or an antigen binding portion thereof, is vedolizumab, or an antigen binding portion thereof.

[0022] In some embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab or an antigen-binding portion thereof) comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2), and wherein the method produces a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof having reduced levels of BP2. In certain embodiments, the method produces a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof having less than 2%, less than 1.5%, less than 1%, or less than 0.7% BP2.

[0023] In some embodiments, the 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 a Chinese hamster ovary (CHO) cell culture. In certain embodiments, the CHO cell culture comprises CHO cells lacking expression of dihydrofolate reductase (DHFR). In certain embodiments, the CHO cell culture comprises CHO cells lacking expression of glutamine synthetase (GS).

[0024] In some embodiments, the method further comprises purifying the composition comprising the humanized anti-α4β7 antibody, or an antigen-binding portion thereof, from mammalian host cell proteins (HCP) using one or more chromatographic separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, ceramic hydroxyapatite (CHT) chromatography, and hydrophobic interaction chromatography (HIC).

[0025] In certain embodiments, an affinity chromatography resin comprising protein A is used to purify a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof.

[0026] In certain embodiments, the composition comprising the humanized anti-α4β7 antibody, or antigen binding portion thereof, is purified using affinity chromatography prior to incubation to reduce basic isoforms. In certain embodiments, the composition comprising the humanized anti-α4β7 antibody, or antigen binding portion thereof, is purified using affinity chromatography after incubation.

[0027] In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is purified using cation exchange chromatography. In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is purified using cation exchange chromatography prior to incubation. In certain embodiments, a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is purified using cation exchange chromatography after incubation.

[0028] In certain embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is purified using anion exchange chromatography. In certain embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is purified using anion exchange chromatography prior to incubation. In certain embodiments, the composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof is purified using anion exchange chromatography after incubation.

[0029] In certain embodiments, CHT chromatography is used to purify a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof. In certain embodiments, CHT chromatography is used to purify a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof prior to incubation. In certain embodiments, CHT chromatography is used to purify a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof after incubation.

[0030] In some embodiments, the method comprises incorporating the composition into a pharmaceutical formulation.

[0031] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In specific embodiments, the lyophilized pharmaceutical formulation is a dried lyophilized pharmaceutical formulation. In some such embodiments, the method further comprises the step of reconstituting the dried lyophilized pharmaceutical formulation with a liquid to make it suitable for administration.

[0032] In alternative embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some such embodiments, the liquid pharmaceutical formulation is suitable for subcutaneous administration to a human.

[0033] In another embodiment, the present invention provides a method for purifying a humanized anti-α4β7 antibody, or an antigen-binding portion thereof, having reduced levels of basic isoform species, wherein the humanized anti-α4β7 antibody, or the antigen-binding portion thereof, is maintained at or above pH 6.5 for at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the total time between initial recovery of the humanized anti-α4β7 antibody, or the antigen-binding portion thereof, from a cell culture harvest by ultrafiltration / diafiltration (UF / DF) and formulation of the humanized anti-α4β7 antibody, or the antigen-binding portion thereof, in a pharmaceutically acceptable carrier. By maintaining the pH of the humanized anti-α4β7 antibody, or antigen-binding portion thereof, at or above pH 6.5 during the majority of the purification process, basic isoform species of the humanized anti-α4β7 antibody, or antigen-binding portion thereof, can be reduced relative to an equivalent purification process in which the humanized anti-α4β7 antibody, or antigen-binding portion thereof, is at a pH below pH 6.5 for a substantial period of time (e.g., greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, or greater than 40% of the total time between initial recovery of the humanized anti-α4β7 antibody, or antigen-binding portion thereof, from a cell culture harvest by ultrafiltration / diafiltration (UF / DF) and formulation of the anti-α4β7 antibody, or antigen-binding portion thereof, in a pharmaceutically acceptable carrier).

[0034] On the other hand, the present invention provides a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab) with reduced levels of basic isoform species. In some embodiments, the basic isoform species of the humanized anti-α4β7 antibody or its antigen-binding portion constitute less than 15%, 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% of the humanized anti-α4β7 antibody or its antigen-binding portion present in the composition. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding portion having reduced levels of basic isoform species is produced using the methods described herein. In some embodiments, a composition comprising a humanized anti-α4β7 antibody or its antigen-binding portion is produced by a combination of the methods of the present invention.

[0035] On the other hand, provided herein is a method for producing a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab), the method comprising: (a) contacting a sample containing the humanized anti-α4β7 antibody or its antigen-binding portion and a host cell protein (HCP) with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a conductivity of 11 mS / cm or less, such that the HCP binds to the anion exchange resin; (b) collecting a flow through material from the anion exchange resin, wherein the flow through material comprises the humanized anti-α4β7 antibody or its antigen-binding portion.

[0036] In another aspect, the present invention provides a method for producing a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab), the method comprising: (a) contacting a sample containing a humanized anti-α4β7 antibody or an antigen-binding portion thereof and a host cell protein (HCP) with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a pH of 11 mS / cm or less (e.g., about 11 mS / cm or less, about 10 mS / cm or less, about 9 mS / cm or less). / cm or less, about 8 mS / cm or less, about 7 mS / cm or less, about 6 mS / 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) to allow the HCP to bind to the anion exchange resin; (b) contacting the anion exchange resin with an elution buffer; and (c) collecting the flow-through from the anion exchange resin, wherein the flow-through comprises a humanized anti-α4β7 antibody or an antigen-binding portion thereof.

[0037] In some embodiments of the above aspects, the method is a method of producing a composition comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof (e.g., vedolizumab) having a reduced amount of HCP, and the flow-through comprises a humanized anti-α4β7 antibody, or an antigen-binding portion thereof (e.g., vedolizumab) and a reduced amount of HCP.

[0038] In some embodiments, the loading buffer has a conductivity of 9 mS / cm to 11 mS / cm (eg, about 9 mS / cm to about 11 mS / cm or about 10 mS / cm to about 11 mS / cm).

[0039] In some embodiments, the loading buffer has a conductivity of less than 11 mS / cm (e.g., less than 11 mS / cm, less than 10 mS / cm, less than 9 mS / cm, less than 8 mS / cm, less than 7 mS / cm, less than 6 mS / cm, less than 5 mS / cm, less than 4 mS / cm, less than 3 mS / cm, or less than 2 mS / cm).

[0040] In some embodiments, the loading buffer has a conductivity of about 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm, or 11 mS / cm.

[0041] In some embodiments, HCP is Chinese hamster ovary (CHO) cell protein. In certain embodiments, HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression. In certain embodiments, HCP is derived from CHO cells lacking glutamine synthetase (GS) expression.

[0042] In some embodiments, the anion exchange resin is washed with wash buffer. In some embodiments, the wash buffer has a conductivity of 11mS / cm or less. In some embodiments, the wash buffer has a conductivity of 9mS / cm to 11mS / cm. In some embodiments, the wash buffer has a conductivity less than 11mS / cm. In some embodiments, the wash buffer has a conductivity of about 9mS / cm, 9.5mS / cm, 10mS / cm, 10.5mS / cm or 11mS / cm. In some embodiments, the wash buffer has the same conductivity as the loading buffer.

[0043] In some embodiments, the loading buffer comprises sodium chloride and / or sodium phosphate.

[0044] In some embodiments, the loading buffer comprises 20-150 mM salt, 50-125 mM salt, or 75-100 mM salt. In one embodiment, the salt comprises sodium chloride and / or sodium phosphate. For example, the buffer may comprise about 20 mM, about 30 mM, about 40 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 sodium chloride. Additionally, or alternatively, the buffer may comprise about 20 mM, about 30 mM, about 40 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 sodium phosphate.

[0045] In some embodiments, the wash buffer comprises sodium chloride and / or sodium phosphate.

[0046] In some embodiments, the wash buffer comprises 20-150 mM salt, 50-125 mM salt, or 75-100 mM salt. In one embodiment, the salt comprises sodium chloride and / or sodium phosphate. For example, the buffer may comprise about 20 mM, about 30 mM, about 40 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 sodium chloride. Additionally, or alternatively, the buffer may comprise about 20 mM, about 30 mM, about 40 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 sodium phosphate.

[0047] In some embodiments, the conductivity of the wash buffer is equal to or lower than the conductivity of the load buffer. In some embodiments, the wash buffer has the same conductivity as the load buffer.

[0048] In some embodiments, the anion exchange resin is formatted as an anion exchange column or anion exchange membrane.

[0049] In some embodiments, the anion exchange resin comprises quaternary amine functional groups.

[0050] In some embodiments, the sample containing humanized anti-α4β7 antibody or antigen-binding portion thereof (e.g., vedolizumab) and HCP is derived from a mammalian cell culture after one or more chromatographic separation steps. In certain embodiments, the one or more chromatographic separation steps include one or more steps selected from the group consisting of affinity chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography (HIC), and ceramic hydroxyapatite (CHT) chromatography.

[0051] In some embodiments, the amount of HCP in the flowthrough is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

[0052] In some embodiments, the amount of HCP in the flow-through is reduced by 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 than about 98%) relative to the amount of HCP in the flow-through produced when the same sample is subjected to the method with a loading buffer having a conductivity greater than 12 mS / cm.

[0053] In some embodiments, the method further comprises treating the flow-through material by a process comprising ultrafiltration and / or diafiltration to exchange the buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients.

[0054] In some embodiments, the method comprises incorporating the composition into a pharmaceutical formulation.

[0055] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In specific embodiments, the lyophilized pharmaceutical formulation is a dried lyophilized pharmaceutical formulation. In some such embodiments, the method further comprises the step of reconstituting the dried lyophilized pharmaceutical formulation with a liquid to make it suitable for administration.

[0056] In alternative embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some such embodiments, the liquid pharmaceutical formulation is suitable for subcutaneous administration to a human.

[0057] On the other hand, provided herein is a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab) produced by any method of the present invention. Also provided herein is a composition comprising a humanized anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab), wherein the composition can be obtained by any method of the present invention. In some embodiments, the amount of HCP in the composition is 8ppm or less, 7.5ppm or less, 7ppm or less, 6.5ppm or less, 6ppm or less, 5.5ppm or less, 5ppm or less, 4.5ppm or less, 4ppm or less, 3.5ppm or less, 3ppm or less, 2.5ppm or less, or 2ppm or less. In another embodiment, the basic isotype species of the humanized anti-α4β7 antibody, or antigen-binding portion thereof (e.g., vedolizumab) constitute less than 16%, less than 15%, 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% of the antibody species present in the composition.

[0058] In some embodiments, a composition comprising a humanized anti-α4β7 antibody, or antigen-binding portion thereof (eg, vedolizumab) is produced by a combination of the methods of the invention.

[0059] On the other hand, the present invention relates to a method for increasing the yield of a humanized anti-α4β7 antibody or its antigen-binding portion (e.g., vedolizumab) recovered after elution from a mixed mode chromatography resin, the method comprising equilibrating the mixed mode chromatography resin with an equilibration buffer, loading a solution comprising the anti-α4β7 antibody or its antigen-binding portion and a loading buffer onto the mixed mode chromatography resin so that the anti-α4β7 antibody or its antigen-binding portion binds to the mixed mode chromatography resin, washing the mixed mode chromatography resin with a wash buffer, and eluting the anti-α4β7 antibody or its antigen-binding portion from the mixed mode chromatography resin with an elution buffer, wherein the equilibration buffer, the loading buffer and / or the wash buffer have a pH of 7.0 or below.

[0060] In one embodiment, the equilibration buffer, loading buffer, and / or wash buffer have a pH of 6.0-7.0. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer have a pH of 6.5-7.0. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer have a pH of 6.6-6.8.

[0061] In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a salt concentration of 30mM to 70mM. In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a salt concentration of 40mM to 70mM. In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a salt concentration of 50mM to 65mM. In another embodiment, loading buffer and / or washing buffer have a salt concentration of 55mM-65mM. In another embodiment, salt includes sodium chloride and / or sodium phosphate.

[0062] In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a sodium chloride concentration of 30mM to 70mM. In another embodiment, the loading buffer and / or washing buffer have a sodium chloride concentration of 40mM to 70mM. In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a sodium chloride concentration of 40mM to 60mM. In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a sodium chloride concentration of 45mM-55mM. In another embodiment, the balancing buffer, loading buffer and / or washing buffer have a sodium chloride concentration of about 50mM. In another embodiment, the balancing buffer, loading buffer and / or washing buffer further comprise sodium phosphate.

[0063] In some embodiments, the equilibration buffer, loading buffer, and / or washing buffer have the same pH value. In some embodiments, the equilibration buffer, loading buffer, and / or washing buffer have the same salt concentration. In some embodiments, the equilibration buffer, loading buffer, and / or washing buffer can be the same buffer. In some embodiments of the foregoing aspects, the mixed mode resin can be a ceramic hydroxyapatite resin, such as a CHT resin.

[0064] In another aspect, provided herein is a low basic species composition comprising an anti-α4β7 antibody, wherein the composition comprises 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 of the anti-α4β7 antibody, wherein the basic isoform species has a net positive charge relative to the major isoform of the anti-α4β7 antibody, and wherein the 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. In some embodiments, the basic isoform species can be quantified by cation exchange (CEX) chromatography. For example, in some embodiments, the basic isoform species can be quantified by determining the relative area of ​​a peak that elutes more slowly from a cation exchange (CEX) resin than a peak corresponding to a major isoform.

[0065] Thus, in another aspect, provided herein is a low basic species composition comprising an anti-α4β7 antibody, wherein the composition comprises 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 of the anti-α4β7 antibody, wherein the basic isoform species have a net positive charge relative to the major isoform of the anti-α4β7 antibody and can be quantified by determining the relative area of ​​a peak that elutes slower from a cation exchange (CEX) resin than the peak corresponding to the major isoform, and wherein the 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.

[0066] In some embodiments, the composition comprises a first basic isoform peak (BP1) and a 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 comprises less than 1% BP2. In some embodiments, the composition comprises less than 0.7% BP2.

[0067] 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. In some embodiments, the ratio of BP1 to BP2 is at least 10.

[0068] In some embodiments, the composition comprises less than 8% of the total basic isoform species of anti-α4β7 antibodies. In some embodiments, the composition comprises less than 7% of the total basic isoform species of anti-α4β7 antibodies. In some embodiments, the composition comprises less than 6% of the total basic isoform species of anti-α4β7 antibodies. In some embodiments, the composition comprises less than 5% of the total basic isoform species of anti-α4β7 antibodies.

[0069] On the other hand, provided herein is a pharmaceutical composition comprising a composition provided herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pH of the pharmaceutical composition is between 6.0-7.0. In some embodiments, the pH of the pharmaceutical composition is about pH 6.3. In other embodiments, the pH of the pharmaceutical composition is pH 6.3 to pH 6.5.

[0070] In some embodiments, the pharmaceutical composition further comprises an amino acid. In some embodiments, the amino acid is arginine or histidine.

[0071] In some embodiments, the pharmaceutical composition further comprises a sugar. In certain embodiments, the sugar is sucrose or trehalose.

[0072] In some embodiments, the pharmaceutical composition comprises arginine, histidine, and sucrose. In some embodiments, the pharmaceutical composition comprises arginine, histidine, sucrose, and polysorbate 80.

[0073] In some embodiments, the pharmaceutical composition comprises at least 200 mg, at least 250 mg, or at least 300 mg of anti-α4β7 antibody. In some embodiments, the pharmaceutical composition comprises about 300 mg of anti-α4β7 antibody.

[0074] In some embodiments, the anti-α4β7 antibody is vedolizumab, or an antigen-binding portion thereof.

[0075] In another aspect, provided herein is a method for producing a low basic species composition comprising an anti-α4β7 antibody having 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, the method comprising providing a composition comprising an anti-α4β7 antibody having a pH greater than pH 6.3; and incubating the composition comprising the anti-α4β7 antibody for a period of time greater than 10 hours; thereby producing a low basic species composition comprising an anti-α4β7 antibody having 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. In some embodiments, the 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.

[0076] In another aspect, provided herein is a method of producing a low basic species composition comprising an anti-α4β7 antibody having 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 isotype species, the method comprising providing a composition comprising vedolizumab having a pH greater than pH 6.3; and incubating the composition comprising vedolizumab for a period of time sufficient to reduce the level of basic vedolizumab isotype species in the composition; thereby producing a low basic species composition comprising an anti-α4β7 antibody having 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 isotype species. In some embodiments, the 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.

[0077] Typically, the basic isotype species has a net positive charge relative to the main isotype of the anti-α4β7 antibody. In some embodiments, the level of the basic isotype species can be quantified using cation exchange chromatography (CEX). In some embodiments, the level of the basic isotype species can be quantified by determining the relative area of ​​the peak eluted from the cation exchange (CEX) resin more slowly than the peak corresponding to the main isotype. In some embodiments, the composition comprises a first basic isotype peak (BP1) and a second basic isotype 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 comprises less than 1% BP2. In some embodiments, the composition comprises less than 0.7% BP2.

[0078] 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. In some embodiments, the ratio of BP1 to BP2 is at least 10.

[0079] In some embodiments, the composition comprises less than 8% of the total basic isoform species of anti-α4β7 antibodies. In some embodiments, the composition comprises less than 7% of the total basic isoform species of anti-α4β7 antibodies. In some embodiments, the composition comprises less than 6% of the total basic isoform species of anti-α4β7 antibodies. In some embodiments, the composition comprises less than 5% of the total basic isoform species of anti-α4β7 antibodies.

[0080] In some embodiments, provided herein are compositions comprising an anti-α4β7 antibody obtainable by the aforementioned methods. In some embodiments, the antibody is vedolizumab or an antigen-binding portion thereof.

[0081] In some embodiments, provided herein are compositions comprising an anti-α4β7 antibody obtained by the aforementioned methods. In some embodiments, the antibody is vedolizumab or an antigen-binding portion thereof.

[0082] On the other hand, provided herein is a method for treating a disease or condition in a human subject, wherein the method comprises administering to the subject an amount of an anti-α4β7 antibody (e.g., vedolizumab) provided herein in an amount effective to treat a disease or condition in a human subject. In some embodiments, the pharmaceutical composition comprises a vedolizumab composition having a reduced level of basic vedolizumab isotype species and / or a reduced host cell protein level as provided herein and / or as produced according to the methods provided herein. In some embodiments, the anti-α4β7 antibody comprises a heavy chain variable region containing SEQ ID NO: 1 and a light chain variable region containing SEQ ID NO: 2.

[0083] In one embodiment, the disease or disorder is inflammatory bowel disease (IBD). In some embodiments, IBD is ulcerative colitis, Crohn's disease, ileitis, celiac disease, nontropical sprue, enteropathy associated with seronegative arthropathy, microscopic colitis or collagenous colitis, eosinophilic gastroenteritis, or pouchitis following proctocolectomy and ileoanal anastomosis. In some embodiments, inflammatory bowel disease is Crohn's disease or ulcerative colitis. Other diseases that can be treated include, for example, primary sclerosing cholangitis (PSC) and graft versus host disease (GVHD).

[0084] In addition, the present invention also includes the following embodiments:

[0085] 1. A method of producing a composition comprising vedolizumab having reduced levels of basic vedolizumab isotype species, the method comprising:

[0086] providing a composition comprising vedolizumab having a pH greater than pH 6.5; and

[0087] incubating the composition comprising vedolizumab for a period of greater than 10 hours;

[0088] Thereby generating a composition comprising vedolizumab having reduced levels of basic vedolizumab isotype species.

[0089] 2. The method of item 1, wherein the incubation is performed at ambient temperature.

[0090] 3. The method according to item 1, wherein the incubation is performed at 15°C-30°C.

[0091] 4. The method according to item 1, wherein the incubation is carried out at 20°C-25°C.

[0092] 5. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is provided at a pH of about 6.5-8.5.

[0093] 6. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is provided at a pH of about 7.0-8.0.

[0094] 7. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is provided at a pH of about 7.0-7.5.

[0095] 8. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is provided at a pH of about pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4 or pH 8.5.

[0096] 9. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 10 to 120 hours.

[0097] 10. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 120 hours.

[0098] 11. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 96 hours.

[0099] 12. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 72 hours.

[0100] 13. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 12 to 48 hours.

[0101] 14. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of at least 12 hours.

[0102] 15. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 120 hours.

[0103] 16. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 96 hours.

[0104] 17. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 72 hours.

[0105] 18. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 24 to 48 hours.

[0106] 19. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours or about 120 hours.

[0107] 20. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

[0108] 21. The method of item 20, wherein the mammalian cell culture is a Chinese Hamster Ovary (CHO) cell culture.

[0109] 22. The method of item 21, wherein the CHO cell culture comprises CHO cells lacking expression of dihydrofolate reductase (DHFR).

[0110] 23. The method of item 21, wherein the CHO cell culture comprises CHO cells lacking expression of glutamine synthetase (GS).

[0111] 24. The method of any of the preceding clauses, wherein the method further comprises purifying the composition comprising vedolizumab from mammalian host cell proteins (HCP) using one or more chromatographic separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

[0112] 25. The method of item 24, wherein the composition comprising vedolizumab is purified using an affinity chromatography resin comprising protein A.

[0113] 26. The method of item 25, wherein the composition comprising vedolizumab is purified using affinity chromatography prior to the incubation.

[0114] 27. The method of item 25, wherein the composition comprising vedolizumab is purified using affinity chromatography after the incubation.

[0115] 28. The method of item 24, wherein the composition comprising vedolizumab is purified using cation exchange chromatography.

[0116] 29. The method of item 28, wherein the composition comprising vedolizumab is purified using cation exchange chromatography prior to the incubation.

[0117] 30. The method of item 28, wherein the composition comprising vedolizumab is purified using cation exchange chromatography after the incubation.

[0118] 31. The method of item 24, wherein the composition comprising vedolizumab is purified using anion exchange chromatography.

[0119] 32. The method of item 31, wherein the composition comprising vedolizumab is purified using anion exchange chromatography prior to the incubation.

[0120] 33. The method of item 31, wherein after said incubation said composition comprising vedolizumab is purified using anion exchange chromatography.

[0121] 34. The method of item 24, wherein the composition comprising vedolizumab is purified using CHT chromatography.

[0122] 35. The method of item 34, wherein the composition comprising vedolizumab is purified using CHT chromatography prior to the incubation.

[0123] 36. The method of item 34, wherein after said incubation said composition comprising vedolizumab is purified using CHT chromatography.

[0124] 37. A composition comprising vedolizumab, wherein the composition is produced by the method of any one of items 1-36.

[0125] 38. The composition of item 37, wherein the basic vedolizumab isotype species constitute less than 10% of the vedolizumab species present in the composition.

[0126] 39. A method of producing a composition comprising vedolizumab having reduced amounts of host cell proteins (HCPs), the method comprising:

[0127] (a) contacting a sample containing vedolizumab and HCP with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a conductivity of 11 mS / cm or less, such that the HCP binds to the anion exchange resin; and

[0128] (b) collecting flow-through material from the anion exchange resin,

[0129] wherein the flow-through material comprises vedolizumab and a reduced amount of HCP.

[0130] 40. The method of item 39, wherein the loading buffer has a conductivity of 9 to 11 mS / cm.

[0131] 41. The method of item 39, wherein the loading buffer has a conductivity of 10 mS / cm or less.

[0132] 42. The method of item 39, wherein the loading buffer has a conductivity of 9 mS / cm or less.

[0133] 43. The method of item 39, wherein the loading buffer has a conductivity of about 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm or 11 mS / cm.

[0134] 44. The method of any one of items 39 to 42, wherein the HCP is Chinese Hamster Ovary (CHO) cell protein.

[0135] 45. The method of item 44, wherein the HCP is derived from CHO cells lacking expression of dihydrofolate reductase (DHFR).

[0136] 46. ​​The method of item 44, wherein the HCP is derived from CHO cells lacking glutamine synthetase (GS) expression.

[0137] 47. The method according to any of items 39 to 46, further comprising contacting the anion exchange resin with a wash buffer.

[0138] 48. The method of item 47, wherein the wash buffer has a conductivity of less than 11 mS / cm.

[0139] 49. The method of item 47, wherein the wash buffer has a conductivity of 9 to 11 mS / cm.

[0140] 50. The method of item 47, wherein the wash buffer has the same conductivity as the load buffer.

[0141] 51. The method according to any one of items 39-50, wherein the loading buffer comprises sodium chloride and / or sodium phosphate.

[0142] 52. The method according to any of items 39 to 50, wherein the wash buffer comprises sodium chloride and / or sodium phosphate.

[0143] 53. The method of any of items 39-52, wherein the anion exchange resin is formatted as an anion exchange column or an anion exchange membrane.

[0144] 54. The process according to any one of items 39 to 53, wherein the anion exchange resin comprises quaternary amine functional groups.

[0145] 55. The method according to any of items 39 to 54, wherein the sample containing vedolizumab and HCP is derived from a mammalian cell culture after one or more chromatographic separation steps.

[0146] 56. The method of item 55, wherein the one or more chromatographic separation steps comprise one or more steps selected from the group consisting of affinity chromatography, cation exchange chromatography and ceramic hydroxyapatite (CHT) chromatography.

[0147] 57. The method of any one of items 39 to 56, wherein the amount of HCP in the eluate is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

[0148] 58. The method of any one of items 39-57, wherein the amount of HCP in the flow-through material is reduced by at least 50% relative to the amount of HCP in the flow-through material produced when the method is performed using the same sample with a loading buffer having a conductivity greater than 12 mS / cm.

[0149] 59. The method of any one of items 39 to 58, wherein the method further comprises treating the flow-through material by a process comprising ultrafiltration and / or diafiltration to exchange the elution buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients.

[0150] 60. A composition comprising vedolizumab produced by the method of any one of items 39-59.

[0151] 61. The composition of item 60, wherein the amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

[0152] 62. A method for increasing the yield of vedolizumab recovered after elution from a mixed mode chromatography resin, the method comprising equilibrating the mixed mode chromatography resin with an equilibration buffer, loading a solution comprising vedolizumab and a loading buffer onto the mixed mode chromatography resin so that the vedolizumab binds to the mixed mode chromatography resin, washing the mixed mode chromatography resin with a wash buffer, and eluting the vedolizumab from the mixed mode chromatography resin with an elution buffer, wherein the equilibration buffer, the loading buffer and / or the wash buffer have a pH of 7.0 or below.

[0153] 63. The method of item 62, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a pH of 6.0-7.0.

[0154] 64. The method of item 62, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a pH of 6.5-7.0.

[0155] 65. The method of item 62, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a pH of 6.6-6.8.

[0156] 66. The method according to any one of items 62 to 65, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 30 mM to 70 mM.

[0157] 67. The method of item 66, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 40 mM to 70 mM.

[0158] 68. The method of item 66, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 50 mM to 65 mM.

[0159] 69. The method of item 66, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 55 mM-65 mM.

[0160] 70. The method according to any of items 66-69, wherein the salt comprises sodium chloride and / or sodium phosphate.

[0161] 71. The method according to any one of items 62 to 65, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 30 mM to 70 mM.

[0162] 72. The method of item 71, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 40 mM to 70 mM.

[0163] 73. The method of item 71, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 40 mM to 60 mM.

[0164] 74. The method of item 71, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 45 mM-55 mM.

[0165] 75. The method of item 71, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of about 50 mM.

[0166] 76. The method according to any one of items 71 to 75, wherein the equilibration buffer, the loading buffer and / or the wash buffer further comprises sodium phosphate.

[0167] 77. The method according to any one of items 62 to 76, wherein the equilibration buffer, the loading buffer and / or the wash buffer have the same pH.

[0168] 78. The method according to any one of items 62 to 77, wherein the equilibration buffer, the loading buffer and / or the wash buffer have the same salt concentration.

[0169] 79. The method according to any one of items 62 to 76, wherein the equilibration buffer, the loading buffer and / or the wash buffer are the same buffer.

[0170] 80. The method of any of items 62 to 79, wherein the mixed mode resin is a ceramic hydroxyapatite resin.

[0171] In addition, the present invention also includes the following embodiments:

[0172] 1. A method for producing a composition comprising vedolizumab, the method comprising:

[0173] providing a composition comprising vedolizumab having a pH greater than pH 6.5; and

[0174] incubating the composition comprising vedolizumab for a period of at least 20 minutes to 10 hours;

[0175] Thereby a composition comprising vedolizumab is produced.

[0176] 2. A method as described in item 1, wherein the method is a method for producing a composition comprising vedolizumab with reduced levels of basic vedolizumab isotype species, and wherein the method produces a composition comprising vedolizumab with reduced levels of basic vedolizumab isotype species.

[0177] 3. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having <16%, <15%, <14%, <13%, <12%, <11% or <10% basic vedolizumab isotype species.

[0178] 4. A method as described in any of items 1-3, wherein the incubation is performed during the purification of vedolizumab, and wherein the incubation is performed before (a) ultrafiltration / diafiltration (UF / DF) of the antibody or (b) before formulating the antibody in a pharmaceutically acceptable buffer.

[0179] 5. A method as described in any of items 1-4, wherein the incubation is carried out at ambient temperature.

[0180] 6. The method of any one of items 1 to 4, wherein the incubation is carried out at 15°C-30°C, or wherein the incubation is carried out at 20°C-25°C.

[0181] 7. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about 6.5-8.5.

[0182] 8. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about 7.0-8.0.

[0183] 9. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about 7.0-7.5.

[0184] 10. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4 or pH 8.5.

[0185] 11. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 10-120 hours.

[0186] 12. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 12-120 hours.

[0187] 13. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 12-96 hours.

[0188] 14. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 12-72 hours.

[0189] 15. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 12-48 hours.

[0190] 16. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of at least 12 hours.

[0191] 17. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 24-120 hours.

[0192] 18. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 24-96 hours.

[0193] 19. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 24-72 hours.

[0194] 20. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 24-48 hours.

[0195] 21. The method of any of the preceding items, wherein the composition comprising vedolizumab is incubated for a period of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours or about 120 hours.

[0196] 22. A method for purifying a humanized anti-α4β7 antibody or an antigen-binding portion thereof from a clarified cell culture harvest, the method comprising

[0197] (i) providing a clarified cell culture harvest obtained from a culture of a recombinant host cell expressing the anti-α4β7 antibody or antigen-binding portion thereof, and

[0198] (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 below for no longer than 24 hours,

[0199] The anti-α4β7 antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:5.

[0200] 23. A method as described in claim 22, wherein the anti-α4β7 antibody or its antigen-binding portion has reduced levels of basic isotype species (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding portion produced by the same method, wherein the antibody is exposed to a pH of 4.0 or a pH below 4.0 (e.g., pH 3.6-4.0) for a longer period of time, i.e., greater than 24 hours.

[0201] 24. The method of item 22 or 23, wherein the anti-α4β7 antibody or antigen binding portion thereof is vedolizumab or an antigen binding portion thereof.

[0202] 25. The method of any of the preceding items, wherein the composition comprising vedolizumab or an antigen-binding portion thereof comprises a first basic isotype peak (BP1) and a second basic isotype peak (BP2), and wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having a reduced level of BP2.

[0203] 26. The method of claim 25, wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having less than 2%, less than 1.5%, less than 1%, or less than 0.7% BP2.

[0204] 27. The method of any of the preceding items, wherein the composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

[0205] 28. The method of claim 27, wherein the mammalian cell culture is a Chinese Hamster Ovary (CHO) cell culture.

[0206] 29. A method as described in claim 28, wherein the CHO cell culture comprises CHO cells lacking expression of dihydrofolate reductase (DHFR).

[0207] 30. The method of claim 28, wherein the CHO cell culture comprises CHO cells lacking expression of glutamine synthetase (GS).

[0208] 31. The method of any of the preceding items, wherein the method further comprises purifying the composition comprising vedolizumab from mammalian host cell proteins (HCP) using one or more chromatographic separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

[0209] 32. The method of claim 31, wherein the composition comprising vedolizumab is purified using an affinity chromatography resin comprising protein A.

[0210] 33. A method as described in claim 32, wherein the composition comprising vedolizumab is purified using affinity chromatography prior to the incubation.

[0211] 34. The method of claim 32, wherein the composition comprising vedolizumab is purified using affinity chromatography after the incubation.

[0212] 35. The method of claim 31, wherein the composition comprising vedolizumab is purified using cation exchange chromatography.

[0213] 36. A method as described in claim 35, wherein the composition comprising vedolizumab is purified using cation exchange chromatography prior to the incubation.

[0214] 37. A method as described in claim 35, wherein the composition comprising vedolizumab is purified using cation exchange chromatography after the incubation.

[0215] 38. The method of claim 31, wherein the composition comprising vedolizumab is purified using anion exchange chromatography.

[0216] 39. The method of claim 38, wherein the composition comprising vedolizumab is purified using anion exchange chromatography prior to the incubation.

[0217] 40. The method of claim 38, wherein the composition comprising vedolizumab is purified using anion exchange chromatography after the incubation.

[0218] 41. The method of claim 31, wherein the composition comprising vedolizumab is purified using CHT chromatography.

[0219] 42. A method as described in claim 41, wherein the composition comprising vedolizumab is purified using CHT chromatography prior to the incubation.

[0220] 43. A method as described in claim 41, wherein the composition comprising vedolizumab is purified using CHT chromatography after the incubation.

[0221] 44. The method of any one of items 1-43, wherein the method comprises incorporating the composition into a pharmaceutical formulation.

[0222] 45. The method of claim 44, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

[0223] 46. ​​The method of claim 45, wherein the lyophilized pharmaceutical formulation is a dried lyophilized pharmaceutical formulation.

[0224] 47. The method of claim 46, further comprising the step of reconstituting the dried lyophilized pharmaceutical formulation with a liquid to render it suitable for administration.

[0225] 48. The method of claim 44, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

[0226] 49. A composition comprising vedolizumab, wherein the composition is produced by the method of any one of items 1-48.

[0227] 50. A composition comprising vedolizumab, wherein the composition is obtainable by the method of any one of items 1-48.

[0228] 51. A composition as described in claim 49 or 50, wherein the basic vedolizumab isoform species constitute less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the vedolizumab species present in the composition.

[0229] 52. A low basic species composition comprising an anti-α4β7 antibody, wherein the composition comprises 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 of the anti-α4β7 antibody, wherein the basic isoform species have a net positive charge relative to the major isoform of the anti-α4β7 antibody and can be quantified by determining the relative area of ​​a peak that elutes slower from a cation exchange (CEX) resin than the peak corresponding to the major isoform, and wherein the 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.

[0230] 53. The composition of claim 52, wherein the composition comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2).

[0231] 54. A composition as described in claim 53, wherein the composition contains less than 2% BP2.

[0232] 55. A composition as described in claim 53, wherein the composition contains less than 1.5% BP2.

[0233] 56. A composition as described in claim 53, wherein the composition contains less than 1% BP2.

[0234] 57. A composition as described in claim 53, wherein the composition contains less than 0.7% BP2.

[0235] 58. A composition as described in claim 53, wherein the ratio of BP1 to BP2 is at least 3.

[0236] 59. A composition as described in claim 53, wherein the ratio of BP1 to BP2 is at least 5.

[0237] 60. A composition as described in claim 53, wherein the ratio of BP1 to BP2 is at least 7.

[0238] 61. A composition as described in claim 53, wherein the ratio of BP1 to BP2 is at least 10.

[0239] 62. A pharmaceutical composition comprising the composition of any one of items 52-61 and a pharmaceutically acceptable carrier or excipient.

[0240] 63. A method of producing a composition comprising vedolizumab, the method comprising:

[0241] (a) contacting a sample containing vedolizumab and a host cell protein (HCP) with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a conductivity of 11 mS / cm or less, such that the HCP binds to the anion exchange resin; and

[0242] (b) collecting flow-through material from the anion exchange resin,

[0243] The eluate contains vedolizumab.

[0244] 64. A method as described in item 63, wherein the method is a method of producing a composition comprising vedolizumab with a reduced amount of HCP, and the flow-through material comprises vedolizumab and a reduced amount of HCP.

[0245] 65. A method as described in item 63 or 64, wherein the loading buffer has a conductivity of 9 mS / cm to 11 mS / cm.

[0246] 66. A method as described in item 63 or 64, wherein the loading buffer has a conductivity of 10 mS / cm or less.

[0247] 67. A method as described in item 63 or 64, wherein the loading buffer has a conductivity of 9 mS / cm or less.

[0248] 68. A method as described in item 63 or 64, wherein the loading buffer has a conductivity of about 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm or 11 mS / cm.

[0249] 69. A method as described in any of items 63-68, wherein the HCP is Chinese Hamster Ovary (CHO) cell protein.

[0250] 70. A method as described in claim 69, wherein the HCP is derived from CHO cells lacking expression of dihydrofolate reductase (DHFR).

[0251] 71. A method as described in claim 69, wherein the HCP is derived from CHO cells lacking glutamine synthetase (GS) expression.

[0252] 72. The method of any one of items 63-71, further comprising contacting the anion exchange resin with a wash buffer.

[0253] 73. A method as described in item 72, wherein the wash buffer has a conductivity of less than 11 mS / cm.

[0254] 74. A method as described in item 72, wherein the wash buffer has a conductivity of 9 mS / cm to 11 mS / cm.

[0255] 75. A method as described in item 72, wherein the wash buffer has the same conductivity as the loading buffer.

[0256] 76. A method as described in any of items 63-75, wherein the loading buffer comprises sodium chloride and / or sodium phosphate.

[0257] 77. A method as described in any of items 63-75, wherein the wash buffer comprises sodium chloride and / or sodium phosphate.

[0258] 78. A method as described in any of items 63-77, wherein the anion exchange resin is formatted as an anion exchange column or an anion exchange membrane.

[0259] 79. A method as described in any of items 63-78, wherein the anion exchange resin comprises a quaternary amine functional group.

[0260] 80. The method of any one of items 63-79, wherein the sample containing vedolizumab and HCP is derived from a mammalian cell culture after one or more chromatographic separation steps.

[0261] 81. A method as described in claim 80, wherein the one or more chromatographic separation steps include one or more steps selected from the group consisting of affinity chromatography, cation exchange chromatography and ceramic hydroxyapatite (CHT) chromatography.

[0262] 82. A method as described in any of items 63-81, wherein the amount of HCP in the flow-through material is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

[0263] 83. A method as described in any of items 63-82, wherein the amount of HCP in the flow-through material is reduced by at least 50% relative to the amount of HCP in the flow-through material produced when the method is performed using the same sample with a loading buffer having a conductivity greater than 12 mS / cm.

[0264] 84. A method as described in any of items 63-83, wherein the method further comprises treating the flow-through material by a process comprising ultrafiltration and / or diafiltration to exchange the elution buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients.

[0265] 85. A method as described in any of items 63-84, wherein the method comprises incorporating the composition into a pharmaceutical formulation.

[0266] 86. A method as described in claim 85, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

[0267] 87. The method of claim 86, wherein the lyophilized pharmaceutical formulation is a dried lyophilized pharmaceutical formulation.

[0268] 88. The method of claim 87, further comprising the step of reconstituting the dried lyophilized drug formulation with a liquid to render it suitable for administration.

[0269] 89. The method of claim 85, wherein the drug formulation is a liquid drug formulation.

[0270] 90. The method of item 89 or 48, wherein the liquid drug formulation is suitable for subcutaneous administration to a human.

[0271] 91. A composition comprising vedolizumab produced by the method of any one of items 63-90.

[0272] 92. A composition comprising vedolizumab, wherein the composition is obtainable by the method of any one of items 63-90.

[0273] 93. A composition as described in item 91 or 92, wherein the amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

[0274] 94. A method for increasing the yield of vedolizumab recovered after elution from a mixed mode chromatography resin, the method comprising equilibrating the mixed mode chromatography resin with an equilibration buffer, loading a solution comprising vedolizumab and a loading buffer onto the mixed mode chromatography resin so that the vedolizumab binds to the mixed mode chromatography resin, washing the mixed mode chromatography resin with a wash buffer, and eluting the vedolizumab from the mixed mode chromatography resin with an elution buffer, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a pH of 7.0 or below.

[0275] 95. A method as described in item 94, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a pH of 6.0-7.0.

[0276] 96. A method as described in item 94, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a pH of 6.5-7.0.

[0277] 97. A method as described in item 94, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a pH of 6.6-6.8.

[0278] 98. A method as described in any of items 94-97, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a salt concentration of 30 mM to 70 mM.

[0279] 99. A method as described in item 98, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a salt concentration of 40 mM to 70 mM.

[0280] 100. A method as described in item 98, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a salt concentration of 50 mM to 65 mM.

[0281] 101. A method as described in item 98, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a salt concentration of 55mM-65mM.

[0282] 102. A method as described in any of items 98-101, wherein the salt comprises sodium chloride and / or sodium phosphate.

[0283] 103. A method as described in any of items 94-97, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a sodium chloride concentration of 30 mM to 70 mM.

[0284] 104. A method as described in item 103, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a sodium chloride concentration of 40 mM to 70 mM.

[0285] 105. A method as described in item 103, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a sodium chloride concentration of 40 mM to 60 mM.

[0286] 106. A method as described in item 103, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a sodium chloride concentration of 45mM-55mM.

[0287] 107. A method as described in item 103, wherein the equilibration buffer, the loading buffer and / or the washing buffer have a sodium chloride concentration of approximately 50 mM.

[0288] 108. A method as described in any of items 103-107, wherein the equilibration buffer, the loading buffer and / or the washing buffer further comprises sodium phosphate.

[0289] 109. A method as described in any of items 94-108, wherein the equilibration buffer, the loading buffer and / or the washing buffer have the same pH.

[0290] 110. A method as described in any of items 94-109, wherein the equilibration buffer, the loading buffer and / or the washing buffer have the same salt concentration.

[0291] 111. A method as described in any of items 94-108, wherein the equilibration buffer, the loading buffer and / or the washing buffer are the same buffer.

[0292] 112. A method as described in any of items 94-111, wherein the mixed mode resin is a ceramic hydroxyapatite resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0293] Figure 1A cation exchange (CEX)-high performance liquid chromatography (HPLC) profile of vedolizumab is depicted, with the indicated peaks representing acidic species, basic species, and major isoforms of vedolizumab.

[0294] Figure 2 Depicted are the elution profiles of vedolizumab purified using standard ceramic hydroxyapatite (CHT) equilibration and wash buffers after loading 27 mg / ml protein or 35 mg / ml protein on a CHT column.

[0295] Figure 3 Depicted are the elution profiles of vedolizumab purified using standard CHT equilibration and wash buffers or reduced pH buffers after loading 38 mg / ml protein on a CHT column. DETAILED DESCRIPTION

[0296] Provided herein are methods for purifying anti-α4β7 integrin antibodies such as vedolizumab from liquid solutions, e.g., clarified harvests from mammalian cell cultures. The present invention relates in particular to purification methods for controlling the amount of product-related substances (e.g., basic and / or acidic isoform species) and / or process-related impurities (e.g., host cell proteins (HCPs), host cell nucleic acids, viruses, chromatographic materials, and / or culture medium components) present in purified preparations of anti-α4β7 integrin antibodies or antigen-binding fragments thereof (e.g., vedolizumab). Vedolizumab is a relatively hydrophobic antibody, which presents a challenge to purification, particularly when the antibody is produced in large quantities at the purity levels required for therapeutic use.

[0297] I. Definitions

[0298] In order to more readily understand the present invention, certain terms are first defined.

[0299] The cell surface molecule "α4β7 integrin" or "α4β7" (used interchangeably throughout) is a heterodimer of the α4 chain (CD49D, ITGA4) and the β7 chain (ITGB7). Human α4-integrin and β7-integrin genes (GenBank (National Center for Biotechnology Information, Bethesda, Md.) RefSeq accession numbers NM_000885 and NM_000889, respectively) are expressed by B and T lymphocytes, particularly memory CD4+ lymphocytes. As a typical feature of many integrins, α4β7 can exist in a resting or activated state. Ligands for α4β7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM (e.g., MAdCAM-1)). Antibodies that bind to α4β7 integrin are referred to herein as "anti-α4β7 antibodies".

[0300] As used herein, an antibody or antigen-binding fragment thereof having "binding specificity to the α4β7 complex" binds to α4β7 but does not bind to α4β1 or α4β2. E B7. Vedolizumab is an example of an antibody having binding specificity for the α4β7 complex.

[0301] The term "about" means that the following value is not an exact value but the center point of a range of + / -5% of the value. If the value is a relative value given as a percentage, the term "about" also means that the following value is not an exact value but the center point of a range of + / -5% of the value, whereby the upper limit of the range cannot exceed the value 100%.

[0302] As used herein, the term one or more "aggregates" refers to the association of two or more antibodies or antibody fragments. For example, the aggregate can be a dimer, trimer, tetramer or a multimer greater than a tetramer of an antibody and / or antibody fragment. Antibody aggregates can be soluble or insoluble. The association between the aggregated molecules can be covalent or non-covalent, regardless of the mechanism by which they are associated. The association can be a direct association between the aggregated molecules, or an indirect association by other molecules that connect them together. Examples of the latter include, but are not limited to, disulfide bonding with other proteins, hydrophobic association with lipids, charge association with DNA, affinity association with leached protein A, or mixed association with a variety of components. Aggregates may be irreversibly formed during protein expression in cell culture, during protein purification in downstream processing, or during storage. The presence of aggregates in solution can be determined using, for example, size exclusion chromatography (SEC) (e.g., SEC with UV detection, SEC with light scattering detection (SEC-LSD)), field flow fractionation, analytical ultracentrifugation sedimentation velocity, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced).

[0303] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions with high variability, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody has a fragment crystallizable (Fc) region. In certain embodiments, the antibody is of the IgG1 isotype and has a kappa light chain.

[0304] As used herein, the term "basic species" or "basic isotype species" refers to a variant of an antibody, or an antigen-binding portion thereof (e.g., vedolizumab), characterized by an overall basic charge. The basic species of an antibody, or an antigen-binding portion thereof, can be detected by various methods known in the art, such as cation exchange-high performance liquid chromatography (CEX-HPLC), CEX-mass spectrometry, or isoelectric focusing. Basic species of an antibody may include, but are not limited to, charge variants, structural variants, and / or fragment variants. In some embodiments, a composition comprising an antibody, or an antigen-binding portion thereof, may comprise more than one type of basic isotype species. In some embodiments, multiple basic isotype species can be identified based on differences in retention times during CEX-HPLC separation. For example, when a composition comprising an antibody (e.g., vedolizumab) is analyzed using CEX-HPLC, one or more basic isotype peaks can be identified, each peak representing one or more basic isotype species of the antibody, such as Figure 1 For example, in some embodiments, a basic isoform species is an isoform of the antibody in which an aspartic acid residue has undergone isomerization to a succinimide. Host cell impurities or other impurities not associated with the antibody, or antigen-binding portion thereof, are not considered "basic species" or "basic isoform species" of the antibody, or antigen-binding portion thereof, by primary sequence.

[0305] As used herein, the term "buffer" refers to an aqueous solution that resists pH changes through the action of its acid-base conjugate components. Buffers are used to establish a specific set of conditions, such as biochemical conditions, to mediate control of a processing step or a chromatographic support such as a chromatographic resin or membrane.

[0306] "CDRs" or "complementarity determining regions" are regions of hypervariability interspersed with more conserved regions called "framework regions" (FRs).

[0307] As used herein, the term "antigen binding fragment" or "antigen binding portion" of an antibody refers to Fab, Fab', F(ab')2 and Fv fragments, single-chain antibodies, functional heavy chain antibodies (nanoantibodies), and any portion of an antibody that is specific for at least one desired epitope that competes with an intact antibody for specific binding (e.g., an isolated portion of a complementary determining region with sufficient framework sequence to specifically bind to an epitope). Antigen binding fragments can be produced by recombinant techniques, or by enzymatic or chemical cleavage of antibodies.

[0308] As used herein, "chromatographic support" refers to a solid or porous matrix having a specific chemical composition or a specific three-dimensional structure or in which specific chemical groups or macromolecules can be fixed to perform chromatography, including affinity chromatography, gel filtration (size exclusion chromatography) or ion exchange chromatography. Examples of chromatographic supports include, but are not limited to, resins (e.g., agarose) or membranes. As used herein, "chromatographic housing" refers to a structure containing a chromatographic support. Examples of chromatographic housings include columns or cartridges, or other containers.

[0309] As used herein, the term "clarified harvest" refers to a liquid material containing a protein of interest (e.g., an anti-α4β7 antibody) extracted from a cell culture (e.g., a fermentation bioreactor) after undergoing one or more process steps to remove solid particles (e.g., cell debris and particulate impurities) from the material. After cell culture, the harvest is typically purified using separation techniques such as centrifugation and filtration to remove cells and cell debris. The initial clarification, particulate removal step produces a "clarified harvest" that can be used, for example, in subsequent chromatography steps (downstream processing). The clarified harvest is typically the starting material for downstream processing, such as the downstream processing steps described herein.

[0310] As used herein, the terms "culture" and "cell culture" generally refer to the (upstream) process in which cells grow under controlled conditions, typically outside of their natural environment. "Culturing" cells refers to contacting cells with a cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation. In certain embodiments, cell culture refers to methods for producing and maintaining a host cell population capable of producing a recombinant protein of interest (e.g., anti-α4β7 antibody), as well as methods and techniques for producing and collecting proteins of interest. For example, once an expression vector is incorporated into a suitable host (e.g., a host cell in culture), the host can be maintained under conditions suitable for expressing the relevant nucleotide coding sequence and collecting and purifying the desired recombinant protein. "Cell culture" may also refer to a solution containing cells.

[0311] As used herein, the term "downstream process" refers to one or more techniques used to purify a protein of interest (e.g., an antibody) following an upstream process. For example, downstream process techniques include purification of a protein product using, for example, affinity chromatography (including protein A affinity chromatography), ion exchange chromatography (such as anion or cation exchange chromatography), size exclusion chromatography, mixed mode chromatography, hydrophobic interaction chromatography (HIC), or displacement chromatography.

[0312] As used herein, the term "elution solution" or "eluent" refers to an aqueous liquid formulated to displace a protein of interest, e.g., an antibody, from a chromatography support (e.g., a resin or membrane). In one embodiment, the elution solution has different biochemical properties than the equilibration and / or wash solutions such that the protein of interest (e.g., an antibody) preferentially associates with the elution solution rather than the chromatography support (e.g., a resin or membrane).

[0313] As used herein, the term "equilibrium solution" refers to an aqueous liquid formulated to establish initial operating conditions for a process step or chromatographic support (e.g., a chromatographic operation). The equilibrium solution is used to prepare, for example, a solid phase, such as a chromatographic support (e.g., a resin or membrane), which is used to load a protein of interest (e.g., an antibody).

[0314] As used herein, "flow-through" for example in relation to a chromatography step, refers to the process by which proteins elute while impurities bind and remain associated with the chromatography support during loading and washing.

[0315] The term "high molecular weight" or "HMW" is used to refer to an antibody complex having a molecular weight greater than a monomeric antibody. In one embodiment, the HMW aggregate has a molecular weight greater than about 147 kDa. The presence of high molecular weight aggregates can be determined by standard methods known in the art, such as size exclusion chromatography (SEC).

[0316] The non-human (e.g., rodent) antibody of "humanized" form is a chimeric antibody containing the minimum sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which the residues from the hypervariable region of the receptor are replaced by the residues from the hypervariable region with the desired specificity, affinity and ability of a non-human species (donor antibody), such as a mouse, rat, rabbit or non-human primate. In some cases, the framework region (FR) residues of a human antibody are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in a residue not seen in a receptor antibody or in a donor antibody. These modifications are made so that the antibody performance is further improved. Generally, a humanized antibody will include substantially all at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable CDR loops of a non-human antibody, and all or substantially all of the FRs are FRs of a human antibody sequence. A humanized antibody will optionally also include at least a portion of an antibody constant region (Fc), typically at least a portion of a constant region of a human antibody. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0317] As used herein, the term "impurity" with respect to impurities contained in a solution containing an antibody to be purified includes both process-related impurities and product-related impurities. As used herein, the term "process-related impurities" refers to an impurity (or impurities) present in a composition (e.g., solution) containing a protein but not derived from the protein itself. For example, process-related impurities include, but are not limited to, cell culture medium components, host cell components (such as proteins (HCPs), host cell nucleic acids, or lipid-containing subcellular structures or fragments thereof), viruses, trace metals or ions from buffers, and leachable materials from material processing containers or chromatographic supports. Process-related impurities (upstream and / or downstream processing) of proteins (e.g., antibodies) may be formed during preparation. As used herein, the term "host cell impurities" refers to any protein, nucleic acid contaminant, lipid contaminant, or by-product introduced by a host cell line, cell culture fluid, or cell culture. The term "host cell protein" refers to a protein by-product introduced by a host cell line, cell culture fluid, or cell culture. Examples of impurities include, but are not limited to, Chinese hamster ovary protein (CHOP), E. coli protein, yeast protein, simian COS protein, or myeloma cell protein (e.g., NS0 protein (mouse plasmacytoma cells derived from BALB / c mice)). Host cell proteins do not include proteins of interest produced in a host cell expression system. For example, when CHO cells are used to produce recombinant antibodies or fragments thereof, the term "host cell protein" encompasses proteins derived from CHO cells, rather than recombinant antibodies or fragments thereof. As used herein, the term "product-related impurities" includes impurities derived from the protein of interest (e.g., antibody) itself. For example, product-related impurities include, but are not limited to, aggregates, misfolded species, oxidized or deamidated species, or low molecular weight fragments of the antibody of interest.

[0318] As used herein, the term "recombinant antibody" refers to an antibody produced as a result of transcription and translation of one or more genes carried on one or more recombinant expression vectors that have been introduced into a host cell (e.g., a mammalian cell). In certain embodiments, the recombinant protein is an antibody belonging to an isotype selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD or IgE. In certain embodiments, the recombinant antibody is IgG1.

[0319] The term "recombinant host cell" (used interchangeably with the term "host cell" herein) includes cells into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to a particular subject cell, but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may in fact be different from the parent cell, but are still included within the scope of the term "host cell" as used herein. In addition, it should be understood that, unless otherwise indicated, when the term "cell" is used, such as a host cell or a mammalian cell or a mammalian host cell, a cell population is intended to be included.

[0320] "Substantially purified" with respect to a desired protein means that a purified sample comprising the protein contains 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 97.5%, at least 98%, at least 98.5%, or at least 99% of the desired recombinant protein with less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5% impurities.

[0321] As used herein, in the context of protein (e.g., antibody) production, the term "upstream processes" refers to activities involved in producing and collecting proteins (e.g., antibodies) from host cells (e.g., when cells are cultured to produce the protein (e.g., antibody) of interest).

[0322] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0323] As used herein, the term "washing liquid" or "washing solution" refers to an aqueous liquid formulated to displace unbound contaminants from a chromatographic support (such as a resin or membrane). In some embodiments, the washing liquid is passed over a solid support (e.g., a resin or membrane) after loading a protein (e.g., an antibody of interest) and before eluting the protein of interest (e.g., an antibody). In one embodiment, the washing liquid has biochemical properties similar to those of an equilibrium solution.

[0324] II. Anti-α4β7 Integrin Antibodies

[0325] The methods disclosed herein can be used to produce large quantities of highly purified compositions comprising anti-α4β7 integrin antibodies. It will be apparent that any of the methods for producing anti-α4β7 integrin antibodies described herein can be used alone or in combination. In an exemplary embodiment, the antibody is vedolizumab, or an antibody having one or more antigen binding regions of vedolizumab. Vedolizumab is also referred to by its trade name (Takeda Pharmaceuticals, Inc.). Vedolizumab is a humanized antibody comprising a human IgG1 framework and constant regions and antigen-binding CDRs from the murine antibody Act-1. Vedolizumab CDRs, variable regions, and mutant Fc regions (mutated to eliminate Fc effector functions) are described in U.S. Pat. No. 7,147,851, which is incorporated herein by reference in its entirety.

[0326] Vedolizumab is a humanized monoclonal antibody that specifically binds to the α4β7 integrin (e.g., the α4β7 complex). Vedolizumab blocks the interaction of the α4β7 integrin with the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and inhibits the migration of memory T lymphocytes across the endothelium into the inflamed gastrointestinal parenchymal tissue. Vedolizumab does not bind to or inhibit the function of integrins α4β1 or αEβ7, nor does it antagonize the interaction of the α4 integrin with the vascular cell adhesion molecule-1 (VCAM-1).

[0327] The α4β7 integrin is expressed on the surface of a discrete subset of memory T lymphocytes that preferentially migrate into the gastrointestinal tract. MAdCAM-1 is primarily expressed on intestinal endothelial cells and plays a key role in the homing of T lymphocytes to intestinal lymphoid tissues. The interaction of the α4β7 integrin with MAdCAM-1 is believed to be an important contributor to mucosal inflammation, such as the chronic inflammation that is a hallmark of ulcerative colitis and Crohn's disease. Vedolizumab can be used to treat inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, pouchitis (including chronic pouchitis), graft-versus-host disease, and HIV.

[0328] The heavy chain variable region of vedolizumab is provided herein as SEQ ID NO: 1, and the light chain variable region of vedolizumab is provided herein as SEQ ID NO: 5. Vedolizumab comprises a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4. Vedolizumab comprises a light chain variable region comprising CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 8. In one embodiment, the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 9 and a light chain comprising an amino acid sequence of SEQ ID NO: 10. The sequences of vedolizumab and vedolizumab are also described in U.S. Patent Publication No. 2014 / 0341885 and U.S. Patent Publication No. 2014-0377251, each of which is expressly incorporated herein by reference in its entirety. The methods disclosed herein can be performed using antibodies comprising the binding regions (e.g., CDRs) or variable regions set forth above.

[0329] The method of the present invention can be used to produce anti-α4β7 antibodies, in particular vedolizumab or antibodies having the binding region (ie, CDR) or variable region of vedolizumab in mammalian cells.

[0330] Exemplary strategies for antibody generation

[0331] In certain embodiments, the methods described herein can be combined with one or more additional steps to facilitate the production and / or purification of vedolizumab, including one or more steps described below. In order to produce recombinant proteins such as vedolizumab for a long time and at high yield, mammalian host cells can be engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab). Exemplary cell culture processes and considerations for producing monoclonal antibodies such as vedolizumab are described below: Li et al. (2010) mAbs 2:5,466-477 and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685, the entire contents of which are incorporated herein by reference.

[0332] In certain embodiments, the primary recovery can be performed by sequentially employing the steps of reducing pH, centrifugation, and filtration to remove cells and cell debris (including HCP) from the production bioreactor harvest. In certain embodiments, the invention relates to subjecting the sample mixture from the primary recovery to one or more of affinity chromatography, anion exchange (AEX), cation exchange (CEX), hydrophobic interaction chromatography (HIC), ceramic hydroxyapatite chromatography (CHT), and / or mixed mode (MM) purification steps. In some embodiments, the order of steps can affect the resulting quality of the antibody composition by adjusting the level of aggregates, impurities, or isotypes.

[0333] In an exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), mixed mode, cation exchange, anion exchange.

[0334] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), mixed mode, anion exchange, cation exchange.

[0335] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), cation exchange, mixed mode, anion exchange.

[0336] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), anion exchange, mixed mode, cation exchange.

[0337] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: mixed mode, affinity chromatography (eg, protein A), anion exchange, cation exchange.

[0338] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), CHT chromatography, cation exchange, anion exchange.

[0339] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), CHT chromatography, anion exchange, cation exchange.

[0340] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), cation exchange, CHT chromatography, anion exchange.

[0341] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), hydrophobic interaction chromatography, cation exchange, anion exchange.

[0342] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), hydrophobic interaction chromatography, anion exchange, cation exchange.

[0343] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), anion exchange, hydrophobic interaction chromatography, cation exchange.

[0344] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), cation exchange, hydrophobic interaction chromatography, anion exchange.

[0345] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), cation exchange, anion exchange, hydrophobic interaction chromatography.

[0346] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: affinity chromatography (eg, protein A), anion exchange, cation exchange, hydrophobic interaction chromatography.

[0347] In another exemplary embodiment, a composition comprising vedolizumab can be purified using a process comprising the following steps in the following order: hydrophobic interaction chromatography, affinity chromatography (eg, protein A), anion exchange, cation exchange.

[0348] It should be understood that the purification steps do not have to be immediately adjacent to each other; various other process steps (such as filtration or viral reduction steps) can be inserted between the chromatography steps without interfering with the effect of the order of the chromatography steps on the charge profile.

[0349] To adjust the level of basic isoform species present in the vedolizumab composition, an incubation step may be incorporated between any of the foregoing purification steps as described herein. Additionally or alternatively, the purification process may be adapted to minimize the duration of exposure of the antibody to low pH conditions, as described herein.

[0350] To adjust the levels of host cell proteins present in the vedolizumab composition, AEX can be performed at any stage of the vedolizumab purification process employing anion exchange chromatography using a low conductivity buffer as described herein.

[0351] To adjust the yield of vedolizumab in a purification process comprising CHT, the CHT conditions described herein may be used at any stage of the vedolizumab purification process employing ceramic hydroxyapatite chromatography.

[0352] Certain embodiments of the invention will include further purification steps. Examples of additional purification procedures that may be performed before, during, or after ion exchange chromatography include ethanol precipitation, isoelectric focusing, reverse phase HPLC, silica gel chromatography, heparin chromatography Sepharose, TM , further anion exchange chromatography and / or further cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography (for example, using protein G, antibodies, specific substrates, ligands or antigens as capture agents).

[0353] In certain embodiments, the unbound flow-through and wash fractions may be further fractionated, and combinations of fractions that provide the desired product purity may be combined.

[0354] In certain embodiments, protein concentration can be adjusted to achieve differential partitioning behavior between antibody product and product-related substances so that purity and / or yield can be further improved. In certain embodiments, loading can be performed with different protein concentrations during the loading operation to improve the product quality / yield of any particular purification step.

[0355] In certain embodiments, column temperatures can be independently varied to improve the separation efficiency and / or yield of any particular purification step.

[0356] In certain embodiments, the loading and washing solution matrices may be different or consist of a mixture of chemicals while achieving similar "resin interaction" behavior, such that the novel separations described above may be achieved. For example, but not by way of limitation, the loading and washing solutions may differ in ionic strength or pH while maintaining substantially similar functionality in terms of product rinsing achieved during the washing steps. In certain embodiments, additives such as amino acids, sugars, PEG, etc. may be added to the loading or washing steps to adjust the partitioning behavior, thereby achieving separation efficiency and / or yield.

[0357] In certain embodiments, the loading and washing steps can be controlled by measuring the level of product-related impurities / substances in the column effluent or the collection pool or both in-line, online or off-line to achieve the target product quality and / or yield. In certain embodiments, the loading concentration can be dynamically controlled by diluting in-line or in batches or continuously with a solution or other solution to achieve the distribution required for improved separation efficiency and / or yield.

[0358] Certain embodiments of the present invention employ ultrafiltration and diafiltration steps to further concentrate and formulate the protein of interest, such as an antibody product. Ultrafiltration is described in detail in the following: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996); and Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9). One filtration process is tangential flow filtration, as described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalogue" pages 177-202 (Bedford, Mass., 1995 / 96). Ultrafiltration is generally considered to mean filtration using a filter with a pore size of less than 0.1 μm. By employing a filter with such a small pore size, the volume of the sample can be reduced by allowing the sample solution to permeate through the filter membrane pores while retaining the protein (such as an antibody) above the membrane surface.

[0359] Diafiltration is a method of using a membrane filter to remove and exchange salts, sugars, and non-aqueous solvents, separating them from bound species, to remove low molecular weight species and / or to cause rapid changes in ions and / or pH environments. Microsolutes can be most effectively removed by adding solvent to the diafiltered solution at a rate approximately equal to the osmotic flow rate. This washes away micro-species from the solution at a constant volume, effectively purifying the retained protein of interest. In certain embodiments of the invention, a diafiltration step is optionally used to exchange various solutions used in conjunction with the present invention, as well as to remove impurities from the protein preparation, prior to further chromatography or other purification steps.

[0360] Ultrafiltration / diafiltration (UF / DF) of vedolizumab can be performed in a selected device and membrane, such as polyethersulfone or regenerated cellulose, e.g. or Membrane, in In one embodiment, UF is performed using a cellulose membrane cast on a microporous polyethylene membrane with a molecular weight cutoff of 30 kDa.

[0361] III. Preparation of Vedolizumab Compositions Containing Altered Levels of Basic Isotype Species

[0362] In addition to the major or main isoform of vedolizumab, preparations of vedolizumab from mammalian host cells typically also contain small amounts of acidic and / or basic isoforms. Acidic and basic isoforms can be quantified by methods known in the art, including, for example, cation exchange-high pressure liquid chromatography (CEX-HPLC). Acidic and basic vedolizumab isoforms can be separated from the major isoforms based on the retention time differences on the CEX resin. Typically, as Figure 1 As depicted, the acidic vedolizumab isotype species present in the vedolizumab liquid formulation have a shorter retention time relative to the main isotype, while the basic vedolizumab isotype species have a longer retention time relative to the main isotype. The vedolizumab formulation may contain more than one acidic species and / or more than one basic species, whose charge varies slightly, and therefore elutes from the CEX resin at different retention times. Multiple basic peaks are mentioned herein with respect to their retention times on the CEX resin, whereby the first basic isotype peak eluted from the CEX resin after the main isotype peak is referred to herein as "basic peak 1", the second basic isotype peak eluted from the CEX resin after the main isotype peak is referred to herein as "basic peak 2", the third basic isotype peak eluted from the CEX resin after the main isotype peak is referred to herein as "basic peak 3", and so on.

[0363] In pharmaceutical antibody preparations, it may be necessary to maximize the percentage of antibodies present as the main isotype while minimizing the percentage of basic and / or acidic isotypes. On the one hand, the invention provides a method for adjusting the percentage of basic vedolizumab isotypes in a composition comprising vedolizumab. The method is based on the surprising discovery that the distribution of vedolizumab isotypes can be adjusted by changes in pH. The basic isotypes of vedolizumab are particularly sensitive to pH fluctuations. As described herein, this pH-dependent regulation of isotype distribution is at least partially driven by the fluctuations in the level of the basic peak 2 and the accompanying fluctuations in the level of the main isotype of vedolizumab.

[0364] It is not intended to be bound by theory, and at least in part based on the findings provided herein, it is believed that there are at least two basic isotype species in some vedolizumab preparations. The first species, eluted from CEX resin as "basic peak 1", is due to the presence of lysine residues at the carboxyl end of the IgG heavy chain. The second species, eluted from CEX resin as "basic peak 2", is due to the isomerization of one or more aspartic acid residues in the antibody to form a succinimide intermediate. In certain embodiments, one or more aspartic acid residues have undergone isomerization to form a succinimide intermediate. Glycine or serine residues (adjacent and near an amino acid at the carboxyl end) adjacent to aspartic acid at "n+1 position" can be conducive to the isomerization of aspartic acid residues to succinimide. In some embodiments, the identification of vedolizumab at SEQ ID NO:1 residue 102 comprising succinimide instead of aspartic acid allows to reduce, minimize or remove this basic isotype variant in vedolizumab preparations. In some embodiments, the basic isoform variant can be removed by a method including, for example, fractionating a preparation containing vedolizumab (e.g., on a CEX resin) and removing (or failing to collect) those fractions containing succinimide instead of aspartic acid at residue 102 of SEQ ID NO: 1, which can also be identified as the fraction of vedolizumab eluted from the CEX resin as "basic peak 2". In some embodiments, during the production and purification of vedolizumab, the level of the basic isoform variant (referred to herein as "succinimide variant" or alternatively as "basic isoform peak 2" or "BP2" variant) can be minimized by controlling the pH exposure of the antibody. In some embodiments, a composition comprising a reduced level of such a basic isoform variant may include a corresponding increase in the relative proportion of the major isoform of vedolizumab. Therefore, in some embodiments, a composition comprising a reduced level of such a basic isoform variant may have an increased efficacy relative to a composition comprising an increased level of such a basic isoform variant.

[0365] In one embodiment, provided herein is a method of purifying vedolizumab having reduced levels of basic isoform species, wherein the vedolizumab is maintained at pH 5.5 or above, pH 5.6 or above, pH 5.7 or above, pH 5.8 or above, pH 5.9 or above, pH 6.0 or above, pH 6.1 or above, pH 6.2 or above, pH 6.3 or above, pH 6.4 or above, or pH 6.5 or above. 6.5, continuing for at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the total time between initial recovery of vedolizumab from the cell culture harvest by ultrafiltration / diafiltration (UF / DF) and formulation of the vedolizumab in a pharmaceutically acceptable carrier. By maintaining vedolizumab at or above pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5 during the majority of the purification process, the vedolizumab can be purified by evaporation at a pH lower than pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5, relative to a purification process in which vedolizumab is maintained at a pH lower than pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5. The basic vedolizumab isoform species are reduced at a pH of 5.5-6.5 for a substantial period of time, for example, greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, or greater than 40% of the total time between initial recovery of vedolizumab from a cell culture harvest by ultrafiltration / diafiltration (UF / DF) and formulation of vedolizumab in a pharmaceutically acceptable carrier. In some embodiments, the method is performed at a commercial manufacturing scale, for example, using a vedolizumab formulation derived from a cell culture harvest produced at a 1000 L scale, a 2000 L scale, a 3000 L scale, a 4000 L scale, or a 5000 L scale (e.g., at least a 3000 L scale).

[0366] Thus, in one aspect, the invention provides a method for producing a low basic species composition of an anti-α4β7 antibody, or an antigen-binding portion thereof, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing the anti-α4β7 antibody, or an antigen-binding portion thereof, and (ii) purifying the anti-α4β7 antibody, or the antigen-binding portion thereof, from the cell culture harvest, wherein the antibody is exposed to a pH of 3.5 or below (e.g., pH 2.5-3.5, a pH of below 3.0, or a pH of below 3.5) for no more than 20 minutes, no more than 30 minutes, no more than 45 minutes, no more than 1 hour, no more than 3 hours, no more than 5 hours, no more than 7 hours, no more than 10 hours, or no more than 12 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising the anti-α4β7 antibody, or the antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 3.5 or below (e.g., pH 2.5-3.5, a pH of below 3.0, or a pH of below 3.5). 2.5-3.5, pH below 3.0 or pH below 3.5) for a longer time, i.e., greater than 20 minutes, greater than 30 minutes, greater than 45 minutes, greater than 1 hour, greater than 3 hours, greater than 5 hours, greater than 7 hours, greater than 10 hours or greater than 12 hours. In some embodiments, the low alkaline species composition comprises a lower BP2 level relative to the control. In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding portion comprises one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography and a combination thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding portion is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell. In some embodiments, the low basic species composition comprises 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 isoform species. In some embodiments, the low basic species composition comprises less than 4%, less than 3%, less than 2%, or less than 1% basic isoform peak 2.

[0367] In another aspect, the present invention provides a method for producing a low basic species composition of an anti-α4β7 antibody, or an antigen-binding portion thereof, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing the anti-α4β7 antibody, or an antigen-binding portion thereof, and (ii) purifying the anti-α4β7 antibody, or the antigen-binding portion thereof, from the cell culture harvest, wherein the antibody is exposed to a pH of 4.0 or below (e.g., pH 3.6 to 4.0) for no more than 20 minutes, no more than 30 minutes, no more than 45 minutes, no more than 1 hour, no more than 3 hours, no more than 5 hours, no more than 10 hours, no more than 12 hours, no more than 15 hours, no more than 18 hours, or no more than 24 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or the antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.0 or below (e.g., pH 3.6 to 4.0). 3.6 to 4.0) for a longer time, i.e., greater than 20 minutes, greater than 30 minutes, greater than 45 minutes, greater than 1 hour, greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 15 hours, greater than 18 hours or greater than 24 hours. In some embodiments, the low alkaline species composition comprises a lower BP2 level relative to the control. In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding portion comprises one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and a combination thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding portion is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell. In some embodiments, the low basic species composition comprises 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 isoform species. In some embodiments, the low basic species composition comprises less than 4%, less than 3%, less than 2%, or less than 1% basic isoform peak 2.

[0368] In another aspect, the present invention provides a method for producing a low basic species composition of an anti-α4β7 antibody or an antigen-binding portion thereof, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of a recombinant host cell expressing the anti-α4β7 antibody or an antigen-binding portion thereof, (ii) and purifying the anti-α4β7 antibody or the antigen-binding portion thereof from the cell culture harvest, wherein the antibody is exposed to a pH of 4.5 or below (e.g., pH 4.1 to 4.5) for no more than 3 hours, no more than 5 hours, no more than 10 hours, or no more than 12 hours, no more than 18 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, or no more than 96 hours, wherein the anti-α4β7 antibody, or an antigen-binding portion thereof, has a reduced level of basic isotype species as compared to a control (determined by CEX), wherein the control is a composition comprising an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.5 or a pH below 4.5 (e.g., pH 4.1 to 4.5) for a longer period of time, i.e., greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 72 hours, or greater than 96 hours. In some embodiments, the low basic species composition comprises a lower level of BP2 relative to the control. In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding portion comprises one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and a combination thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding portion is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell. In some embodiments, the low alkaline species composition comprises 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% alkaline isoform species. In some embodiments, the low alkaline species composition comprises less than 4%, less than 3%, less than 2%, or less than 1% alkaline isoform peak 2.

[0369] In another aspect, the present invention provides a method for producing a low basic species composition of an anti-α4β7 antibody, or an antigen-binding portion thereof, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing the anti-α4β7 antibody, or an antigen-binding portion thereof, and (ii) purifying the anti-α4β7 antibody, or the antigen-binding portion thereof, from the cell culture harvest, wherein the antibody is exposed to a pH of 5.0 or less (e.g., pH 4.6 to 5.0) for no more than 3 hours, no more than 5 hours, no more than 10 hours, or no more than 12 hours, no more than 18 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, or no more than 96 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or the antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 5.0 or less (e.g., pH 4.6 to 5.0). 4.6 to 5.0) for a longer period of time, i.e., greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 72 hours or greater than 96 hours. In some embodiments, the low alkaline species composition comprises a lower BP2 level relative to the control. In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding portion comprises one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and a combination thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding portion is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell. In some embodiments, the low basic species composition comprises 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 isoform species. In some embodiments, the low basic species composition comprises less than 4%, less than 3%, less than 2%, or less than 1% basic isoform peak 2.

[0370] In another aspect, the present invention provides a method for producing a low basic species composition of an anti-α4β7 antibody, or an antigen-binding portion thereof, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing the anti-α4β7 antibody, or an antigen-binding portion thereof, and (ii) purifying the anti-α4β7 antibody, or the antigen-binding portion thereof, from the cell culture harvest, wherein the antibody is exposed to a pH of 5.5 or less (e.g., pH 5.1 to 5.5) for no more than 3 hours, no more than 5 hours, no more than 10 hours, or no more than 12 hours, no more than 18 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, or no more than 96 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or the antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 5.5 or less (e.g., pH 5.1 to 5.5). 5.1 to 5.5) for a longer period of time, i.e., greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 72 hours or greater than 96 hours. In some embodiments, the low alkaline species composition comprises a lower BP2 level relative to the control. In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding portion is vedolizumab or its antigen-binding portion. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding portion comprises one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and a combination thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding portion is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell. In some embodiments, the low basic species composition comprises 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 isoform species. In some embodiments, the low basic species composition comprises less than 4%, less than 3%, less than 2%, or less than 1% basic isoform peak 2.

[0371] In some embodiments, the methods provided herein are performed at a commercial manufacturing scale, e.g., using a vedolizumab formulation derived from a cell culture harvest produced at a 1000L scale, a 2000L scale, a 3000L scale, a 4000L scale, or a 5000L scale (e.g., at least a 3000L scale).

[0372] In some aspects, the methods described herein can be used to regulate the level of a basic vedolizumab isoform in a composition (e.g., a liquid composition) comprising vedolizumab. In some embodiments, the methods can be used to produce a vedolizumab composition having a low level of a basic vedolizumab species.

[0373] In one aspect, the invention provides a method of reducing the level of a basic vedolizumab isoform species in a composition comprising vedolizumab by incubating the composition at a pH greater than pH 6.5 for a period of time sufficient to reduce the level of the basic vedolizumab isoform species. In one embodiment, the method is performed at ambient temperature, e.g., 20° C.-25° C. In other embodiments, the method is performed at 2° C.-8° C.

[0374] In another aspect, the present invention provides a method of producing a composition comprising vedolizumab having reduced levels of basic vedolizumab isotype species. The method may include providing a composition comprising vedolizumab having a pH of or above pH 6.5, and incubating the composition comprising vedolizumab for a period of time sufficient to reduce the level of basic vedolizumab isotype species, thereby producing a composition comprising vedolizumab having reduced levels of basic vedolizumab isotype species.

[0375] In order to effectively reduce the level of basic vedolizumab isoform species in the vedolizumab composition, incubation is preferably performed at a pH of pH 6.5 or above.

[0376] In an exemplary embodiment, the pH of the vedolizumab composition is about pH 6.5-9.0. For example, the pH of the vedolizumab composition can be in the range of about pH 6.5-9.0, pH 6.5-8.5, pH 6.5-8.0, pH 6.5-7.5, or pH 6.5-7.0. Alternatively, the pH of the vedolizumab composition can be in the range of about pH 7.0-9.0, pH 7.5-9.0, pH 8.0-9.0, or pH 8.5-9.0. In other embodiments, the pH of the vedolizumab composition may be in the range of about pH 6.5-7.5, e.g., pH 6.6-7.3, pH 6.6-7.5, pH 6.7-7.5, pH 6.8-7.5, pH 6.9-7.5, pH 7.0-7.5, pH 7.1-7.5, pH 7.2-7.5, pH 7.3-7.5, or pH 7.4-7.5. In other embodiments, the pH of the vedolizumab composition may be in the range of about pH 6.5-7.5, pH 6.5-7.4, pH 6.5-7.3, pH 6.5-7.2, pH 6.5-7.1, pH 6.5-7.0, pH 6.5-6.9, pH 6.5-6.8, pH 6.5-6.75, or pH 6.5-6.6. In other embodiments, the pH of the vedolizumab composition can be in the range of about 7.0-7.5. In other embodiments, the pH of the vedolizumab composition can be in the range of about 7.5-8.0. In other embodiments, the pH of the vedolizumab composition can be in the range of about 8.0-8.5. In exemplary embodiments, the pH of the vedolizumab composition can be about pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9 or pH 9.0.

[0377] The composition comprising vedotin under the above-mentioned pH (e.g., 6.5 or a pH higher than 6.5) can be incubated for a time period sufficient to reduce the percentage of alkaline vedotin isotype species in the vedotin composition. In an exemplary embodiment, incubation occurs for 20 minutes or longer, 30 minutes or longer, 45 minutes or longer, 1 hour or longer, 1.5 hours or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 8 hours or longer, 10 hours or longer, 12 hours or longer, 15 hours or longer, 18 hours or longer, 24 hours or longer, 48 hours or longer, 72 hours or longer, 96 hours or longer, 120 hours or longer, 144 hours or longer, or 168 hours or longer. In some embodiments, incubation occurs for a time period of about 0.5 days or longer, 1 day or longer, 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, or 7 days or longer. In some embodiments, hatching can occur from about 20 minutes to about 1 hour, from about 20 minutes to about 1 hour, from about 20 minutes to about 2 hours, from about 20 minutes to about 3 hours, from about 1 hour to about 3 hours, from about 1 hour to about 5 hours, or from about 5 hours to about 8 hours. In some embodiments, hatching can occur from about 8 hours to about 168 hours (7 days) or longer. In some embodiments, hatching can occur for about 8-168 hours, such as about 8-144 hours (6 days), about 8-120 hours (5 days), about 8-96 hours (4 days), about 8-72 hours (3 days), about 8-48 hours (2 days), about 8-36 hours, about 8-24 hours (1 day), about 8-18 hours, about 8-12 hours, about 15-36 hours, or about 8-10 hours. In other embodiments, hatching can occur for about 10 hours to about 168 hours or longer, for example, about 10-168 hours, about 12-168 hours, about 18-168 hours, about 24-168 hours, about 36-168 hours, about 48-168 hours, about 72-168 hours, about 96-168 hours, about 120-168 hours, or about 144-168 hours. In some embodiments, hatching can occur for about 0.5-7 days, for example, about 0.5-5 days, about 0.5-4 days, about 0.5-3 days, about 0.5-2 days, or about 0.5-1 days. In some embodiments, hatching can occur for about 1-5 days, about 1-3 days, or about 1-2 days. In exemplary embodiments, the hatching at pH 6.5 or more than pH 6.5 occurs for a period of 1-2 days, 1-3 days, or 1-5 days. In other exemplary embodiments, the incubation time at pH 6.5 or above is 1 / 3 of the total purification time. > 25%, for example, the duration starting with providing clarified cell culture harvest and ending with purified antibody UF / DF.

[0378] In other embodiments, incubation occurs to be enough to reduce the percentage of basic vedolizumab isotypes in the composition by 1% or more, for example 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, 15% 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 more, or 100% or more time period. In an exemplary embodiment, incubation occurs to be enough to reduce the percentage of basic vedolizumab isotype species in the composition by 1%-5% time period. In other embodiments, incubation occurs for a time period sufficient to reduce the percentage of basic vedolizumab isotype species in the composition by 1-10%. In other embodiments, incubation occurs for a time period sufficient to reduce the percentage of basic vedolizumab isotype species in the composition by 2-10%. In other embodiments, incubation occurs for a time period sufficient to reduce the percentage of basic vedolizumab isotype species in the composition by 5-10%. In other embodiments, incubation occurs for a time period sufficient to reduce the percentage of basic vedolizumab isotype species in the composition by 2-20%. In other embodiments, incubation occurs for a time period sufficient to reduce the percentage of basic vedolizumab isotype species in the composition by 5-20%.

[0379] In other embodiments, the incubation occurs for a period of time sufficient to produce a low basic species composition comprising an anti-α4β7 antibody (e.g., vedolizumab), wherein the composition has 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% of the total basic isoform species. In some embodiments, the method comprises incubating the composition at a pH greater than pH 6.3 for a period of time sufficient to achieve a level of basic isoform peak 2 of less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the method comprises hydrating a composition comprising vedolizumab at a pH greater than 6.3 (e.g., pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, or pH 8.0). and incubating the composition comprising an anti-α4β7 antibody (e.g., vedolizumab) for a period of 20 minutes or more, such as 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 or more, 72 hours or more, 96 hours or more, 120 hours or more, 144 hours or more, or 168 hours or more.

[0380] In some embodiments, during the incubation period, the pH of the vedolizumab composition can be maintained at or above pH 6.3. In other embodiments, during the incubation period, the pH of the vedolizumab composition can be maintained at or above pH 6.5. In some embodiments, the vedolizumab composition is maintained at approximately the same pH for the duration of the incubation period.

[0381] Incubation can be carried out at any suitable temperature. For example, incubation can be carried out at a temperature in the range of about 0°C-40°C. In another embodiment, incubation can be carried out at a temperature in the range of about 1°C-37°C. In some embodiments, incubation is carried out at a temperature in the range of about 0°C-4°C or in the range of 4°C-8°C. In other embodiments, incubation is carried out at ambient temperature. For example, incubation can be carried out at a temperature in the range of about 20°C-25°C. In other embodiments, incubation is carried out at 37°C or about 37°C. In some embodiments, incubation is carried out at a temperature in the range of about 1°C-25°C. In some embodiments, incubation is carried out at a temperature in the range of about 5°C-18°C. In some embodiments, incubation is carried out at a temperature in the range of about 15°C-30°C. In some embodiments, incubation is carried out at a temperature in the range of about 33°C-37°C. In an exemplary embodiment, incubation is carried out at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0382] The aforementioned method may optionally include additional steps for adjusting the pH of the vedolizumab composition. For example, if it is necessary to reduce the percentage of alkaline vedolizumab species in the composition comprising vedolizumab, the method may include the step of raising the pH of the composition before incubation. For example, if the composition comprising vedolizumab is at a pH lower than 6.5, the method may include the step of raising the pH of the composition to 6.5 or a pH higher than 6.5. On the contrary, if it is necessary to increase the percentage of alkaline vedolizumab species in the composition comprising vedolizumab, the method may include the step of reducing the pH of the composition before incubation. For example, if the pH of the composition comprising vedolizumab is at 6.5 or higher than 6.5, the method may include the step of reducing the pH of the composition to a pH lower than 6.5.

[0383] The composition comprising vedolizumab can be a liquid solution. In some embodiments, the composition comprising vedolizumab is derived from a host cell for producing vedolizumab or its 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, such as a CHO cell lacking dihydrofolate reductase (DHFR) expression or a CHO cell lacking glutamine synthetase (GS) expression.

[0384] After separating antibody from cell culture, the aforementioned method can be incorporated into the small-scale or large-scale process of vedotin purification.In certain embodiments, the first recovery of vedotin from host cell culture (for example, bioreactor harvest) can adopt the step of centrifugation and filtration.Can be before or after centrifugation and / or before or after filtration, vedotin compositions are carried out to method described herein, to reduce the level of basic isotype species in the corresponding purification stage composition.After initial recovery, the downstream process steps that can be used for purifying vedotin from process related impurities and / or product related impurities include but are not limited to affinity chromatography (such as protein A chromatography), depth filtration, cation exchange (CEX), anion exchange (AEX), mixed mode chromatography (MM), ceramic hydroxyapatite chromatography (CHT), hydrophobic interaction chromatography (HIC), ultrafiltration, and / or diafiltration.Can be before or after any downstream process step, vedotin compositions are carried out to method described herein, to reduce the level of basic isotype species in the corresponding purification stage composition.

[0385] For example, the vedolizumab composition may be incubated as described herein before or after affinity chromatography.In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., affinity chromatography loading material or affinity chromatography eluate) to a pH of pH 6.5 or above.

[0386] In another embodiment, the vedolizumab composition may be incubated as described herein before or after depth filtration.In one embodiment, the method may include adjusting the pH of the vedolizumab composition to a pH at or above pH 6.5 before or after depth filtration.

[0387] In another embodiment, the vedolizumab composition may be incubated as described herein before or after cation exchange (CEX). In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., CEX loading material or CEX eluate) to a pH of pH 6.5 or above.

[0388] In another embodiment, the vedolizumab composition may be incubated as described herein before or after anion exchange (AEX). In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., AEX loading material or AEX flow-through) to a pH of pH 6.5 or above.

[0389] In another embodiment, the vedolizumab composition may be incubated as described herein before or after mixed mode chromatography (MM). In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., MM loading material or MM eluate) to a pH of pH 6.5 or above.

[0390] In another embodiment, the vedolizumab composition may be incubated as described herein before or after ceramic hydroxyapatite chromatography (CHT). In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., CHT loading material or CHT eluate) to a pH of pH 6.5 or above.

[0391] In another embodiment, the vedolizumab composition may be incubated as described herein before or after hydrophobic interaction chromatography (HIC). In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., HIC loading material or HIC eluate) to a pH of pH 6.5 or above.

[0392] In another embodiment, the vedolizumab composition may be incubated as described herein before or after ultrafiltration and / or diafiltration (UF / DF). In one embodiment, the method may include adjusting the pH of the vedolizumab composition to a pH of pH 6.5 or above before or after UF / DF.

[0393] IV. Vedolizumab Compositions Containing Reduced Basic Isoform Species

[0394] In some aspects, the invention provides a vedotin composition comprising a reduced alkaline vedotin isotype species level. In some embodiments, the composition (e.g., referring to part III and embodiments) with reduced alkaline isotype species level can be obtained by the method provided herein. In some embodiments, the composition (e.g., referring to part III) with reduced alkaline isotype species level is produced by the method provided herein. Therefore, in one aspect, there is provided herein a composition comprising vedotin, wherein the composition is produced by the method especially including the following: the composition comprising vedotin is hatched at a pH greater than pH 6.5 to be sufficient to reduce the time of the level of alkaline isotype species in the composition. After incubation, the composition comprising vedotin can be optionally subjected to further purification steps, and the purification steps are designed to, for example, reduce the level of impurities in the process source and / or the impurities in the product source in the composition.

[0395] In another aspect, provided herein is a composition comprising vedolizumab or an antigen-binding portion thereof, wherein the composition is obtainable by the above method, e.g., by limiting the duration of exposure of the antibody to low pH conditions during the purification process.

[0396] In some embodiments, provided herein is a composition comprising vedolizumab, or an antigen-binding portion thereof, wherein the composition is obtainable by a method comprising, inter alia, (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab, or an antigen-binding portion thereof, and (ii) purifying vedolizumab, or an antigen-binding portion thereof, from the cell culture harvest, wherein the antibody, or the antigen-binding portion thereof, is exposed to a pH of 3.5 or below (e.g., pH 2.5-3.5, a pH of below 3.0, or a pH of below 3.5) for no more than 20 minutes, no more than 30 minutes, no more than 45 minutes, no more than 1 hour, no more than 3 hours, no more than 5 hours, no more than 7 hours, no more than 10 hours, or no more than 12 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 3.5 or below (e.g., a pH of 2.5-3.5, a pH below 3.0 or a pH below 3.5) for a longer period of time, i.e., greater than 20 minutes, greater than 30 minutes, greater than 45 minutes, greater than 1 hour, greater than 3 hours, greater than 5 hours, greater than 7 hours, greater than 10 hours or greater than 12 hours.

[0397] In some embodiments, provided herein is a composition comprising vedolizumab, or an antigen-binding portion thereof, wherein the composition is obtainable by a method comprising, inter alia, (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab, or an antigen-binding portion thereof, and (ii) purifying vedolizumab, or an antigen-binding portion thereof, from the cell culture harvest, wherein the antibody, or the antigen-binding portion thereof, is exposed to a pH of 4.0 or below (e.g., pH 3.6 to 4.0) for no more than 20 minutes, no more than 30 minutes, no more than 45 minutes, no more than 1 hour, no more than 3 hours, no more than 5 hours, no more than 10 hours, no more than 12 hours, no more than 15 hours, no more than 18 hours, or no more than 24 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.0 or below (e.g., pH 3.6 to 4.0). 3.6 to 4.0) for a longer period of time, i.e., greater than 20 minutes, greater than 30 minutes, greater than 45 minutes, greater than 1 hour, greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 15 hours, greater than 18 hours or greater than 24 hours.

[0398] In another aspect, provided herein is a composition comprising vedolizumab, or an antigen-binding portion thereof, wherein the composition is obtainable by a method comprising, inter alia, (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab, or an antigen-binding portion thereof, and (ii) purifying vedolizumab, or an antigen-binding portion thereof, from the cell culture harvest, wherein the antibody, or the antigen-binding portion thereof, is exposed to a pH of 4.5 or below (e.g., pH 4.1 to 4.5) for no more than 3 hours, no more than 5 hours, no more than 10 hours, or no more than 12 hours, no more than 18 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, or no more than 96 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (as determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.5 or below (e.g., pH 4.5). 4.1 to 4.5) for a longer period of time, i.e., greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 72 hours or greater than 96 hours.

[0399] In another aspect, provided herein is a composition comprising vedolizumab, or an antigen-binding portion thereof, wherein the composition is obtainable by a method comprising, inter alia, (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab, or an antigen-binding portion thereof, and (ii) purifying vedolizumab, or an antigen-binding portion thereof, from the cell culture harvest, wherein the antibody, or the antigen-binding portion thereof, is exposed to a pH of 5.0 or below (e.g., pH 4.6 to 5.0) for no more than 3 hours, no more than 5 hours, no more than 10 hours, or no more than 12 hours, no more than 18 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, or no more than 96 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 5.0 or below (e.g., pH 4.6 to 5.0). 4.6 to 5.0) for a longer period of time, i.e., greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 72 hours or greater than 96 hours.

[0400] In another aspect, provided herein is a composition comprising vedolizumab, or an antigen-binding portion thereof, wherein the composition is obtainable by a method comprising, inter alia, (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab, or an antigen-binding portion thereof, and (ii) purifying vedolizumab, or an antigen-binding portion thereof, from the cell culture harvest, wherein the antibody, or the antigen-binding portion thereof, is exposed to a pH of 5.5 or below (e.g., pH 5.1 to 5.5) for no more than 3 hours, no more than 5 hours, no more than 10 hours, or no more than 12 hours, no more than 18 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, or no more than 96 hours, wherein the anti-α4β7 antibody, or the antigen-binding portion thereof, has a reduced level of basic isotype species (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or an antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 5.5 or below (e.g., pH 5.1 to 5.5). 5.1 to 5.5) for a longer period of time, i.e., greater than 3 hours, greater than 5 hours, greater than 10 hours, greater than 12 hours, greater than 18 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 72 hours or greater than 96 hours.

[0401] In some embodiments of the foregoing aspects, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:5.

[0402] In some embodiments of the foregoing aspects, the step of purifying the anti-α4β7 antibody, or antigen-binding portion thereof, comprises one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, and combinations thereof.

[0403] In some embodiments of the foregoing aspects, the host cell expressing the anti-α4β7 antibody, or antigen binding portion thereof, is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell.

[0404] In some embodiments of the foregoing aspects, the composition comprises 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 isoform species.

[0405] In some embodiments of the foregoing aspects, the composition comprises less than 4%, less than 3%, less than 2%, or less than 1% Basic Isoform Peak 2.

[0406] In some embodiments, provided herein is a vedolizumab composition, wherein the basic species of vedolizumab account for 15% or less of the vedolizumab isoforms in the composition. For example, in some embodiments, provided herein is a vedolizumab composition comprising 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% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the basic isoform species.

[0407] In some embodiments, the level of basic isoform species in the vedolizumab composition is about 1% to 15%, 1% to 14%, 1% to 13%, 1% to 12%, 1% to 11%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2%. In other embodiments, the level of basic isoform species in the vedolizumab composition is about 2% to 11%, 3% to 11%, 4% to 11%, 5% to 11%, 6% to 11%, 7% to 11%, 8% to 11%, 9% to 11%, or 10% to 11%. In other embodiments, the level of basic isotype species in the vedolizumab composition is about 1% to 10%, 2% to 10%, 3% to 10%, 4% to 10%, 5% to 10%, 6% to 10%, 7% to 10%, 8% to 10%, or 9% to 10%. In other embodiments, the level of basic isotype species in the vedolizumab composition is about 1% to 9%, 2% to 9%, 3% to 9%, 4% to 9%, 5% to 9%, 6% to 9%, 7% to 9%, or 8% to 9%. In other embodiments, the level of basic isotype species in the vedolizumab composition is about 1% to 8%, 2% to 8%, 3% to 8%, 4% to 8%, 5% to 8%, 6% to 8%, or 7% to 8%. In other embodiments, the level of basic isotype species in the vedolizumab composition is about 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, or 6% to 7%. In other embodiments, the level of basic isotype species in the vedolizumab composition is about 1% to 6%, 2% to 6%, 3% to 6%, 4% to 6%, or 5% to 6%. In other embodiments, the level of basic isotype species in the vedolizumab composition is about 1% to 5%, 2% to 5%, 3% to 5%, or 4% to 5%. In an exemplary embodiment, the level of basic isotype species in the vedolizumab composition is about 5% or less.

[0408] In some embodiments, the percentage of basic isoform species in the vedolizumab composition is reduced by 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, 15% 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 more, or 100% or more, relative to the percentage of basic isoform species in a vedolizumab composition produced by the same method as described herein that was not incubated at a pH greater than pH 6.5.

[0409] The percentage of basic isoform species in a composition comprising vedolizumab can be determined by any suitable method, including but not limited to CEX-HPLC. In some embodiments, provided herein is a low basic species composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab), wherein the composition comprises 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% of the total basic isoform species of the anti-α4β7 antibody or its antigen-binding portion, wherein the basic isoform species has a net positive charge relative to the major isoform of the anti-α4β7 antibody or its antigen-binding portion as determined by CEX, wherein the anti-α4β7 antibody or its antigen-binding portion comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 5. In some embodiments, the composition comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2). In some such embodiments, the composition comprises less than 2% BP2, less than 1.5% BP2, less than 1% BP2, or less than 0.7% BP2. In some embodiments, the composition comprises 1.5% to 2.5% BP2, 1.2% to 2.2%, 1% to 2% BP2, 1% to 1.8% BP2, 1% to 1.6% BP2, 1% to 1.5% BP2, 0.8% to 1.8% BP2, 0.8% to 1.6% BP2, 0.8% to 1.4% BP2, 0.8% to 1.2% BP2, 0.8% to 1% BP2, 0.7% to 1.7% BP2. 2. 0.7% to 1.5% BP2, 0.7% to 1.3% BP2, 0.7% to 1% BP2, 0.6% to 1.6% BP2, 0.6% to 1.4% BP2, 0.6% to 1.2% BP2, 0.6% to 1% BP2, 0.6% to 0.8% BP2, 0.5% to 1.5% BP2, 0.5% to 1.3% BP2, 0.5% to 1% BP2, or 0.5% to 0.8% BP2.

[0410] 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 10). In some embodiments, the ratio of BP1 to BP2 is 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 12, 3 to 11, 3 to 9, 3 to 6, 3 to 5, 3 to 4, 4 to 12, 4 to 11, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 6, 6 to 12, 6 to 11, 6 to 10, 6 to 8, 6 to 7, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 7 to 8, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 12, 9 to 11, or 9 to 10.

[0411] In some embodiments, the low basic species composition comprises less than 8% (e.g., less than 7%, less than 6%, or less than 5%) of the total basic isotype species of an anti-α4β7 antibody (e.g., vedolizumab). In some embodiments, the low basic species composition comprises 4% to 8%, 4% to 7.5%, 4% to 7%, 4% to 6.5%, 4% to 6%, 4% to 5.5%, 4% to 5%, 5% to 8%, 5% to 7.5%, 5% to 7%, 5% to 6.5%, 5% to 6%, 6% to 8%, 6% to 7.5%, or 6% to 7% of the total basic isotype species of an anti-α4β7 antibody (e.g., vedolizumab).

[0412] In some embodiments, the aforementioned compositions may be incorporated into a pharmaceutical composition comprising an anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab) and a pharmaceutically acceptable carrier or excipient. Therefore, in some embodiments, provided herein is a pharmaceutical composition comprising a low-alkaline species of anti-α4β7 antibody or an antigen-binding portion thereof (e.g., vedolizumab) and a pharmaceutically acceptable carrier. The antibody formulation may be maintained as a liquid or lyophilized into a dry antibody formulation. On the one hand, the dry, lyophilized antibody formulation is provided in a single-dose vial containing 150 mg, 180 mg, 240 mg, 300 mg, 360 mg, 450 mg or 600 mg of anti-α4β7 antibody, and can be reconstituted with a liquid (e.g., sterile water) for administration. On the other hand, the anti-α4β7 antibody (e.g., vedolizumab) is in a stable liquid pharmaceutical composition stored in a container (e.g., a vial, a syringe, or a cartridge) at about 2°C-8°C until it is administered to a subject in need. In some embodiments, the syringe or cartridge can provide a single dose of 54 mg, 108 mg, 160 mg, or 216 mg of the antibody. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody may contain one or more excipients, including but not limited to amino acids (e.g., arginine, histidine, and / or histidine monohydrochloride), sugars (e.g., sucrose), surfactants (e.g., polysorbate 80), and / or buffers (e.g., citrate, phosphate, etc.). In one embodiment, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody contains L-arginine, L-histidine, L-histidine monohydrochloride, sucrose, and / or polysorbate 80. In another embodiment, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody contains citrate, arginine, histidine, and / or polysorbate 80. Other formulations and uses of anti-α4β7 antibodies are described, for example, in U.S. Pat. No. 9,764,033 and U.S. Pat. No. 10,040,855. The entire contents of each of the foregoing patents are incorporated herein by reference.

[0413] V. Preparation of Vedolizumab Compositions Containing Reduced Levels of Host Cell Proteins

[0414] In some aspects, the invention provides a method for producing a composition comprising vedolizumab with a reduced amount of host cell proteins. The method is based on the surprising discovery that the use of an AEX buffer (e.g., a loading buffer) with a reduced conductivity relative to standard operating conditions produces a vedolizumab composition with a reduced host cell protein (HCP) level relative to the vedolizumab composition produced under standard operating conditions.

[0415] Thus, in one aspect, the invention provides a method of producing a composition comprising vedolizumab with reduced amounts of host cell proteins (HCPs), wherein the method involves contacting a sample containing vedolizumab and HCPs with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a reduced conductivity relative to standard buffer conditions, and collecting a flow-through material from the anion exchange resin, wherein the flow-through comprises vedolizumab and reduced amounts of HCPs. In an exemplary embodiment, AEX is performed in a flow-through mode, wherein vedolizumab does not bind to the AEX resin and is collected in the flow-through material without a separate elution step.

[0416] The standard operating range of buffer conductivity for anion exchange (AEX) (e.g., by anion exchange Q membrane adsorbent) is about 11-15 mS / cm (average of about 13.6 mS / cm). Thus, in some embodiments, standard AEX buffer conditions include a loading buffer having a conductivity of about 11 mS / cm or greater, about 12 mS / cm or greater, about 13 mS / cm or greater, about 14 mS / cm or greater, or about 15 mS / cm or greater. In some embodiments, the standard buffer conductivity is about 11 mS / cm to about 12 mS / cm, about 11 mS / cm to about 13 mS / cm, about 11 mS / cm to about 14 mS / cm, or about 11 mS / cm to about 15 mS / cm. In some embodiments, the standard buffer conductivity is about 14 mS / cm to about 15 mS / cm, about 13 mS / cm to about 15 mS / cm, about 12 mS / cm to about 15 mS / cm, or about 11 mS / cm to about 15 mS / cm. In some embodiments, the standard buffer conductivity is about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, or about 15 mS / cm.

[0417] Relative to standard AEX buffer conditions, the AEX buffer (e.g., AEX loading buffer) with reduced conductivity used in the methods described herein has a reduced conductivity. For example, in certain embodiments, the loading buffer with reduced conductivity has a conductivity of about 15 mS / cm or less, about 14 mS / cm or less, about 13 mS / cm or less, about 12 mS / cm or less, about 11 mS / cm or less, about 10 mS / cm or less, about 9 mS / cm or less, about 8 mS / cm or less, about 7 mS / cm or less, about 6 mS / 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. In some embodiments, the loading buffer with reduced conductivity has a conductivity of about 11 mS / cm or less.

[0418] In certain embodiments, the loading buffer with reduced conductivity has a conductivity of about 1 mS / cm to about 11 mS / cm, about 2 mS / cm to about 11 mS / cm, about 3 mS / cm to about 11 mS / cm, about 4 mS / cm to about 11 mS / cm, about 5 mS / cm to about 11 mS / cm, about 6 mS / cm to about 11 mS / cm, about 7 mS / cm to about 11 mS / cm, about 8 mS / cm to about 11 mS / cm, about 9 mS / cm to about 11 mS / cm, or about 10 mS / cm to about 11 mS / cm, including ranges within one or more of the foregoing. In some embodiments, the loading buffer with reduced conductivity has a conductivity of about 11 mS / cm to about 12 mS / cm, about 11 mS / cm to about 13 mS / cm, about 11 mS / cm to about 14 mS / cm, or about 11 mS / cm to about 14.5 mS / cm, including ranges within one or more of the foregoing. In certain embodiments, the loading buffer with reduced conductivity has a conductivity of about 1 mS / cm to about 2 mS / cm, about 1 mS / cm to about 3 mS / cm, about 1 mS / cm to about 4 mS / cm, about 1 mS / cm to about 5 mS / cm, about 1 mS / cm to about 6 mS / cm, about 1 mS / cm to about 7 mS / cm, about 1 mS / cm to about 8 mS / cm, about 1 mS / cm to about 9 mS / cm, about 1 mS / cm to about 10 mS / cm, or about 1 mS / cm to about 11 mS / cm, including ranges within one or more of the foregoing.

[0419] In certain embodiments, the loading buffer with reduced conductivity has a conductivity of about 1 mS / cm, about 1.5 mS / cm, about 2 mS / cm, about 2.5 mS / cm, about 3 mS / cm, about 3.5 mS / cm, about 4 mS / cm, about 4.5 mS / cm, about 5 mS / cm, about 5.5 mS / cm, about 6 mS / cm, about 6.5 mS / cm, about 7 mS / cm, about 7. 5 mS / cm, about 8 mS / cm, about 8.5 mS / cm, 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm, 11 mS / cm, 11.5 mS / cm, 12 mS / cm, 12.5 mS / cm, 13 mS / cm, 13.5 mS / cm, 14 mS / cm, 14.5 mS / cm, or 15 mS / cm.

[0420] In some embodiments, the method may further include applying a wash buffer to the AEX resin after applying a loading buffer containing vedolizumab. In some embodiments, the wash buffer has the same conductivity as the loading buffer. In other embodiments, the wash buffer has an increased conductivity relative to the loading buffer. In other embodiments, the wash buffer has a reduced conductivity relative to the loading buffer.

[0421] In some embodiments, the loading buffer comprises sodium chloride and / or sodium phosphate. In an exemplary embodiment, the loading buffer contains 40-70mM NaCl (e.g., 40mM, 45mM, 50mM, 55mM, 60mM, 65mM or 70mM NaCl). In some embodiments, the loading buffer comprises 55-65mM NaCl. Additionally or alternatively, the loading buffer may comprise sodium phosphate. In an exemplary embodiment, the loading buffer contains 20-50mM sodium phosphate (e.g., 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM sodium phosphate). In some embodiments, the loading buffer comprises 35-45mM sodium phosphate. In some embodiments, the pH of the loading buffer is pH 6.5 or higher than pH 6.5, for example, pH 6.5-8.5, pH 7.0-7.5, pH6.8-7.4, etc. In some embodiments, the pH of the loading buffer is about 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, 8.4, or 8.5.

[0422] In some embodiments, provided herein is a method for purifying an α4β7 antibody (e.g., vedolizumab), the method comprising contacting a solution comprising the antibody with an AEX resin balanced in a low conductivity buffer in a flow-through mode, and collecting the flow-through material. In one embodiment, the low conductivity solution comprises a mixture of NaCl and a buffer having a pH of 6.5-8.5. In some embodiments, the low conductivity solution has a conductivity of 5 to 15 mS / cm, 5 to 11 mS / cm, 7 to 10 mS / cm, or about 10 mS / cm.

[0423] In some embodiments, the anion exchange resin is formatted as an anion exchange membrane. In some embodiments, the anion exchange resin is formatted as an anion exchange chromatographic column. In some embodiments, the anion exchange resin comprises a quaternary amine functional group. Exemplary AEX resins include, but are not limited to, Mustang Q (Pall Corporation, PortWashington, NY), Sartobind Q (Sartorius GmbH, Goettingen, Germany). Other exemplary AEX resins include, for example, Eshmuno Q resin (EMD Millipore, Burlington, MA) and Nuvia Q resin (Bio-Rad, Hercules, CA).

[0424] HCP can be derived from a host cell for producing vedolizumab or its antigen-binding portion thereof. For example, in some embodiments, vedolizumab is produced in Chinese hamster ovary (CHO) cells, and HCP is a CHO cell protein. In certain embodiments, HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression. In certain embodiments, HCP is derived from CHO cells lacking glutamine synthetase (GS) expression.

[0425] In certain embodiments, the amount of HCP in the flowthrough is about 8 ppm or less, about 7.5 ppm or less, about 7 ppm or less, about 6.5 ppm or less, about 6 ppm or less, about 5.5 ppm or less, about 5 ppm or less, about 4.5 ppm or less, about 4 ppm or less, about 3.5 ppm or less, about 3 ppm or less, about 2.5 ppm or less, about 2 ppm or less, about 1.5 ppm or less, or about 1 ppm or less.

[0426] In some embodiments, the amount of HCP in the flow-through is 1ppm to 8ppm, 2ppm to 8ppm, 3ppm to 8ppm, 4ppm to 8ppm, 5ppm to 8ppm, 6ppm to 8ppm or 7ppm to 8ppm, including the range within one or more of the above. In some embodiments, the amount of HCP in the flow-through is 1ppm to 2ppm, 1ppm to 3ppm, 1ppm to 4ppm, 1ppm to 5ppm, 1ppm to 6ppm, 1ppm to 7ppm or 1ppm to 8ppm, including the range within one or more of the above. In some embodiments, the amount of HCP in the flow-through is 1ppm to 3ppm, 3ppm to 5ppm or 5ppm to 7ppm.

[0427] In certain embodiments, the amount of HCP in the flow-through is reduced by 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 greater than about 98% relative to the amount of HCP in the flow-through material produced when the same sample is subjected to the method using a loading buffer having a standard conductivity (e.g., a conductivity of 11-15 mS / cm or above).

[0428] In certain embodiments, the amount of HCP in the flow-through is reduced by about 0.5% to about 50%, about 1% to about 50%, about 2% to about 50%, about 5% to about 50%, about 10% to about 50%, about 15% to about 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%. In certain embodiments, the amount of HCP in the flow-through is reduced by about 50% to about 55%, about 50% to 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 50% to about 98%, relative to the amount of HCP in the flow-through produced when the method is performed using the same sample with a loading buffer having a standard conductivity (e.g., about 11-15 mS / cm).

[0429] In certain embodiments, the amount of HCP in the flow-through is reduced by about 1% to about 10%, 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 about 98%. In certain embodiments, the amount of HCP in the flow-through is reduced by 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 ... From about 5% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 98%.

[0430] The sample containing vedolizumab and HCP is derived from mammalian cell culture, optionally after one or more additional purification steps (including, for example, affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, ceramic hydroxyapatite (CHT) chromatography, and mixed mode chromatography, or a combination thereof). In addition, after the AEX method described herein, the HCP level in the sample containing vedolizumab can be optionally further reduced by one or more additional purification steps (including, for example, affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, ceramic hydroxyapatite (CHT) chromatography, and mixed mode chromatography, or a combination thereof).

[0431] Therefore, in some embodiments, the method described herein may include one or more additional purification steps to further reduce the HCP level in the sample containing vedolizumab. In one embodiment, the present invention provides a method for reducing the HCP level in the composition containing vedolizumab using the AEX method described herein, and the method further includes one or more additional purification steps. The one or more additional purification steps may be performed before or after the AEX method described herein. In some embodiments, the one or more additional purification steps include one or more chromatographic separations. In an exemplary embodiment, the one or more additional purification steps include affinity chromatography (e.g., protein A chromatography), cation exchange chromatography, hydrophobic interaction chromatography, ceramic hydroxyapatite (CHT) chromatography or mixed mode chromatography or a combination thereof.

[0432] In an exemplary embodiment, the present invention provides a method for reducing the level of HCP in a composition comprising vedolizumab, the method comprising providing a composition comprising vedolizumab and HCP, purifying vedolizumab from HCP by performing affinity chromatography, mixed mode chromatography, and / or cation exchange chromatography, and further purifying vedolizumab from HCP by performing the AEX method described herein. In one embodiment, the loading material for the AEX method described herein comprises a cation exchange eluate. The foregoing method can be used to produce a composition comprising vedolizumab using an anion exchange buffer having a conductivity of 11-15mS / cm or higher than 11-15mS / cm, the composition having a reduced level of HCP relative to the level of HCP present in the composition produced by performing the same method.

[0433] In another exemplary embodiment, the present invention provides a method for reducing the level of HCP in a composition comprising vedolizumab, the method comprising providing a composition comprising vedolizumab and HCP, purifying vedolizumab from HCP by performing affinity chromatography, cation exchange chromatography and / or hydroxyapatite chromatography (e.g., ceramic hydroxyapatite (CHT) chromatography), and further purifying vedolizumab from HCP by performing the AEX method described herein. In one embodiment, the loading material for the AEX method described herein includes a hydroxyapatite chromatography eluate. The foregoing method can be used to produce a composition comprising vedolizumab using an anion exchange buffer having a conductivity of 11-15mS / cm or higher than 11-15mS / cm, the composition having a reduced level of HCP relative to the level of HCP present in a composition produced by performing the same method.

[0434] Vedolizumab content and / or host cell protein content can be measured by any method known in the art, including but not limited to HCP ELISA, chromatography (e.g., SEC), analytical ultracentrifugation, light scattering (DLS or MALLS), mass spectrometry (e.g., MALDI-TOF MS), or nanoscale measurement (such as nanoparticle trajectory analysis NTA, NanoSight Ltd, Wiltshire, UK).

[0435] In some embodiments, the method further comprises treating the AEX flow-through material by a process comprising ultrafiltration and / or diafiltration to exchange the elution buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients.

[0436] VI. Vedolizumab Compositions Containing Reduced Host Cell Proteins

[0437] In some aspects, the present invention provides a vedolizumab composition comprising a reduced host cell protein. In some embodiments, the composition with reduced host cell protein is produced by the method provided herein (e.g., see Part V). Therefore, on the one hand, a composition comprising vedolizumab is provided herein, wherein the composition is produced in the following manner: a sample containing vedolizumab and HCP is contacted with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a reduced conductivity relative to a standard buffer condition; and a flow-through material is collected from an anion exchange resin, wherein the flow-through material comprises vedolizumab and a reduced amount of HCP. After eluting from an AEX resin, the flow-through material may be optionally treated to exchange the elution buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients, thereby forming a pharmaceutical composition with a reduced amount of HCP. The buffer exchange step can be performed using standard methods, including, for example, ultrafiltration and / or diafiltration.

[0438] HCP can be derived from a host cell for producing vedolizumab or its antigen-binding portion thereof. For example, in some embodiments, vedolizumab is produced in Chinese hamster ovary (CHO) cells, and HCP is a CHO cell protein. In certain embodiments, HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression. In certain embodiments, HCP is derived from CHO cells lacking glutamine synthetase (GS) expression.

[0439] In certain embodiments, the amount of HCP in the composition is about 8 ppm or less, about 7.5 ppm or less, about 7 ppm or less, about 6.5 ppm or less, about 6 ppm or less, about 5.5 ppm or less, about 5 ppm or less, about 4.5 ppm or less, about 4 ppm or less, about 3.5 ppm or less, about 3 ppm or less, about 2.5 ppm or less, about 2 ppm or less, about 1.5 ppm or less, or about 1 ppm or less.

[0440] In some embodiments, the amount of HCP in the composition is 1ppm to 8ppm, 2ppm to 8ppm, 3ppm to 8ppm, 4ppm to 8ppm, 5ppm to 8ppm, 6ppm to 8ppm, or 7ppm to 8ppm, including the range within one or more of the above. In some embodiments, the amount of HCP in the composition is 1ppm to 2ppm, 1ppm to 3ppm, 1ppm to 4ppm, 1ppm to 5ppm, 1ppm to 6ppm, 1ppm to 7ppm, or 1ppm to 8ppm, including the range within one or more of the above. In some embodiments, the amount of HCP in the composition is 1ppm to 3ppm, 3ppm to 5ppm, or 5ppm to 7ppm.

[0441] In certain embodiments, the amount of HCP in the composition is reduced by 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 greater than about 98%, relative to the amount of HCP in a composition produced by a method performed using the same sample with a loading buffer having a standard conductivity (e.g., a conductivity of 11-15 mS / cm or above).

[0442] In certain embodiments, the amount of HCP in the composition is reduced by about 0.5% to about 50%, about 1% to about 50%, about 2% to about 50%, about 5% to about 50%, about 10% to about 50%, about 15% to about 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%. In certain embodiments, the amount of HCP in the flow-through material is reduced by about 50% to about 55%, about 50% to 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 50% to about 98%, relative to the amount of HCP in a composition produced by a method performed using the same sample with a loading buffer having a standard conductivity (e.g., a conductivity of 11-15 mS / cm or above).

[0443] In certain embodiments, the amount of HCP in the composition is reduced by about 1% to about 10%, 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 about 98%. In certain embodiments, the amount of HCP in the flow-through material is reduced by 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 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 about 98%. In certain embodiments, the amount of HCP in the composition produced when the method is performed using the same sample by using a loading buffer with a standard conductivity (e.g., a conductivity of 11-15mS / cm or higher than 11-15mS / cm) is reduced by 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 30%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, or about 30% to about 30% From about 20% to about 15%; from about 50% to about 40%; from about 60% to about 70%; from about 65% to about 70%; from about 70% to about 75%; from about 75% to about 80%; from about 80% to about 85%; from about 85% to about 90%; from about 90% to about 95%; or from about 95% to about 98%.

[0444] Vedolizumab content and / or host cell protein content can be measured by any method known in the art, including but not limited to HCP ELISA, chromatography (e.g., SEC), analytical ultracentrifugation, light scattering (DLS or MALLS), mass spectrometry (e.g., MALDI-TOF MS), or nanoscale measurement (such as nanoparticle trajectory analysis NTA, NanoSight Ltd, Wiltshire, UK).

[0445] VII. Preparation of Vedolizumab Using Mixed-Mode Chromatography

[0446] Described herein is the buffer for maximizing the productivity of Vedolizumab after eluting from mixed mode chromatographic resin.Under high concentration loading conditions (for example, loading concentration is at least 14g / L, 15g / L, 17g / L, 10g / L, 25g / L, 30g / L, 35g / L or more), protein loss can occur in the washing process of the mixed mode purification using, for example, ceramic hydroxyapatite resin.In order to increase the loading capacity of mixed mode resin (for example, ceramic hydroxyapatite resin), and improve the output of Vedolizumab, it is possible to optimize equilibrium buffer, loading buffer and washing buffer, to reduce the antibody loss during washing column.Surprisingly, the method described herein allows applying a larger loading concentration on mixed mode resin, and increases productivity by 2%-3% relative to standard mixed mode buffer, without changing the product quality (for example, aggregation percentage) in the final eluate.

[0447] Therefore, on the one hand, the present invention provides a method for increasing the yield of the vedolizumab recovered after elution from a mixed mode chromatography resin, the method comprising balancing the mixed mode chromatography resin with an equilibrium buffer, loading a solution comprising vedolizumab and a loading buffer onto the mixed mode chromatography resin so that vedolizumab binds to the mixed mode chromatography resin, washing the mixed mode chromatography resin with a wash buffer, and eluting vedolizumab from the mixed mode chromatography resin with an elution buffer, wherein the equilibrium buffer, the loading buffer and / or the washing buffer have a pH of 7.0 or less than 7.0. The pH of the standard buffer for mixed mode chromatography can be higher, i.e., the pH is 7.2 or higher. In some embodiments of the present invention, an equilibrium buffer having a pH of 7.0 or less than 7.0 is used. In some embodiments, a loading buffer having a pH of 7.0 or less than 7.0 is used. In some embodiments, a washing buffer having a pH of 7.0 or less than 7.0 is used.

[0448] The buffer pH range lower than 7.0 can be used to prevent the loss of the vedotin monoclonal antibody in the washing step process of mixed mode purification.For example, in some embodiments, equilibrium buffer, loading buffer and / or washing buffer may have a pH 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, 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 less than 5.5.For example, 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, 5.6 or 5.5.In some embodiments, the pH of buffer is in the range of about 7.0-5.5.In other embodiments, the pH of buffer is in the range of about 7.0-6.0. In other embodiments, the pH of the buffer is in the range of about 7.0-6.5. In other embodiments, the pH of the buffer is in the range of about 6.8-5.8. In other embodiments, the pH of the buffer is in the range of about 6.8-6.0. In other embodiments, the pH of the buffer is in the range of about 6.8-6.5. In other embodiments, the pH of the buffer is about 6.6-6.8.

[0449] Additionally or alternatively, the buffer for balancing, loading and / or washing the mixed mode chromatography resin for purifying vedotin monoclonal antibody can have a total salt concentration less than 70mM.For example, the buffer can have a salt concentration less than 70mM, less than 65mM, less than 60mM, less than 55mM, less than 50mM, less than 45mM, less than 40mM, less than 35mM or less than 30mM.In some embodiments, the buffer has a salt concentration of about 70mM, about 65mM, about 60mM, about 55mM, about 50mM, about 45mM, about 40mM, about 35mM or about 30mM.In other embodiments, the buffer has a salt concentration within the range of 30-70mM.In some embodiments, the buffer has a salt concentration within the range of 40-65mM.In some embodiments, the buffer has a salt concentration within the range of 45-65mM.In some embodiments, the buffer has a salt concentration within the range of 50-60mM. In some embodiments, the buffer has a salt concentration in the range of 40-50 mM. In some embodiments, the buffer has a salt concentration in the range of 45-55 mM. In some embodiments, the salt present in the buffer for balancing, loading, and / or washing the CHT chromatographic resin for purifying vedolizumab includes sodium chloride and / or sodium phosphate.

[0450] In some embodiments, the buffer for balancing, loading and / or washing the mixed mode chromatography resin for purifying vedotin monoclonal antibody can have a sodium chloride concentration less than 70mM.For example, the buffer can have a sodium chloride concentration less than 70mM, less than 65mM, less than 60mM, less than 55mM, less than 50mM, less than 45mM, less than 40mM, less than 35mM or less than 30mM.In some embodiments, the buffer has a sodium chloride concentration of about 70mM, about 65mM, about 60mM, about 55mM, about 50mM, about 45mM, about 40mM, about 35mM or about 30mM.In other embodiments, the buffer has a sodium chloride concentration within the range of 30-70mM.In some embodiments, the buffer has a sodium chloride concentration within the range of 40-65mM.In some embodiments, the buffer has a sodium chloride concentration within the range of 45-65mM.In some embodiments, the buffer has a sodium chloride concentration within the range of 50-60mM. In some embodiments, the buffer has a sodium chloride concentration in the range of 40-50mM. In some embodiments, the buffer has a sodium chloride concentration in the range of 45-55mM. In some embodiments, the aforementioned sodium chloride buffer may further include sodium phosphate. In some embodiments, the aforementioned sodium chloride buffer may further include 1-30mM sodium phosphate, for example, 5-20mM sodium phosphate, 10-20mM sodium phosphate, etc. In an exemplary embodiment, the sodium chloride buffer may further include a sodium phosphate having a concentration of 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 25mM, or 30mM.

[0451] The above buffers can be used to equilibrate, load and / or wash the mixed mode chromatography resin in the vedolizumab purification process.In an exemplary embodiment, the mixed mode resin can be a ceramic hydroxyapatite resin.

[0452] In some embodiments, two of the balancing buffer, loading buffer and washing buffer have the same pH. In some embodiments, two of the balancing buffer, loading buffer and washing buffer have the same salt concentration. In some embodiments, two of the balancing buffer, loading buffer and washing buffer have the same pH and the same salt concentration. In some embodiments, the balancing buffer, loading buffer and washing buffer all have the same pH. In some embodiments, the balancing buffer, loading buffer and washing buffer all have the same salt concentration. In some embodiments, the balancing buffer, loading buffer and washing buffer all have the same pH and the same salt concentration.

[0453] In some embodiments, the method of increasing the yield of vedolizumab recovered after loading the mixed mode chromatography resin comprises eluting the column at a pH of less than 7, i.e., pH 5.5 to 6.9 or pH 6.5 to 6.8 and a salt (e.g., NaCl) concentration of 40 to 60 mM or 45 to 55 mM. The method may further comprise washing the column at a pH of less than 7, i.e., pH 5.5 to 6.9 or pH 6.5 to 6.8.

[0454] Relative to the yield obtained when balancing, loading, and / or washing steps using a buffer with a pH greater than 7.0 and / or a salt concentration greater than 70mM and / or a sodium chloride concentration greater than 70mM, the buffer and method described herein can improve the yield of the vedolizumab eluted from a mixed mode chromatographic column. In some embodiments, the yield is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20% or more. In some embodiments, relative to a buffer having a pH greater than 7.0, the yield is increased by more than 2%. In some embodiments, relative to a buffer having a pH greater than 7.0, the yield is increased by more than 3%. In some embodiments, relative to a buffer having a pH greater than 7.0, the yield is increased by more than 4%. In some embodiments, relative to a buffer having a pH greater than 7.0, the yield is increased by more than 5%.

[0455] VIII. Methods for Assessing the Purity of Compositions Containing Anti-α4β7 Antibodies

[0456] The purity of a composition comprising an antibody (eg, vedolizumab) can be assessed by any suitable method, including but not limited to the methods described herein.

[0457] (a) Evaluation of basic isoform species

[0458] The present invention provides methods for regulating (e.g., reducing) the level of basic isoform species in a composition comprising vedolizumab or its antigen-binding portion, and methods for regulating (e.g., increasing) the level of the major isoform in a composition comprising vedolizumab. The relative amount of basic vedolizumab isoform species and the relative amount of the major vedolizumab isoform present in the vedolizumab composition can be measured using cation exchange chromatography (CEX), as described in detail in the Examples section. The CEX method fractionates antibody species according to overall surface charge. After dilution with a mobile phase to a low ionic strength, the test sample can be injected onto a CEX column, such as a Dionex Pro-Pac TMWCX-10 column (Thermo Fisher Scientific, Waltham, MA (USA)), which is balanced in a suitable buffer (e.g., 10 mM sodium phosphate, pH 6.6). Antibodies can be eluted using a sodium chloride gradient in the same buffer. Protein elution can be monitored at 280 nm, and peaks are designated as acidic, alkaline, or major isotype categories. Acidic peaks are eluted from the column with a retention time shorter than the major isotype peak, and alkaline peaks are eluted from the column with a retention time longer than the major isotype peak. The major isotype percentage, the sum of the percentage of acidic species, and the sum of the percentage of alkaline species are reported. The major isotype retention time of the sample is compared with the retention time of the reference standard to determine consistency. In certain embodiments, a CEX-HPLC method is used. For example, a composition comprising vedolizumab and its acidic and / or alkaline species can be separated using cation exchange chromatography as described above, and the eluted peak is subsequently analyzed using HPLC. In one embodiment, HPLC can be performed using an Agilent 1200 HPLC system (Agilent, Santa Clara, CA). Quantification is based on the relative area percentage of the detected peaks. Figure 1 CEX-HPLC curves of exemplary vedolizumab formulations are provided in .

[0459] In one embodiment, the CEX assay method comprises diluting the test sample to low ionic strength, injecting onto a CEX column equilibrated in 10 mM sodium phosphate (pH 6.6), eluting the column with a NaCl gradient in the buffer, monitoring the peaks at 280 nm and assigning the peaks as acidic peaks, major peaks, or basic peaks, wherein the acidic peak elutes first with the shortest retention time, the major peak elutes second, and the basic peak elutes with the longest retention time, and quantifying the peak areas, and the amounts thereof are calculated as a percentage of all peak areas.

[0460] (b) Assessment of host cell protein levels

[0461] The present invention provides a method for regulating (e.g., reducing) the level of residual host cell proteins in a composition comprising vedolizumab or its antigen-binding portion thereof. In some embodiments, an enzyme-linked immunosorbent assay (ELISA) can be used to measure the amount of host cell proteins present in the vedolizumab composition using standard techniques. Many ELISA kits designed for this purpose are commercially available, such as the CHOHCP ELISA kit 3G from Cygnus Technologies (Southport, NC (USA)). Fixed polyclonal anti-CHO HCP antibodies can be used to capture host cell proteins in a test sample. The captured protein can then be detected using a suitable detection agent, for example, a horseradish peroxidase labeled form of the same antibody. In this exemplary embodiment, the amount of peroxidase captured by colorimetric measurement of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) at 450nm is directly proportional to the concentration of CHO HCP. HCP concentration can be determined by comparing with a CHO HCP standard curve (such as included in a test kit) and reported as a percentage of the total level of protein in an antibody preparation. In another embodiment, the rabbit-rabbit ("RaRa") method illustrated herein can be used to determine HCP. Polyclonal anti-CHO HCP antibodies are produced by immunizing rabbits with harvested materials made with naked cells (nullcell) using manufacturing conditions similar to those of vedolizumab, and the antibodies are affinity purified. Host CHO cell proteins in vedolizumab samples are captured using fixed polyclonal anti-CHO HCP antibodies, then biotin-labeled forms of the same antibodies are used, followed by detection using horseradish peroxidase-conjugated streptavidin. The amount of peroxidase captured is measured colorimetrically at 450nm using peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is directly proportional to the concentration of CHO HCP. HCP concentration is determined by comparing with the CHO HCP standard curve included in the test kit, and is reported as a percentage of total protein. In another embodiment, the rabbit-goat ("RaGo") method illustrated herein can be used to determine HCP. The polyclonal anti-CHO HCP antibodies were generated by immunizing rabbits and goats with harvested material from naked cell manufacturing using a manufacturing process similar to that of vedolizumab. The antibody pools were independently affinity purified. Host cell proteins in the MLN0002 test sample were captured using immobilized polyclonal rabbit anti-CHO HCP antibodies and then detected by sequential addition of goat anti-CHO affinity purified antibodies and donkey anti-goat IgG reagents (labeled with horseradish peroxidase). The amount of captured peroxidase was measured colorimetrically at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) and was proportional to the concentration of CHO HCP.HCP concentrations were determined by comparison to the CHO HCP standard curve included in the test kit and reported as concentration relative to total vedolizumab (ng / mg).

[0462] A CHO HCP assay suitable for use in conjunction with the various embodiments provided herein comprises capturing HCP using a polyclonal anti-CHO HCP antibody, detecting HCP after binding to a horseradish peroxidase-labeled form of the polyclonal anti-CHO HCP antibody, which converts the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a substrate that is colorimetrically quantified at 450 nm. In one embodiment, a HCP ELISA suitable for use in conjunction with the various embodiments provided herein is an ELISA method that captures HCP using an immobilized polyclonal anti-CHO HCP antibody, preferably a CHO HCP ELISA kit 3G provided with Cygnus Technologies (Southport, NC (USA)), whereby the captured protein is detected by the same antibody in a horseradish peroxidase-labeled form, and the amount of captured peroxidase is measured colorimetrically at 450 nm using 3,3',5,5'-tetramethylbenzidine (TMB), followed by determining the HCP concentration by comparison with a CHO HCP standard curve.

[0463] (c) Evaluating size variations

[0464] In certain embodiments, the level of aggregates, monomers and fragments in the chromatographic samples produced using the technology described herein is analyzed. In each embodiment described herein, size exclusion chromatography (SEC) can be used to determine the relative level of monomers, high molecular weight (HMW) aggregates and low molecular weight (LMW) degradation products present in a population of an antibody or its antigen-binding portion (e.g., Vedolizumab). The SEC method provides separation of antibody monomers from HMW species and LMW degradation products based on size. Commercially available SEC columns can be used to analyze test samples and reference standards using appropriate buffers. For example, in some embodiments, SEC analysis can be performed using a G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA (USA)) or two G3000 SWxl columns connected in series and an isocratic phosphate-sodium chloride buffer system (pH 6.8). The elution of protein species is monitored at 280nm. The main peak (monomer) and total peak area are assessed to determine purity. Sample purity (%) (calculated as monomer%), HMW aggregate %, and / or LMW degradation product % are reported.

[0465] In one embodiment, SEC analysis involves injecting the sample onto two G3000SWxl columns connected in series and running in an isocratic phosphate-sodium chloride buffer system (pH 6.8), where the elution of the protein species is monitored at 280 nm and the main peak (monomer) and total peak areas are measured.

[0466] IX. Pharmaceutical compositions and uses thereof

[0467] The compositions comprising vedolizumab provided herein, for example, compositions comprising vedolizumab with reduced levels of basic isotype species and / or compositions comprising vedolizumab with reduced levels of host cell proteins can be incorporated into pharmaceutical formulations for therapeutic use. Pharmaceutical formulations comprising vedolizumab can be prepared by any suitable method.

[0468] In one aspect, the pharmaceutical formulation comprising the compositions described herein is a lyophilized pharmaceutical formulation. In one aspect, the lyophilized formulation can be stored in a container (e.g., a vial) as a single dose. The container (e.g., a vial) is stored refrigerated, e.g., at about 2°C-8°C or at room temperature, e.g., about 20°C to 35°C, about 25°C, or about 30°C, until it is administered to a subject in need. The vial can be, for example, a 10, 20, or 50cc vial. A container (e.g., a vial) can contain about 90 to 115 mg, about 95 to 105 mg, at least about 100 mg, about 135 to 160 mg, about 145 to 155 mg, at least about 150 mg, about 180 to 220 mg, about 190 to 210 mg, about 195 to 205 mg, at least about 200 mg, about 280 to 320 mg, about 290 to 310 mg, at least about 300 mg, about 380 to 420 mg, about 390 to 410 mg, at least about 400 mg, about 580 to 620 mg, about 590 to 610 mg, or at least about 600 mg of an anti-α4β7 antibody. In one aspect, a vial contains about 200 mg of an anti-α4β7 antibody. The vial may contain enough anti-α4β7 antibody (e.g., vedolizumab) to allow delivery (e.g., be manufactured to deliver) 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. For example, the vial may contain about 15%, about 12%, about 10%, or about 8% more anti-α4β7 antibody than the dosage amount.

[0469] In some embodiments, the compositions described herein are formulated as dry, lyophilized pharmaceutical formulations that can be reconstituted with a liquid (e.g., sterile water) for administration. Administration of the reconstituted formulation can be performed by parenteral injection via one of the above-described routes. Intravenous injection can be performed by infusion, such as by further dilution with sterile isotonic saline, buffer (e.g., phosphate-buffered saline), or Ringer's (lactic acid or glucose) solution.

[0470] In some embodiments, the compositions described herein are formulated as liquid pharmaceutical formulations suitable for subcutaneous administration to humans. In some embodiments, the anti-α4β7 antibody is administered by subcutaneous injection, for example, at a dose of about 54 mg, 108 mg, or about 165 mg, or about 216 mg, about every two, three, or four weeks after the start of therapy or after the third subsequent dose.

[0471] In another aspect, the compositions described herein comprising an anti-α4β7 antibody (e.g., vedolizumab) are in a stable liquid pharmaceutical composition stored in a container (e.g., a vial, a syringe, or a cartridge) at about 2°C-8°C until it is administered to a subject in need thereof. In some embodiments, the stable liquid pharmaceutical composition of the anti-α4β7 antibody comprises about 0% to 5.0%, 0% to 2%, ≤2%, ≤1%, ≤0.6%, or ≤0.5% aggregates. The syringe or cartridge can be a 1 mL or 2 mL container (e.g., for a 160 mg / mL dose) or more than 2 ml (e.g., for a higher dose (at least 320 mg or 400 mg or more)). The syringe or cartridge may contain at least about 20 mg, at least about 50 mg, at least about 70 mg, at least about 80 mg, at least about 100 mg, at least about 108 mg, at least about 120 mg, at least about 155 mg, at least about 180 mg, at least about 200 mg, at least about 240 mg, at least about 300 mg, at least about 360 mg, at least about 400 mg, or at least about 500 mg of anti-α4β7 antibody. In some embodiments, the container (e.g., syringe or cartridge) can be manufactured to deliver about 20 to 120 mg, about 40 mg to 70 mg, about 45 to 65 mg, about 50 to 57 mg, or about 54 mg of anti-α4β7 antibody (e.g., vedolizumab). In other embodiments, the syringe or cartridge can be manufactured to deliver about 90 to 120 mg, about 95 to 115 mg, about 100 to 112 mg, or about 108 mg of an anti-α4β7 antibody (e.g., vedolizumab). In other embodiments, the syringe or cartridge can be manufactured to deliver about 140 to 250 mg, about 150 to 200 mg, about 160 to 170 mg, about 160 to 250 mg, about 175 mg to 210 mg, or about 160 mg, about 165 mg, about 180 mg, or about 200 mg of an anti-α4β7 antibody (e.g., vedolizumab).

[0472] Containers that can be used to store and freeze the purified compositions described herein include polycarbonate bottles (for IV formulations) or PETG bottles (for subcutaneous formulations). After the formulation is aliquoted into bottles, it can be frozen (e.g., at -60 degrees Celsius or lower).

[0473] The pharmaceutical composition may comprise any of the vedolizumab compositions provided herein, e.g., a composition comprising vedolizumab having reduced levels of basic isoform species, and / or a composition comprising vedolizumab and a pharmaceutically acceptable carrier or excipient having reduced levels of a host cell protein. In some embodiments, the pH of the pharmaceutical composition is between 6.0-7.0, such as pH 6.0-6.2, pH 6.0-6.4, pH 6.0-6.6, pH 6.0-6.8, pH 6.1-6.3, pH 6.1-6.5, pH 6.1-6.7, pH 6.1-6.9, pH 6.2-6.4, pH 6.2-6.6, pH 6.2-6.8, pH 6.2-7.0, pH 6.3-6.5, pH 6.3-6.7, pH 6.3-6.9, pH 6.4-6.6, pH 6.4-6.8, pH 6.4-7.0, pH 6.5-6.7, pH 6.5-6.9, pH 6.6-6.8, pH 6.6-7.0, pH In some embodiments, the pH of the pharmaceutical composition is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0474] The pharmaceutical composition may be additionally supplemented with amino acids or sugars. In some embodiments, the pharmaceutical composition further comprises amino acids, such as arginine or histidine. In some embodiments, the pharmaceutical composition further comprises sugars, such as sucrose or trehalose. In some embodiments, the pharmaceutical composition of the anti-α4β7 antibody provided herein comprises arginine, histidine, and / or polysorbate 80. In some embodiments, the pharmaceutical composition of the anti-α4β7 antibody provided herein comprises citrate, arginine, histidine and / or polysorbate 80.

[0475] The compositions comprising vedolizumab provided herein (e.g., compositions comprising vedolizumab having reduced levels of basic isoform species) and / or compositions comprising vedolizumab having reduced levels of host cell proteins can be used in methods of inhibiting integrin α4β7 activity in vitro or in vivo.

[0476] In one aspect, the compositions described herein can be used to treat a disease or condition in a subject, comprising administering to the subject a composition comprising an anti-α4β7 antibody in an amount effective to treat a disease or condition in a human. The human subject can be an adult (e.g., 18 years of age or older), a teenager, or a child. The human subject can be a person 65 years of age or older.

[0477] In one embodiment, the compositions described herein are used to treat subjects who may have lacked adequate response, loss of response or intolerance to treatment with immunomodulators, TNF-α antagonists or combinations thereof. Subjects may have previously received treatment with at least one corticosteroid (e.g., prednisone) and have insufficient, intolerance or dependence on corticosteroids for corticosteroid treatment (e.g., inflammatory bowel disease). Insufficient response to corticosteroids refers to signs and symptoms of persistent active disease despite a history of at least one 4-week induction regimen including a dose equivalent to oral prednisone 30mg daily for 2 weeks or intravenous injection for 1 week. Loss of response to corticosteroids refers to two attempts to gradually reduce corticosteroids to a dose equivalent to oral prednisone 10mg daily and all failed. Intolerance to corticosteroids includes a history of Cushing's syndrome, osteopenia / osteoporosis, hyperglycemia, insomnia and / or infection.

[0478] The immunomodulator can be, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. An inadequate response to an immunomodulator is defined as persistent signs and symptoms of active disease despite at least one 8-week regimen or history of oral azathioprine (greater than or equal to 1.5 mg / kg), 6-mercaptopurine (greater than or equal to 0.75 mg / kg), or methotrexate (greater than or equal to 12.5 mg / week). Intolerance to immunomodulators includes, but is not limited to, nausea / vomiting, abdominal pain, pancreatitis, LFT abnormalities, lymphocytopenia, TPMT gene mutations, and / or infection.

[0479] TNFα antagonists are, for example, agents that inhibit the biological activity of TNFα and preferably bind to TNFα, such as monoclonal antibodies, such as REMICADE (infliximab), HUMIRA (adalimumab), CIMZIA (certolizumab pegol), SIMPONI (golimumab); or circulating receptor fusion proteins, such as ENBREL (etanercept). An inadequate response to a TNF-α antagonist refers to signs and symptoms of persistent active disease despite a history of at least one of a 4-week induction regimen of infliximab 5 mg / kg IV, 2 doses at least 2 weeks apart; adalimumab once 80 mg subcutaneous dose, followed by 40 mg once at least two weeks apart; or 400 mg subcutaneous certolizumab pegol, 2 doses at least 2 weeks apart. Loss of response to a TNF-α antagonist refers to the recurrence of symptoms during maintenance dosing after a previous clinical benefit. Intolerance to TNFα antagonists includes, but is not limited to, infusion-related reactions, demyelination, congestive heart failure, and / or infection.

[0480] As used herein with respect to subjects with ulcerative colitis, loss of maintenance of remission refers to an increase in the Mayo score of at least 3 points and an increase in the modified Baron score of at least 2.

[0481] In one embodiment, diseases that can be treated accordingly include, but are not limited to, inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis, celiac disease, non-tropical sprue, enteropathy associated with seronegative arthropathy, microscopic colitis or collagenous colitis, eosinophilic gastroenteritis, or pouchitis following proctocolectomy and ileoanal anastomosis. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Other diseases that can be treated include, for example, primary sclerosing cholangitis (PSC) and graft versus host disease (GVHD).

[0482] The ulcerative colitis may be moderately to severely active ulcerative colitis (e.g., Mayo score of 6 to 12, endoscopic subscore of 2 or 3). In patients with moderately to severely active ulcerative colitis, treatment may result in induction and maintenance of clinical response, induction and maintenance of clinical remission, or mucosal healing. Treatment may also result in patients reducing, eliminating, or reducing and eliminating the use of corticosteroids (e.g., remission without corticosteroids).

[0483] Crohn's disease can be moderate to severe active Crohn's disease (e.g., Crohn's disease activity index (CDAI) score of 220 to 450). Treatment can achieve a clinical response or achieve clinical remission in patients with moderate to severe active Crohn's disease. Treatment can also result in a patient reducing, eliminating, or reducing and eliminating the use of corticosteroids (e.g., remission without corticosteroids).

[0484] Pancreatitis and insulin-dependent diabetes are other diseases that can be treated using the compositions of the present invention. It is reported that MAdCAM (e.g., MAdCAM-1) is expressed by some blood vessels in the exocrine pancreas from NOD (non-obese diabetic) mice and from BALB / c and SJL mice. It is reported that the expression of MAdCAM (e.g., MAdCAM-1) is induced on the endothelium in the inflamed islets of the pancreas of NOD mice, and MAdCAM (e.g., MAdCAM-1) is the main addressin expressed by the NOD islet endothelium in the early stages of insulitis (Hanninen, A. et al., J. Clin. Invest., 92: 2509-2515 (1993)). Treatment of NOD mice with anti-MAdCAM or anti-β7 antibodies can prevent the development of diabetes (Yang et al., Diabetes, 46: 1542-1547 (1997)). Furthermore, accumulation of lymphocytes expressing α4β7 in pancreatic islets was observed, and MAdCAM-1 has been implicated in the binding of lymphoma cells via α4β7 to blood vessels from inflamed islets (Hanninen, A. et al., J. Clin. Invest., 92:2509-2515 (1993)) or to the gastrointestinal tract in mantle cell lymphoma (Geissmann et al., Am. J. Pathol., 153:1701-1705 (1998)).

[0485] Examples of inflammatory diseases associated with mucosal tissue that can be treated using the compositions of the invention include cholecystitis; cholangitis (Adams and Eksteen Nature Reviews 6:244-251 (2006); Grant et al., Hepatology 33:1065-1072 (2001)), such as primary sclerosing cholangitis; Behcet's disease, for example, of the intestine or pericholangitis (tissue surrounding the bile duct and liver); and graft-versus-host disease (e.g., graft-versus-host disease in the gastrointestinal tract (e.g., after a bone marrow transplant) (Petrovic et al. Blood 103:1542-1547 (2004)). As seen in Crohn's disease, inflammation often extends beyond the mucosal surface, so chronic inflammatory diseases (such as sarcoidosis, chronic gastritis, such as autoimmune gastritis (Katakai et al., Int. Immunol., 14:167-175 (2002))) and other idiopathic conditions can be treated.

[0486] The invention also relates to methods of inhibiting leukocyte infiltration of mucosal tissue. The invention also relates to methods for treating cancer (e.g., α4β7 positive tumors such as lymphoma). Other examples of inflammatory diseases associated with mucosal tissue that can be treated using the formulations of the invention include mastitis (breast) and irritable bowel syndrome.

[0487] Diseases or pathogens whose etiology utilizes the interaction of MAdCAM (eg, MAdCAM-1) with α4β7 can be treated with anti-α4β7 antibodies in the formulations described herein. Examples of such diseases include immunodeficiency disorders, such as those caused by human immunodeficiency virus (see, e.g., WO2008140602).

[0488] The compositions of the present invention are administered with an effective amount of anti-α4β7 antibodies that inhibit the binding of α4β7 integrin to its ligand. For therapy, an effective amount will be sufficient to achieve the desired therapeutic (including preventive) effect (such as an amount sufficient to reduce or prevent α4β7 integrin-mediated binding and / or signaling, thereby inhibiting leukocyte adhesion and infiltration and / or related cellular responses). An effective amount of anti-α4β7 antibodies, such as an effective titer sufficient to maintain saturation (e.g., neutralization) of α4β7 integrin, can induce clinical response or remission of inflammatory bowel disease. An effective amount of anti-α4β7 antibodies can lead to mucosal healing of ulcerative colitis or Crohn's disease. The formulations of the present invention can be administered in unit doses or multiple doses. The dosage can be determined by methods known in the art and can depend on, for example, the age, sensitivity, tolerance and overall health of the individual. Examples of modes of administration include topical routes, such as nasal or inhalation or transdermal administration; enteral routes, such as through a feeding tube or suppository; and parenteral routes, such as intravenous, intramuscular, subcutaneous, intraarterial, intraperitoneal or intravitreal administration. In one embodiment, the total dose is 165 mg. In another embodiment, the total dose is 108 mg. In another embodiment, the total dose is 216 mg. In another embodiment, the total dose is 300 mg.

[0489] In some aspects, the dosing regimen for treating a disease described herein (e.g., UC or Crohn's disease) has two phases, an induction phase and a maintenance phase. In the induction phase, the antibody or its antigen-binding fragment is administered in a manner that quickly provides an effective amount of the antibody or its antigen-binding fragment suitable for certain purposes, such as inducing immune tolerance to the antibody or its antigen-binding fragment or inducing clinical responses and improving inflammatory bowel disease symptoms. When the patient is treated with an anti-α4β7 antibody for the first time, when the patient is treated after a long time without receiving therapy (e.g., more than three months, more than four months, more than six months, more than nine months, more than one year, more than eighteen months or more than two years since the anti-α4β7 antibody therapy), or during the maintenance phase of anti-α4β7 antibody treatment when symptoms of inflammatory bowel disease reappear (e.g., relapse after disease remission), the patient can be administered an induction phase treatment. In some embodiments, the induction phase regimen results in a higher average trough serum concentration (e.g., a concentration just before the next administration) than the average steady-state trough serum concentration maintained during the maintenance regimen.

[0490] In the maintenance phase, the antibody or its antigen-binding fragment is administered in a manner that continues the response achieved by induction therapy with a stable level of the antibody or its antigen-binding fragment. The maintenance regimen can prevent the symptoms of inflammatory bowel disease from reappearing or recurring. The maintenance regimen can provide convenience for patients (e.g., a simple dosing regimen) or require infrequent courses of treatment. In some embodiments, the maintenance regimen may include administering an anti-α4β7 antibody or its antigen-binding fragment (e.g., in a formulation described herein) by a strategy selected from the group consisting of: low doses, infrequent administration, self-administration, and a combination of any of the foregoing.

[0491] In one embodiment, for example, in the induction phase of therapy, the dosing regimen provides an effective amount of an anti-α4β7 antibody or antigen-binding fragment in a formulation described herein for inducing remission of inflammatory bowel disease in a human patient. The duration of the induction period can be about four weeks, about five weeks, about six weeks, about seven weeks, or about eight weeks of treatment. In some embodiments, the induction regimen can utilize a strategy selected from the group consisting of: high-dose anti-α4β7 antibodies or antigen-binding fragments thereof, frequent administration of anti-α4β7 antibodies or antigen-binding fragments thereof, and a combination of high-dose anti-α4β7 antibodies or antigen-binding fragments thereof and frequent administration of anti-α4β7 antibodies or antigen-binding fragments thereof (e.g., in a formulation described herein). Induction administration can be one or more times more than one dose, such as at least two doses. In the induction phase, a dose can be administered once a day, every other day, twice a week, once a week, once every ten days, once every two weeks, or once every three weeks. In some embodiments, the induction dose is administered within the first two weeks of therapy with an anti-α4β7 antibody. In one embodiment, induction dosing can be performed once at the start of treatment (day 0) and once about two weeks after the start of treatment. In another embodiment, the induction phase duration is six weeks. In another embodiment, the induction phase duration is six weeks, and multiple induction doses are administered during the first two weeks.

[0492] In some embodiments, for example, when starting treatment for patients with severe inflammatory bowel disease (e.g., in patients who have failed anti-TNFα therapy), the induction period requires a longer duration than patients with mild or moderate disease. In some embodiments, the induction period for patients with severe disease may have a duration of at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks. In one embodiment, the induction dosing regimen for patients with severe disease may include a dose at week 0 (beginning of treatment), a dose at week 2, and a dose at week 6. In another embodiment, the induction dosing regimen for patients with severe disease may include a dose at week 0 (beginning of treatment), a dose at week 2, a dose at week 6, and a dose at week 10.

[0493] The dosage can be applied once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 10 weeks. Higher or more frequent dosages, such as every other day, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks, can be used to induce the alleviation of active diseases or for treating new patients, such as inducing tolerance to anti-α4β7 antibodies. Dosages once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks or once every 10 weeks can be used for preventive therapy, such as to maintain the alleviation of chronic disease patients. On the one hand, the treatment regimen is to treat at day 0, about week 2, about week 6 and every 1 or 2 weeks thereafter. In certain aspects, the treatment regimen is to treat at day 0, about week 2, about week 6 and every 8 weeks thereafter. On the other hand, the induction treatment regimen is to treat every other day, for a total of 6 treatments.

[0494] In some aspects, durable clinical remission can be achieved with an optimized dosing regimen, for example, clinical remission that persists to at least two, at least three, at least four visits by a nursing physician over a six-month or one-year period after initiation of treatment.

[0495] In some aspects, a durable clinical response can be achieved with an effective dosing regimen, for example, a clinical response that lasts for at least 6 months, at least 9 months, at least one year after initiation of treatment.

[0496] The present disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way.

[0497] Example

[0498] The following example describes the steps in an exemplary process for purifying vedolizumab from CHO cell culture.

[0499] Example 1. Controlling the Charged Isoform of Vedolizumab

[0500] Vedolizumab has three charged isoforms: acidic, major, and basic. Cation exchange (CEX)-HPLC can be used to quantify the isoform distribution of vedolizumab based on the relative areas of the chromatograms representing the acidic, major, and basic species. Figure 1 Exemplary CEX-HPLC curves depicting these three vedolizumab species are shown in .

[0501] Using CEX-HPLC, the charged isoform distribution of vedolizumab was evaluated after storage under various conditions. As summarized in Table 1, in-process retention during the manufacture of vedolizumab affected the distribution of charged isoform species, with basic isoforms being most affected by retention conditions.

[0502] Table 1. Qualitative changes in basic isoforms in process intermediates

[0503]

[0504] These results show that by keeping the antibody under certain conditions, the ratio of different isotypes of vedolizumab can be predictably adjusted. Basic isotypes tend to increase with the duration of retention at a pH less than about 6.5. In contrast, basic isotypes decrease with the duration of retention at a pH greater than about 6.5. These changes are observed for vedolizumab produced at pilot scale (Table 2) and manufacturing scale (Table 3).

[0505] Table 2. Vedolizumab isotypes during in-process retention - pilot purification

[0506]

[0507] Table 3. Vedolizumab isotypes during in-process retention - manufacturing scale purification

[0508]

[0509]

[0510] Other experiments were carried out to assess the charged isotype distribution of the Vedolizumab derived from GS-CHO cells. At 5 ° C or room temperature, the main species, acidic species and basic species of Vedolizumab were assessed after storage for 0-7 days at multiple pH (i.e., pH 4.7, 5.1, 5.3, 5.7, 5.9, 6.1, 6.5 or 6.9). As shown in Tables 4-11, improved stability was observed at a pH greater than or equal to pH 5.9. After protein A capture and elution, the typical pH range of neutralization (pH about 4.9 to 5.2) is related to the increase in the formation of basic species. Relative to the basic species observed during storage at pH < 5.9, the increase of basic species of Vedolizumab slowed down when stored at pH 5.9 or 6.1. As shown in Tables 10 and 11, the increase of basic species was stopped or even reversed when pH was greater than or equal to pH 6.5.

[0511] Table 4. Vedolizumab isoform distribution during in-process retention at pH 4.7

[0512]

[0513]

[0514] Table 5. Vedolizumab isoform distribution during in-process retention at pH 5.1

[0515] Storage temperature(℃) Retention days Acidity % main% Alkalinity% <![CDATA[T0]]> 0 14.37 71.60 14.02 5℃ 1 14.21 71.38 14.42 5℃ 2 14.09 71.39 14.52 5℃ 3 14.25 71.47 14.28 5℃ 5 14.07 71.19 14.74 5℃ 7 14.15 70.51 15.34 Room temperature 1 13.95 71.20 14.85 Room temperature 2 13.65 69.63 16.72 Room temperature 3 13.55 67.97 18.48 Room temperature 5 13.07 66.01 20.92 Room temperature 7 12.63 63.18 24.19

[0516] Table 6. Vedolizumab isotype distribution during retention in the process at pH 5.3

[0517] Storage temperature(℃) Retention days Acidity % main% Alkalinity% <![CDATA[T0]]> 0 14.23 72.22 13.55 5℃ 1 14.36 71.61 14.03 5℃ 2 14.36 71.02 14.62 5℃ 3 14.21 71.59 14.20 5℃ 5 13.98 71.06 14.96 5℃ 7 14.00 70.94 15.06 Room temperature 1 14.03 71.04 14.94 Room temperature 2 13.83 70.03 16.15 Room temperature 3 13.48 68.68 17.84 Room temperature 5 13.29 66.88 19.84 Room temperature 7 12.77 64.93 22.30

[0518] Table 7. Vedolizumab isotype distribution during retention in the process at pH 5.7

[0519]

[0520]

[0521] Table 8. Vedolizumab isotype distribution during retention in the process at pH 5.9

[0522] Storage temperature(℃) Retention days Acidity % main% Alkalinity% <![CDATA[T0]]> 0 14.62 71.53 13.86 5℃ 1 14.29 71.59 14.13 5℃ 2 14.57 71.57 13.86 5℃ 3 14.44 71.29 14.27 5℃ 5 14.32 71.34 14.34 5℃ 7 14.50 71.29 14.21 Room temperature 1 14.34 71.18 14.48 Room temperature 2 14.31 70.88 14.81 Room temperature 3 14.26 70.46 15.28 Room temperature 5 14.47 68.98 16.55 Room temperature 7 14.09 68.08 17.84

[0523] Table 9. Vedolizumab isotype distribution during retention in the process at pH 6.1

[0524]

[0525]

[0526] Table 10. Vedolizumab isotype distribution during retention in the process at pH 6.5

[0527] Storage temperature(℃) Retention days Acidity % main% Alkalinity% <![CDATA[T0]]> 0 14.49 71.70 13.81 5℃ 3 14.30 72.30 13.40 5℃ 7 14.33 72.03 13.65 Room temperature 1 14.52 71.57 13.91 Room temperature 2 14.64 71.47 13.89 Room temperature 3 14.68 71.72 13.60 Room temperature 5 14.71 71.60 13.69 Room temperature 7 14.86 71.00 14.15

[0528] Table 11. Vedolizumab isotype distribution during retention in the process at pH 6.9

[0529] Storage temperature(℃) Retention days Acidity % main% Alkalinity% <![CDATA[T0]]> 0 14.42 71.34 14.25 5C 3 14.59 71.58 13.82 5C 7 14.71 71.60 13.69 Room temperature 1 14.96 71.56 13.48 Room temperature 2 15.08 71.87 13.05 Room temperature 3 15.09 72.14 12.77 Room temperature 5 15.58 72.39 12.03 Room temperature 7 15.96 71.88 12.16

[0530] This data indicates that the levels of the major isotype and the basic isotype in the vedolizumab formulation can be regulated by pH. In particular, exposure of the antibody to low pH (<pH 5.9) increases the basic isotype species while the major isotype decreases accordingly. As the exposure to decreasing pH continues, the level of the basic isotype species increases more rapidly and to a greater extent. Additionally, this trend can be reversed by exposure to elevated pH, e.g., >pH 6.5.

[0531] Example 2. Reduction of the basic isotype of vedolizumab

[0532] To further evaluate the effect of pH on the formation of vedolizumab basic isoforms, the antibody was exposed to high pH conditions (200 mM Tris, pH 9) and cation exchange (CEX)-HPLC was used to quantify the isoform distribution of vedolizumab. For each condition, the relative amount of each vedolizumab isoform was quantified by determining the relative area under the chromatographic peaks corresponding to the acidic isoform, the major isoform, and the basic isoform.

[0533] As shown in Table 12, three peaks corresponding to basic species of vedolizumab appeared at pH 6.3 (control) (i.e., "basic peak 1", "basic peak 2", and "basic peak 3"). At elevated pH, a significant decrease in basic peak 2 was observed, as shown in Table 12.

[0534] Table 12. Sensitivity of basic peak 2 to elevated pH

[0535] Comparison After exposure to 200 mM Tris HCl (pH 9) name area% area% Basic Peak-1 6.85 6.8 Basic Peak-2 3.09 0.85 Basic Peak-3 0.83 0.56

[0536] The material with the characteristic retention time of basic peak 2 eluted from the CEX resin was collected and subjected to enzymatic digestion and subsequently analyzed using mass spectrometry (MS). The characteristic MS peak of succinimide was present in the basic peak 2 CEX fraction from the control formulation, but was absent in the formulation analyzed after exposure to pH 9. Analysis of the primary amino acid sequence of vedolizumab identified aspartate residues in CDR-H3 of vedolizumab that are located near residues that are favorable for aspartate isomerization to succinimide, i.e., glycine and serine at the n+1 position (CDR-H3: GGY D GWDYAIDY (SEQ ID NO: 4)). This finding suggests that the increase in basic species of vedolizumab observed at low pH may be due to the isomerization of this aspartic acid residue to succinimide. Maintaining the antibody at or near neutral pH can slow or prevent the formation of basic peak 2, and treatment with elevated pH (> pH 6.9) can reverse the isomerization reaction, converting succinimide back to aspartic acid (or isoaspartic acid).

[0537] To further evaluate the effect of pH on basic peak 2, relative peak areas were determined by CEX-HPLC after the antibodies were exposed to different pH conditions (pH 6.5, pH 7, pH 8, pH 8.5, or pH 9). As shown in Table 13, basic peak 2 is highly sensitive to pH and decreases with increasing pH, which is consistent with the findings reported in Example 1.

[0538] Table 13. Loss of Basic Peak 2 at Elevated pH

[0539]

[0540]

[0541] The level of the vedolizumab isoform corresponding to basic peak 2 was then evaluated based on vedolizumab formulations derived from two different CHO cell lines (DHFR-CHO and GS-CHO). As shown in Table 14, a reduction in basic peak 2 was observed in vedolizumab formulations derived from both DHFR-CHO and GS-CHO cell lines.

[0542] Table 14. Loss of basic peak 2 in antibody formulations derived from two different CHO cell lines

[0543]

[0544] Example 3. Effect of anion exchange loading conductivity on host cell protein clearance

[0545] Because it is often desirable to reduce the amount of host cell protein contaminants in therapeutic protein compositions, manufacturing methods for producing vedolizumab compositions with reduced host cell protein content were examined.

[0546] The standard operating range of buffer conductivity for anion exchange (AEX) (e.g., by anion exchange Q membrane adsorbent) is about 11-15 mS / cm (average value of about 13.6 mS / cm). In order to evaluate the impurity clearance under conditions beyond the standard operating range, the impurity clearance was tested in a composition obtained using lower conductivity AEX conditions. Two independent starting preparations (harvest 1 and harvest 2) of vedolizumab clarified harvest were tested. After the conductivity of the loading material was adjusted to standard conductivity (about 13.6 mS / cm) or low conductivity (about 11 mS / cm), all samples were subjected to AEX purification. As shown in Table 15, the HCP level of the low conductivity AEX loading material was reduced compared to the standard conductivity AEX loading material.

[0547] Table 15. HCP clearance using rabbit-goat (RaGo) process-specific ELISA

[0548]

[0549]

[0550] Next, AEX performance (flow-through mode) was tested using a loading buffer with a conductivity range below standard operating conditions (i.e., below 13-15 mS / cm). Table 16 summarizes the loading conditions, conductivity, host cell protein content, and the percentage of acidic and basic isoforms. CHO host cell protein ELISA was used for testing. As shown in Table 16, the amount of host cell protein decreased as the conductivity of the AEX loading buffer decreased.

[0551] Table 16. Conductivity variation study - Q membrane performance

[0552]

[0553] The effect of reduced AEX conductivity on HCP clearance was then assessed by two different HCP ELISA methods. In the first method, rabbit primary antibodies and anti-rabbit secondary antibodies were used in HCP ELISA (RaRa). The second method utilized rabbit primary antibodies and goat secondary antibodies in HCP ELISA (RaGo). Tables 17 and 18 summarize the results of these two HCP ELISA methods after loading with standard or low conductivity AEX, respectively. Both methods have demonstrated that HCP clearance is improved by reducing the conductivity of the AEX loading material.

[0554] Table 17. Standard conductivity AEX loading conditions (about 13.3 mS / cm)

[0555]

[0556]

[0557] Table 18. Low conductivity AEX loading conditions (about 10.0 mS / cm)

[0558]

[0559] The amount of HCP in a recombinant protein preparation is variable. As shown herein, the HCP clearance of a vedolizumab preparation can be improved by reducing conductivity during purification using AEX. Independent of the HCP concentration in the starting material, a lower conductivity AEX condition achieves a greater reduction in HCP than a standard higher conductivity AEX condition.

[0560] Example 4. Effect of equilibration and wash conditions on mixed mode resin loading capacity

[0561] In order to minimize the loss of vedolizumab during the purification process, various purification conditions were examined to identify steps where the antibody product yield decreased. Protein loss was observed during the wash steps of ceramic hydroxyapatite (CHT) purification under high concentration loading conditions. For example, Figure 2 The elution profiles of vedolizumab from a CHT column after loading at 27 mg / ml or 35 mg / ml using standard wash and equilibration buffer (75 mM NaCl, 10 mM sodium phosphate, pH 7.2) were compared. Figure 2 As shown, under high concentration loading conditions (35 mg / ml), the protein began to exit the column during the wash step.

[0562] To increase the loading capacity of vedolizumab on the CHT column and to improve the yield of vedolizumab by reducing the amount of protein loss in the CHT wash step, alternative CHT equilibration buffers, loading buffers, and wash buffers were evaluated. The vedolizumab loading (g / l) on the CHT column and the resulting yield achieved after CHT column operation using a lower pH buffer (50mM NaCl, 10mM sodium phosphate, pH 6.7) for CHT equilibration and washing were determined compared to CHT column operation using a standard CHT buffer (75mM NaCl, 10mM sodium phosphate, pH 7.2) at elevated pH. Figure 3 As shown, there was a large amount of protein loss during the wash step using standard CHT buffer conditions (dashed line), but no protein loss was observed using the low pH buffer (solid line). In addition, as summarized in Table 19, the use of low pH buffers in the equilibration and wash steps allowed for a larger loading volume on the CHT column and resulted in a 2%-3% higher yield in the final eluate without changing the product quality (e.g., percentage of aggregates) than that achieved using standard CHT buffer.

[0563] Table 19. Analytical comparison: standard CHT versus CHT with low pH buffer

[0564]

[0565] Equivalence Case

[0566] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0567] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, materials, methods and embodiments are merely illustrative and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0568] Sequence Listing

[0569]

[0570]

Claims

1. A method for purifying a humanized anti-α4β7 antibody or an antigen-binding portion thereof from a clarified cell culture harvest, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of a recombinant host cell expressing the anti-α4β7 antibody or antigen-binding portion thereof, and (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 below for no longer than 24 hours, The anti-α4β7 antibody or its antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

5.

2. The method of claim 1, wherein the anti-α4β7 antibody, or antigen-binding portion thereof, has reduced levels of basic isotype species (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody, or antigen-binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.0 or below (e.g., pH 3.6-4.0) for a longer period of time, i.e., greater than 24 hours.

3. The method of claim 1 or 2, wherein the anti-α4β7 antibody or antigen binding portion thereof is vedolizumab or an antigen binding portion thereof.

4. The method of any one of claims 1-3, wherein the composition comprising vedolizumab or an antigen-binding portion thereof comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2), and wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having a reduced level of BP2.

5. The method of claim 4, wherein the method produces a composition comprising vedolizumab, or an antigen-binding portion thereof, having less than 2%, less than 1.5%, less than 1%, or less than 0.7% BP2.

6. The method of any one of claims 1-5, wherein the composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

7. The method of claim 6, wherein the mammalian cell culture is a Chinese Hamster Ovary (CHO) cell culture.

8. The method of claim 7, wherein the CHO cell culture comprises CHO cells lacking expression of dihydrofolate reductase (DHFR).

9. The method of claim 7, wherein the CHO cell culture comprises CHO cells lacking expression of glutamine synthetase (GS).

10. The method of any one of claims 1-9, wherein the method further comprises purifying the composition comprising vedolizumab from mammalian host cell proteins (HCP) using one or more chromatographic separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

11. The method of claim 10, wherein the composition comprising vedolizumab is purified using an affinity chromatography resin comprising protein A.

12. The method of claim 11, wherein the composition comprising vedolizumab is purified using affinity chromatography prior to the incubation.

13. The method of claim 11, wherein the composition comprising vedolizumab is purified using affinity chromatography after the incubation.

14. The method of claim 11, wherein the composition comprising vedolizumab is purified using cation exchange chromatography.

15. The method of claim 14, wherein the composition comprising vedolizumab is purified using cation exchange chromatography prior to the incubation.

16. The method of claim 14, wherein the composition comprising vedolizumab is purified using cation exchange chromatography after the incubation.

17. The method of claim 10, wherein the composition comprising vedolizumab is purified using anion exchange chromatography.

18. The method of claim 17, wherein the composition comprising vedolizumab is purified using anion exchange chromatography prior to the incubation.

19. The method of claim 17, wherein the composition comprising vedolizumab is purified using anion exchange chromatography after the incubation.

20. The method of claim 10, wherein the composition comprising vedolizumab is purified using CHT chromatography.

21. The method of claim 20, wherein the composition comprising vedolizumab is purified using CHT chromatography prior to the incubation.

22. The method of claim 20, wherein the composition comprising vedolizumab is purified using CHT chromatography after the incubation.

23. The method of any one of claims 1-22, wherein the method comprises incorporating the composition into a pharmaceutical formulation.

24. The method of claim 23, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

25. The method of claim 24, wherein the lyophilized pharmaceutical formulation is a dried lyophilized pharmaceutical formulation.

26. The method of claim 25, further comprising the step of reconstituting the dried lyophilized pharmaceutical formulation with a liquid to render it suitable for administration.

27. The method of claim 23, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

28. A composition comprising vedolizumab, wherein the composition is produced by the method of any one of claims 1-27.

29. A composition comprising vedolizumab, wherein the composition is obtainable by the method of any one of claims 1-27.

30. The composition of claim 28 or 29, wherein the basic vedolizumab isoform species constitute less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of the vedolizumab species present in the composition.

31. A low basic species composition comprising an anti-α4β7 antibody, wherein the composition comprises 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 of the anti-α4β7 antibody, wherein the basic isoform species have a net positive charge relative to the major isoform of the anti-α4β7 antibody and can be quantified by determining the relative area of ​​a peak that elutes slower from a cation exchange (CEX) resin than the peak corresponding to the major isoform, and wherein the 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.

32. The composition of claim 31, wherein the composition comprises a first basic isoform peak (BP1) and a second basic isoform peak (BP2).

33. The composition of claim 32, wherein the composition comprises less than 2% BP2.

34. The composition of claim 32, wherein the composition comprises less than 1.5% BP2.

35. The composition of claim 32, wherein the composition comprises less than 1% BP2.

36. The composition of claim 32, wherein the composition comprises less than 0.7% BP2.

37. The composition of claim 32, wherein the ratio of BP1 to BP2 is at least 3.

38. The composition of claim 32, wherein the ratio of BP1 to BP2 is at least 5.

39. The composition of claim 32, wherein the ratio of BP1 to BP2 is at least 7.

40. The composition of claim 32, wherein the ratio of BP1 to BP2 is at least 10.

41. A pharmaceutical composition comprising the composition of any one of claims 31-40 and a pharmaceutically acceptable carrier or excipient.

42. A method of producing a composition comprising vedolizumab, the method comprising: (a) contacting a sample containing vedolizumab and a host cell protein (HCP) with an anion exchange resin in the presence of a loading buffer, wherein the loading buffer has a conductivity of 11 mS / cm or less, such that the HCP binds to the anion exchange resin; and (b) collecting flow-through material from the anion exchange resin, The eluate contains vedolizumab.

43. The method of claim 42, wherein the method is a method of producing a composition comprising vedolizumab having a reduced amount of HCPs, and the flow-through material comprises vedolizumab and a reduced amount of HCPs.

44. The method of claim 42 or 43, wherein the loading buffer has a conductivity of 9 to 11 mS / cm.

45. The method of claim 42 or 43, wherein the loading buffer has a conductivity of 10 mS / cm or less.

46. ​​The method of claim 42 or 43, wherein the loading buffer has a conductivity of 9 mS / cm or less.

47. The method of claim 42 or 43, wherein the loading buffer has a conductivity of about 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm, or 11 mS / cm.

48. The method of any one of claims 42-47, wherein the HCP is Chinese Hamster Ovary (CHO) cell protein.

49. The method of claim 48, wherein the HCP is derived from CHO cells lacking expression of dihydrofolate reductase (DHFR).

50. The method of claim 48, wherein the HCP is derived from CHO cells lacking expression of glutamine synthetase (GS).

51. The method of any one of claims 42-50, further comprising contacting the anion exchange resin with a wash buffer.

52. The method of claim 51, wherein the wash buffer has a conductivity of less than 11 mS / cm.

53. The method of claim 51, wherein the wash buffer has a conductivity of 9 to 11 mS / cm.

54. The method of claim 51, wherein the wash buffer has the same conductivity as the loading buffer.

55. The method of any one of claims 42-54, wherein the loading buffer comprises sodium chloride and / or sodium phosphate.

56. The method of any one of claims 42-54, wherein the wash buffer comprises sodium chloride and / or sodium phosphate.

57. The method of any one of claims 42-56, wherein the anion exchange resin is formatted as an anion exchange column or an anion exchange membrane.

58. The method of any one of claims 42-57, wherein the anion exchange resin comprises quaternary amine functional groups.

59. The method of any one of claims 42-58, wherein the sample containing vedolizumab and HCP is derived from a mammalian cell culture after one or more chromatographic separation steps.

60. The method of claim 59, wherein the one or more chromatographic separation steps comprise one or more steps selected from the group consisting of affinity chromatography, cation exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

61. The method of any one of claims 42-60, wherein the amount of HCP in the flow-through material is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

62. The method of any one of claims 42-61, wherein the amount of HCP in the flow-through material is reduced by at least 50% relative to the amount of HCP in the flow-through material produced when the method is performed using the same sample with a loading buffer having a conductivity greater than 12 mS / cm.

63. The method of any one of claims 42-62, wherein the method further comprises treating the flow-through material by a process comprising ultrafiltration and / or diafiltration to exchange the elution buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients.

64. The method of any one of claims 62-63, wherein the method comprises incorporating the composition into a pharmaceutical formulation.

65. The method of claim 64, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

66. The method of claim 65, wherein the lyophilized pharmaceutical formulation is a dried lyophilized pharmaceutical formulation.

67. The method of claim 66, further comprising the step of reconstituting the dried lyophilized pharmaceutical formulation with a liquid to render it suitable for administration.

68. The method of claim 64, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

69. The method of claim 68, wherein the liquid drug formulation is suitable for subcutaneous administration to a human.

70. A composition comprising vedolizumab produced by the method of any one of claims 42-69.

71. A composition comprising vedolizumab, wherein the composition is obtainable by the method of any one of claims 42-69.

72. The composition of claim 70 or 71, wherein the amount of HCP in the composition is 8 ppm or less, 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm 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 2 ppm or less.

73. A method for increasing the yield of vedolizumab recovered after elution from a mixed mode chromatography resin, the method comprising equilibrating the mixed mode chromatography resin with an equilibration buffer, loading a solution comprising vedolizumab and a loading buffer onto the mixed mode chromatography resin so that the vedolizumab binds to the mixed mode chromatography resin, washing the mixed mode chromatography resin with a wash buffer, and eluting the vedolizumab from the mixed mode chromatography resin with an elution buffer, wherein the equilibration buffer, the loading buffer and / or the wash buffer have a pH of 7.0 or below.

74. The method of claim 73, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a pH of 6.0-7.

0.

75. The method of claim 73, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a pH of 6.5-7.

0.

76. The method of claim 73, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a pH of 6.6-6.

8.

77. The method of any one of claims 73-76, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 30 mM to 70 mM.

78. The method of claim 77, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 40 mM to 70 mM.

79. The method of claim 77, wherein the equilibration buffer, the loading buffer and / or the wash buffer have a salt concentration of 50 mM to 65 mM.

80. The method of claim 77, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a salt concentration of 55 mM-65 mM.

81. The method of any one of claims 77-80, wherein the salt comprises sodium chloride and / or sodium phosphate.

82. The method of any one of claims 73-76, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 30 mM to 70 mM.

83. The method of claim 82, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 40 mM to 70 mM.

84. The method of claim 82, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 40 mM to 60 mM.

85. The method of claim 82, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of 45 mM-55 mM.

86. The method of claim 82, wherein the equilibration buffer, the loading buffer and / or the wash buffer has a sodium chloride concentration of about 50 mM.

87. The method of any one of claims 82-86, wherein the equilibration buffer, the loading buffer and / or the wash buffer further comprises sodium phosphate.

88. The method of any one of claims 73-87, wherein the equilibration buffer, the loading buffer and / or the wash buffer have the same pH.

89. The method of any one of claims 73-88, wherein the equilibration buffer, the loading buffer and / or the wash buffer have the same salt concentration.

90. The method of any one of claims 73-87, wherein the equilibration buffer, the loading buffer and / or the wash buffer are the same buffer.

91. The method of any one of claims 73-90, wherein the mixed mode resin is a ceramic hydroxyapatite resin.

Images