Methods of producing anti-alpha4beta7 antibodies

Abstract

Provided herein are methods for purifying anti-a4b7 integrin antibodies, such as vedolizumab, from a liquid solution, e.g., from a mammalian cell culture clarified harvest. The invention relates, inter alia, to purification methods for controlling the amount of product-related substances and / or process-related impurities present in purified preparations of anti-a4b7 integrin antibodies or antigen-binding fragments thereof (e.g., vedolizumab). Also provided are compositions comprising anti-a4b7 antibodies and their use in treating disorders.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for purifying an anti-a4b7 antibody or fragment thereof.

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application 62 / 859,494, filed June 10, 2019. The entire contents of the foregoing application are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 5, 2020, is named T103022_1090WO_SL.txt and is 10,009 bytes in size. BACKGROUND

[0006] Large-scale, economic purification of proteins is an increasingly important issue in the biotechnology industry. Typically, biopharmaceuticals are produced by cell culture using prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian or fungal) cell lines that have been engineered to produce large quantities of the therapeutic protein of interest. Since the cell lines used are living organisms, they must be fed a complex cell culture medium containing sugars, amino acids, and growth factors (sometimes supplied by animal serum preparations). Isolating the desired recombinant therapeutic protein from process-related impurities (including, for example, cell culture medium components, host cell proteins (HCPs), host nucleic acids, and / or chromatography materials) as well as product-related impurities (such as aggregates, misfolded species, or fragments of the protein of interest) to a purity sufficient for use as a human therapeutic is a formidable challenge.

[0007] Product-related impurities and process-related impurities, including aggregates, have the potential to interfere with the purification process, affect the protein during storage, and / or can 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).

[0008] Accordingly, there remains a need in the art for improved methods of purifying therapeutic proteins (e.g., antibodies) while effectively removing impurities, improving the recovery of the protein, and maintaining therapeutic requirements. SUMMARY

[0009] The present application is based, at least in part, on the development of processes for producing anti-a4b7 antibodies or antigen-binding portions thereof. In some embodiments, the anti-a4b7 antibodies or antigen-binding portions thereof are humanized anti-a4b7 antibodies or antigen-binding portions thereof. In any of the aspects and embodiments below, the humanized anti-a4b7 antibodies or antigen-binding portions thereof can comprise a heavy chain variable region comprising a CDR1 set forth in SEQ ID NO: 2, a CDR2 set forth in SEQ ID NO: 3, and a CDR3 set forth in SEQ ID NO: 4, and / or a light chain variable region comprising a CDR1 set forth in SEQ ID NO: 6, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 8. In some embodiments, the humanized anti-a4b7 antibodies or antigen-binding portions thereof comprise a heavy chain variable region of SEQ ID NO: 1 and / or a light chain variable region of SEQ ID NO: 2. In some embodiments, the humanized anti-a4b7 antibodies or antigen-binding portions thereof comprise a heavy chain of SEQ ID NO: 9 and / or a light chain of SEQ ID NO: 10. In exemplary embodiments, the anti-a4b7 antibodies are vedolizumab or antigen-binding portions thereof.

[0010] Thus, in one aspect, the present application provides a method of producing a composition comprising a humanized anti-a4b7 antibody or antigen-binding portion thereof, the method comprising: providing a composition comprising a humanized anti-a4b7 antibody or antigen-binding portion thereof at a pH greater than 6.5; and incubating the composition comprising the humanized anti-a4b7 antibody or antigen-binding portion thereof for a period of time of at least 20 minutes to 10 hours; thereby producing a composition comprising a humanized anti-a4b7 antibody or antigen-binding portion thereof.

[0011] In another aspect, the present application provides a method of producing a composition comprising a humanized anti-a4b7 antibody or antigen-binding portion thereof having reduced levels of basic isoform species, the method comprising: providing a composition comprising a humanized anti-a4b7 antibody or antigen-binding portion thereof at a pH greater than 6.5; and incubating the composition comprising the humanized anti-a4b7 antibody or antigen-binding portion thereof for a period of time sufficient to reduce the levels of basic isoform species of the anti-a4b7 antibody or antigen-binding portion thereof in the composition; thereby producing a composition comprising a humanized anti-a4b7 antibody or antigen-binding portion thereof having reduced levels of basic isoform species.

[0012] In some embodiments of the foregoing aspects, the humanized anti-a4b7 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR1 set forth in SEQ ID NO: 2, a CDR2 set forth in SEQ ID NO: 3, and a CDR3 set forth in SEQ ID NO: 4, and / or a light chain variable region comprising a CDR1 set forth in SEQ ID NO: 6, a CDR2 set forth in SEQ ID NO: 7, and a CDR3 set forth in SEQ ID NO: 8. In some embodiments, the humanized anti-a4b7 antibody, or 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-a4b7 antibody, or 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-a4b7 antibody is vedolizumab or an antigen binding portion thereof.

[0013] In some embodiments, the method produces a composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, having < 16%, < 15%, < 14%, < 13%, < 12%, < 11%, or < 10% of basic isoform species.

[0014] In some embodiments, the incubating is performed during purification of the humanized anti-a4b7 antibody, or antigen binding portion thereof, and wherein the incubating is performed (a) prior to ultrafiltration / diafiltration (UF / DF) of the antibody or (b) prior to formulating the antibody in a pharmaceutically acceptable buffer.

[0015] In some embodiments, the incubating is performed at ambient temperature. In some embodiments, the incubating is performed at 15 °C - 30 °C. In some embodiments, the incubating is performed at 20 °C - 25 °C.

[0016] In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is provided at a pH of about 6.5-8.5. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is provided at a pH of about 7.0-8.0. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is provided at a pH of about 7.0-7.5. In other embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is provided at a pH of about 6.6-7.3. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or 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.

[0017] In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 10 to 120 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 12 to 120 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 12 to 96 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 12 to 72 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 12 to 48 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of at least 12 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or 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-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 24 to 120 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 24 to 96 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 24 to 72 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is incubated for a period of about 24 to 48 hours. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or 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.

[0018] In another aspect, provided herein are methods of purifying a humanized anti-a4b7 antibody, or 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-a4b7 antibody, or antigen binding portion thereof, and (ii) purifying the anti-a4b7 antibody, or antigen binding portion thereof, from the cell culture harvest, wherein the antibody is exposed to a pH of 4.0 or a pH lower than 4.0 for no more than 24 hours, wherein the anti-a4b7 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0019] In some embodiments, the anti-a4b7 antibody, or antigen binding portion thereof, has reduced levels of basic isoform species (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-a4b7 antibody, or antigen binding portion thereof, produced by the same method, wherein the antibody is exposed to a pH of 4.0 or a pH lower than 4.0 (e.g., pH 3.6 to 4.0) for a longer period of time, i.e., greater than 24 hours.

[0020] In some embodiments, the anti-a4b7 antibody, or antigen binding portion thereof, is vedolizumab, or antigen binding portion thereof.

[0021] In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof (e.g., vedolizumab, or 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-a4b7 antibody, or antigen binding portion thereof, having reduced levels of BP2. In certain embodiments, the method produces a composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, having less than 2%, less than 1.5%, less than 1%, or less than 0.7% BP2.

[0022] In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is derived from a mammalian cell culture expressing the humanized anti-a4b7 antibody, or 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 dihydrofolate reductase (DHFR) expression. In certain embodiments, the CHO cell culture comprises CHO cells lacking glutamine synthetase (GS) expression.

[0023] In some embodiments, the method further comprises purifying the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, from mammalian host cell proteins (HCPs) using one or more chromatography 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).

[0024] In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using an affinity chromatography resin comprising Protein A.

[0025] In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using affinity chromatography prior to incubation to reduce basic isoforms. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using affinity chromatography after incubation.

[0026] In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using cation exchange chromatography. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using cation exchange chromatography prior to incubation. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using cation exchange chromatography after incubation.

[0027] In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using anion exchange chromatography. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using anion exchange chromatography prior to incubation. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using anion exchange chromatography after incubation.

[0028] In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using CHT chromatography. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using CHT chromatography prior to incubation. In certain embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is purified using CHT chromatography after incubation.

[0029] In some embodiments, the method comprises incorporating the composition into a pharmaceutical formulation.

[0030] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In particular embodiments, the lyophilized pharmaceutical formulation is a dry lyophilized pharmaceutical formulation. In some such embodiments, the method further comprises the step of reconstituting the dry lyophilized pharmaceutical formulation with a liquid to render it suitable for administration.

[0031] 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.

[0032] In another embodiment, the present application provides a method of purifying a humanized anti-a4b7 antibody, or antigen binding portion thereof, having reduced levels of basic isoform species, wherein the humanized anti-a4b7 antibody, or antigen binding portion thereof, is maintained at pH 6.5 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-a4b7 antibody, or antigen binding portion thereof, from a cell culture harvest by ultrafiltration / diafiltration (UF / DF) to formulation of the humanized anti-a4b7 antibody, or antigen binding portion thereof, in a pharmaceutically acceptable carrier. By maintaining the pH of the humanized anti-a4b7 antibody, or antigen binding portion thereof, at pH 6.5 or above pH 6.5 for a majority of the purification process, the humanized anti-a4b7 antibody, or antigen binding portion thereof, can be reduced relative to an equivalent purification process in which the humanized anti-a4b7 antibody, or antigen binding portion thereof, is maintained at a pH below pH 6.5 for a substantial amount 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-a4b7 antibody, or antigen binding portion thereof, from a cell culture harvest by ultrafiltration / diafiltration (UF / DF) to formulation of the anti-a4b7 antibody, or antigen binding portion thereof, in a pharmaceutically acceptable carrier) of basic isoform species of the humanized anti-a4b7 antibody, or antigen binding portion thereof.

[0033] In another aspect, the present application provides a composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof (e.g., vedolizumab), having reduced levels of basic isoform species. In some embodiments, the humanized anti-a4b7 antibody, or antigen binding portion thereof, has 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 basic isoform species composition present in the humanized anti-a4b7 antibody, or antigen binding portion thereof, in the composition. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, having reduced levels of basic isoform species is produced using the methods described herein. In some embodiments, the composition comprising a humanized anti-a4b7 antibody, or antigen binding portion thereof, is produced by the combination of the methods of the present application.

[0034] In another aspect, provided herein is a method of producing a composition comprising a humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab), the method comprising: (a) contacting a sample comprising a humanized anti-a4b7 antibody or antigen binding portion thereof and host cell proteins (HCPs) 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 HCPs bind to the anion exchange resin; (b) collecting flow through material from the anion exchange resin, wherein the flow through material comprises the humanized anti-a4b7 antibody or antigen binding portion thereof.

[0035] In another aspect, provided herein is a method of producing a composition comprising a humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab), the method comprising: (a) contacting a sample comprising a humanized anti-a4b7 antibody or antigen binding portion thereof and host cell proteins (HCPs) 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 (e.g., 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), such that the HCPs bind to the anion exchange resin; (b) contacting the anion exchange resin with an elution buffer; and (c) collecting flow through from the anion exchange resin, wherein the flow through comprises the humanized anti-a4b7 antibody or antigen binding portion thereof.

[0036] In some embodiments of the above aspects, the method is a method of producing a composition comprising a humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab) having a reduced amount of HCPs, and the flow through comprises the humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab) and a reduced amount of HCPs.

[0037] In some embodiments, the loading buffer has a conductivity of 9 mS / cm to 11 mS / cm (e.g., about 9 mS / cm to about 11 mS / cm or about 10 mS / cm to about 11 mS / cm).

[0038] 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).

[0039] 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.

[0040] In some embodiments, the HCPs are Chinese hamster ovary (CHO) cell proteins. In certain embodiments, the HCPs are derived from CHO cells lacking dihydrofolate reductase (DHFR) expression. In certain embodiments, the HCPs are derived from CHO cells lacking glutamine synthetase (GS) expression.

[0041] In some embodiments, the anion exchange resin is washed with a wash buffer. In some embodiments, the wash buffer has a conductivity of 11 mS / cm or less. In some embodiments, the wash buffer has a conductivity of 9 mS / cm to 11 mS / cm. In some embodiments, the wash buffer has a conductivity of less than 11 mS / cm. In some embodiments, the wash buffer has a conductivity of about 9 mS / cm, 9.5 mS / cm, 10 mS / cm, 10.5 mS / cm, or 11 mS / cm. In some embodiments, the wash buffer has the same conductivity as the loading buffer.

[0042] In some embodiments, the loading buffer comprises sodium chloride and / or sodium phosphate.

[0043] 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 can 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 can 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.

[0044] In some embodiments, the wash buffer comprises sodium chloride and / or sodium phosphate.

[0045] 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 can 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 can 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.

[0046] In some embodiments, the conductivity of the wash buffer is equal to or lower than the conductivity of the loading buffer. In some embodiments, the wash buffer has the same conductivity as the loading buffer.

[0047] In some embodiments, the anion exchange resin is formatted as an anion exchange column or an anion exchange membrane.

[0048] In some embodiments, the anion exchange resin comprises a quaternary amine functional group.

[0049] In some embodiments, the sample comprising the humanized anti-a4b7 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 comprise 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.

[0050] In some embodiments, the amount of HCP in the flow-through 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.

[0051] In some embodiments, the amount of HCPs 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 HCPs in the flow-through produced when the method is performed using the same sample with a loading buffer having an electrical conductivity greater than 12 mS / cm.

[0052] In some embodiments, the method further comprises processing 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.

[0053] In some embodiments, the method comprises incorporating the composition into a pharmaceutical formulation.

[0054] In certain embodiments, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In particular embodiments, the lyophilized pharmaceutical formulation is a dry lyophilized pharmaceutical formulation. In some such embodiments, the method further comprises the step of reconstituting the dry lyophilized pharmaceutical formulation with a liquid to render it suitable for administration.

[0055] 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.

[0056] In another aspect, provided herein is a composition comprising a humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab) produced by any of the methods of the application. Also provided herein is a composition comprising a humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab), wherein the composition is obtainable by any of the methods of the application. In some embodiments, the amount of HCPs 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. In another embodiment, the basic isoform species of the humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab) comprises 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.

[0057] In some embodiments, the composition comprising a humanized anti-a4b7 antibody or antigen binding portion thereof (e.g., vedolizumab) is produced by the combination of the methods of the present application.

[0058] In another aspect, the present application relates to a method of increasing the yield of a humanized anti-a4b7 antibody or antigen binding portion thereof (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-a4b7 antibody or antigen binding portion thereof and a loading buffer onto the mixed mode chromatography resin, allowing the anti-a4b7 antibody or antigen binding portion thereof to bind to the mixed mode chromatography resin, washing the mixed mode chromatography resin with a wash buffer, and eluting the anti-a4b7 antibody or antigen binding portion thereof from the mixed mode chromatography resin with an elution buffer, wherein the equilibration buffer, loading buffer, and / or wash buffer has a pH of 7.0 or below 7.0.

[0059] In one embodiment, the equilibration buffer, loading buffer, and / or wash buffer has a pH of 6.0-7.0. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer has a pH of 6.5-7.0. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer has a pH of 6.6-6.8.

[0060] In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer has a salt concentration of 30 mM to 70 mM. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer has a salt concentration of 40 mM to 70 mM. In another embodiment, the equilibration buffer, loading buffer, and / or wash buffer has a salt concentration of 50 mM to 65 mM. In another embodiment, the loading buffer and / or wash buffer has a salt concentration of 55 mM-65 mM. In another embodiment, the salt comprises sodium chloride and / or sodium phosphate.

[0061] In another embodiment, the equilibration buffer, the loading buffer, and / or the wash buffer has a sodium chloride concentration of 30 mM to 70 mM. In another embodiment, the loading buffer and / or the wash buffer has a sodium chloride concentration of 40 mM to 70 mM. In another embodiment, the equilibration buffer, the loading buffer, and / or the wash buffer has a sodium chloride concentration of 40 mM to 60 mM. In another embodiment, the equilibration buffer, the loading buffer, and / or the wash buffer has a sodium chloride concentration of 45 mM-55 mM. In another embodiment, the equilibration buffer, the loading buffer, and / or the wash buffer has a sodium chloride concentration of about 50 mM. In another embodiment, the equilibration buffer, the loading buffer, and / or the wash buffer further comprises sodium phosphate.

[0062] In some embodiments, the equilibration buffer, the loading buffer, and / or the wash buffer has the same pH. In some embodiments, the equilibration buffer, the loading buffer, and / or the wash buffer has the same salt concentration. In some embodiments, the equilibration buffer, the loading buffer, and / or the wash 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.

[0063] In another aspect, provided herein are low basic species compositions comprising an anti-a4b7 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-a4b7 antibody, wherein the basic isoform species has a net positive charge relative to the predominant isoform of the anti-a4b7 antibody, and wherein the anti-a4b7 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 peaks that elute more slowly from a cation exchange (CEX) resin than peaks corresponding to the predominant isoform.

[0064] Accordingly, in another aspect, provided herein is a low basic species composition comprising an anti-a4b7 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-a4b7 antibody, wherein the basic isoform species has a net positive charge relative to the predominant isoform of the anti-a4b7 antibody and can be quantified by determining the relative area of the peak that elutes more slowly from a cation exchange (CEX) resin than the peak corresponding to the predominant isoform, and wherein the anti-a4b7 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2.

[0065] 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.

[0066] 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.

[0067] In some embodiments, the composition comprises less than 8% of the total basic isoform species of the anti-a4b7 antibody. In some embodiments, the composition comprises less than 7% of the total basic isoform species of the anti-a4b7 antibody. In some embodiments, the composition comprises less than 6% of the total basic isoform species of the anti-a4b7 antibody. In some embodiments, the composition comprises less than 5% of the total basic isoform species of the anti-a4b7 antibody.

[0068] In another aspect, 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.

[0069] In some embodiments, the pharmaceutical composition further comprises an amino acid. In some embodiments, the amino acid is arginine or histidine.

[0070] In some embodiments, the pharmaceutical composition further comprises a sugar. In certain embodiments, the sugar is sucrose or trehalose.

[0071] In some embodiments, the pharmaceutical composition comprises arginine, histidine, and sucrose. In some embodiments, the pharmaceutical composition comprises arginine, histidine, sucrose, and polysorbate 80.

[0072] In some embodiments, the pharmaceutical composition comprises at least 200 mg, at least 250 mg, or at least 300 mg of the anti-a4b7 antibody. In some embodiments, the pharmaceutical composition comprises about 300 mg of the anti-a4b7 antibody.

[0073] In some embodiments, the anti-a4b7 antibody is vedolizumab or an antigen binding portion thereof.

[0074] In another aspect, provided herein is a method of producing a low basic species composition comprising an anti-a4b7 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 total basic isoform species, the method comprising providing a composition comprising an anti-a4b7 antibody at a pH greater than pH 6.3; and incubating the composition comprising the anti-a4b7 antibody for a period of time greater than 10 hours; thereby producing a low basic species composition comprising the anti-a4b7 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 total basic isoform species. In some embodiments, the anti-a4b7 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2.

[0075] In another aspect, provided herein is a method of producing a low basic species composition comprising an anti-a4b7 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 total basic isoform species, the method comprising providing a composition comprising vedolizumab at 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 isoform species in the composition; thereby producing a low basic species composition comprising the anti-a4b7 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 total basic isoform species. In some embodiments, the anti-a4b7 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2.

[0076] Generally, the basic isoform species have a net positive charge relative to the major isoform of the anti-a4b7 antibody. In some embodiments, the level of basic isoform species can be quantified using cation exchange chromatography (CEX). In some embodiments, the level of basic isoform species can be quantified by determining the relative area of peaks that elute more slowly from a cation exchange (CEX) resin than peaks corresponding to the major isoform. 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.

[0077] 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.

[0078] In some embodiments, the composition comprises less than 8% of the total basic isoform species of the anti-a4b7 antibody. In some embodiments, the composition comprises less than 7% of the total basic isoform species of the anti-a4b7 antibody. In some embodiments, the composition comprises less than 6% of the total basic isoform species of the anti-a4b7 antibody. In some embodiments, the composition comprises less than 5% of the total basic isoform species of the anti-a4b7 antibody.

[0079] In some embodiments, provided herein are compositions comprising an anti-a4b7 antibody obtainable by the foregoing methods. In some embodiments, the antibody is vedolizumab or an antigen binding portion thereof.

[0080] In some embodiments, provided herein are compositions comprising an anti-a4b7 antibody obtained by the foregoing methods. In some embodiments, the antibody is vedolizumab or an antigen binding portion thereof.

[0081] In another aspect, provided herein are methods for treating a disease or disorder in a human subject, wherein the method comprises administering to the subject a pharmaceutical composition provided herein comprising an anti-a4b7 antibody (e.g., vedolizumab) in an amount effective to treat the disease or disorder in the human subject. In some embodiments, the pharmaceutical composition comprises a vedolizumab composition having reduced levels of basic vedolizumab isoform species and / or having reduced levels of host cell proteins as provided herein and / or as produced according to the methods provided herein. In some embodiments, the anti-a4b7 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2.

[0082] In one embodiment, the disease or disorder is inflammatory bowel disease (IBD). In some embodiments, the IBD is ulcerative colitis, Crohn’s disease, ileitis, celiac disease, non-tropical sprue, enteropathy associated with seronegative arthropathies, microscopic colitis, or collagenous colitis, eosinophilic gastroenteritis, or pouchitis following proctocolectomy and ileal pouch-anal 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).

[0083] In addition, the present application also includes the following embodiments:

[0084] 1. A method of producing a composition comprising vedolizumab having reduced levels of basic vedolizumab isotype species, the method comprising:

[0085] providing a composition comprising vedolizumab at a pH greater than pH 6.5; and

[0086] incubating the composition comprising vedolizumab for a period of time greater than 10 hours;

[0087] thereby producing a composition comprising vedolizumab having reduced levels of basic vedolizumab isotype species.

[0088] 2. The method of item 1, wherein the incubating is performed at ambient temperature.

[0089] 3. The method of item 1, wherein the incubating is performed at 15°C - 30°C.

[0090] 4. The method of item 1, wherein the incubating is performed at 20°C - 25°C.

[0091] 5. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about 6.5 - 8.5.

[0092] 6. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about 7.0 - 8.0.

[0093] 7. The method of any of the preceding items, wherein the composition comprising vedolizumab is provided at a pH of about 7.0 - 7.5.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 14. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is incubated for a period of at least 12 hours.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 20. The method of any of the preceding clauses, wherein the composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

[0107] 21. The method of clause 20, wherein the mammalian cell culture is a Chinese hamster ovary (CHO) cell culture.

[0108] 22. The method of clause 21, wherein the CHO cell culture comprises CHO cells lacking dihydrofolate reductase (DHFR) expression.

[0109] 23. The method of clause 21, wherein the CHO cell culture comprises CHO cells lacking glutamine synthetase (GS) expression.

[0110] 24. The method of any of the preceding clauses, wherein the method further comprises purifying the composition comprising vedolizumab from mammalian host cell proteins (HCPs) using one or more chromatography separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

[0111] 25. The method of clause 24, wherein the composition comprising vedolizumab is purified using an affinity chromatography resin comprising protein A.

[0112] 26. The method of clause 25, wherein the composition comprising vedolizumab is purified using affinity chromatography prior to the incubation.

[0113] 27. The method of clause 25, wherein the composition comprising vedolizumab is purified using affinity chromatography after the incubation.

[0114] 28. The method of clause 24, wherein the composition comprising vedolizumab is purified using cation exchange chromatography.

[0115] 29. The method of clause 28, wherein the composition comprising vedolizumab is purified using cation exchange chromatography prior to the incubation.

[0116] 30. The method of clause 28, wherein the composition comprising vedolizumab is purified using cation exchange chromatography after the incubation.

[0117] 31. The method of item 24, wherein the composition comprising vedolizumab is purified using anion exchange chromatography.

[0118] 32. The method of item 31, wherein the composition comprising vedolizumab is purified using anion exchange chromatography prior to the incubation.

[0119] 33. The method of item 31, wherein the composition comprising vedolizumab is purified using anion exchange chromatography after the incubation.

[0120] 34. The method of item 24, wherein the composition comprising vedolizumab is purified using CHT chromatography.

[0121] 35. The method of item 34, wherein the composition comprising vedolizumab is purified using CHT chromatography prior to the incubation.

[0122] 36. The method of item 34, wherein the composition comprising vedolizumab is purified using CHT chromatography after the incubation.

[0123] 37. A composition comprising vedolizumab, wherein the composition is produced by the method of any one of items 1-36.

[0124] 38. The composition of item 37, wherein the basic vedolizumab isoform species constitute less than 10% of the vedolizumab species present in the composition.

[0125] 39. A method of producing a composition comprising vedolizumab having a reduced amount of host cell proteins (HCPs), the method comprising:

[0126] (a) 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 conductivity of 11 mS / cm or less, such that HCPs bind to the anion exchange resin; and

[0127] (b) collecting flow-through material from the anion exchange resin,

[0128] wherein the flow-through material comprises vedolizumab and a reduced amount of HCPs.

[0129] 40. The method of item 39, wherein the loading buffer has a conductivity of 9 mS / cm to 11 mS / cm.

[0130] 41. The method of item 39, wherein the loading buffer has a conductivity of 10 mS / cm or less.

[0131] 42. The method of item 39, wherein the loading buffer has a conductivity of 9 mS / cm or less.

[0132] 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.

[0133] 44. The method of any one of items 39-42, wherein the HCP is a Chinese hamster ovary (CHO) cell protein.

[0134] 45. The method of item 44, wherein the HCP is derived from a CHO cell lacking dihydrofolate reductase (DHFR) expression.

[0135] 46. The method of item 44, wherein the HCP is derived from a CHO cell lacking glutamine synthetase (GS) expression.

[0136] 47. The method of any one of items 39-46, further comprising contacting the anion exchange resin with a wash buffer.

[0137] 48. The method of item 47, wherein the wash buffer has a conductivity of less than 11 mS / cm.

[0138] 49. The method of item 47, wherein the wash buffer has a conductivity of 9 mS / cm to 11 mS / cm.

[0139] 50. The method of item 47, wherein the wash buffer has the same conductivity as the loading buffer.

[0140] 51. The method of any one of items 39-50, wherein the loading buffer comprises sodium chloride and / or sodium phosphate.

[0141] 52. The method of any one of items 39-50, wherein the wash buffer comprises sodium chloride and / or sodium phosphate.

[0142] 53. The method of any one of items 39-52, wherein the anion exchange resin is formatted as an anion exchange column or an anion exchange membrane.

[0143] 54. The method of any one of items 39-53, wherein the anion exchange resin comprises a quaternary amine functional group.

[0144] 55. The method of any one of items 39-54, wherein the sample containing vedolizumab and HCPs is derived from a mammalian cell culture following one or more chromatographic separation steps.

[0145] 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.

[0146] 57. The method of any one of items 39-56, 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.

[0147] 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.

[0148] 59. The method of any one of items 39-58, wherein the method further comprises processing 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.

[0149] 60. A composition comprising vedolizumab produced by the method of any one of items 39-59.

[0150] 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.

[0151] 62. A method of 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 such 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 equilibration buffer, loading buffer, and / or wash buffer has a pH of 7.0 or below 7.0.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 66. The method of any one of items 62-65, wherein the equilibration buffer, the loading buffer, and / or the wash buffer has a salt concentration of 30 mM to 70 mM.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 70. The method of any one of items 66-69, wherein the salt comprises sodium chloride and / or sodium phosphate.

[0160] 71. The method of any one of items 62-65, wherein the equilibration buffer, the loading buffer, and / or the wash buffer has a sodium chloride concentration of 30 mM to 70 mM.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 76. The method of any one of items 71-75, wherein the equilibration buffer, the loading buffer, and / or the wash buffer further comprises sodium phosphate.

[0166] 77. The method of any one of items 62-76, wherein the equilibration buffer, the loading buffer, and / or the wash buffer has the same pH.

[0167] 78. The method of any one of items 62-77, wherein the equilibration buffer, the loading buffer, and / or the wash buffer has the same salt concentration.

[0168] 79. The method of any one of items 62-76, wherein the equilibration buffer, the loading buffer, and / or the wash buffer is the same buffer.

[0169] 80. The method of any one of items 62-79, wherein the mixed-mode resin is a ceramic hydroxyapatite resin. BRIEF DESCRIPTION OF DRAWINGS

[0170] Figure 1 Cation exchange (CEX)-high performance liquid chromatography (HPLC) profiles of vedolizumab are depicted, with indicated peaks representing acidic species, basic species, and the primary isoform of vedolizumab.

[0171] Figure 2 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 are depicted.

[0172] Figure 3 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 are depicted. DETAILED DESCRIPTION

[0173] Provided herein are methods for purifying anti-a4b7 integrin antibodies, such as vedolizumab, from a liquid solution, e.g., from a clarified harvest of a mammalian cell culture. The present invention relates, inter alia, 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, chromatography material, and / or media components) present in a purified preparation of an anti-a4b7 integrin antibody or antigen-binding fragment thereof (e.g., vedolizumab). Vedolizumab is a relatively hydrophobic antibody, which presents challenges for purification, particularly when producing the antibody in large quantities at the level of purity required for therapeutic use.

[0174] I. Definitions

[0175] To facilitate the understanding of this invention, a number of terms are defined below.

[0176] The cell surface molecule "a4b7 integrin" or "a4b7" (used interchangeably throughout) is a heterodimer of a4 chain (CD49D, ITGA4) and b7 chain (ITGB7). The human a4-integrin and b7-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 is typical of many integrins, a4b7 can exist in a resting or activated state. Ligands for a4b7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM (e.g., MAdCAM-1)). Antibodies that bind a4b7 integrin are referred to herein as "anti-a4b7 antibodies."

[0177] As used herein, an antibody or antigen-binding fragment thereof having "binding specificity for a4b7 complex" binds a4b7, but does not bind a4b1 or a4b7 integrin. E B7. Vedolizumab is one example of an antibody having binding specificity for a4b7 complex.

[0178] The term "about" indicates that the following value is not an exact value but the center point of a + / - 5% range of the value. If the value is a relative value given in percent, the term "about" also indicates that the following value is not an exact value but the center point of a + / - 5% range of the value, whereby the upper limit of the range cannot exceed the value 100%.

[0179] As used herein, the term "aggregate" refers to the association of two or more antibodies or antibody fragments. For example, an aggregate can be a dimer, trimer, tetramer, or larger polymer of antibodies and / or antibody fragments. Antibody aggregates can be soluble or insoluble. Association between aggregate molecules can be covalent or non-covalent, regardless of the mechanism by which they associate. The association can be direct association between aggregate molecules or indirect association through other molecules that link 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 multiple components. Aggregates may form irreversibly 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, analysis of ultracentrifugation sedimentation rate, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reducing and non-reducing).

[0180] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains linked together by disulfide bonds: two heavy (H) chains and two light (L) chains. 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 highly denaturing regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody has a fragment crystallizable (Fc) region. In some implementations, the antibody is an IgG1 isotype and has a κ light chain.

[0181] As used herein, the term "basic species" or "basic isotype" refers to a variant of an antibody or its antigen-binding moiety (e.g., vedolizumab) characterized by an overall basic charge. The basic species of an antibody or its antigen-binding moiety 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. The basic species of an antibody can include, but is not limited to, charge variants, structural variants, and / or fragment variants. In some embodiments, a composition comprising an antibody or its antigen-binding moiety may contain more than one type of basic isotype. In some embodiments, multiple basic isotypes can be identified based on differences in retention time during CEX-HPLC separation. For example, when analyzing a composition comprising an antibody (e.g., vedolizumab) using CEX-HPLC, one or more basic isotype peaks can be identified, each peak representing one or more basic isotypes of the antibody, such as... Figure 1 As shown. For example, in some embodiments, a basic isotype is an isotype of an antibody in which the aspartic acid residue has been isomerized to succinimide. Host cell impurities or other impurities unrelated to the antibody or its antigen-binding moiety are not considered, on a primary sequence basis, a “basic type” or “basic isotype” of the antibody or its antigen-binding moiety.

[0182] As used herein, the term "buffer solution" refers to an aqueous solution that resists pH changes through the action of its acid-base conjugate components. Buffer solutions are used to establish a specific set of conditions, such as biochemical conditions, to mediate the control of processing steps or chromatographic supports (such as chromatographic resins or membranes).

[0183] “CDR” or “complementary determination zone” are high-variability zones scattered in more conservative areas, called “frame zone” (FR).

[0184] As used herein, the term “antigen-binding fragment” or “antigen-binding moiety” of an antibody refers to Fab, Fab', F(ab')2, and Fv fragments, single-chain antibodies, functional heavy-chain antibodies (nanobodies), and any portion of an antibody that is specific for at least one desired epitope, competing with the intact antibody for specific binding (e.g., a separated portion of the complementarity-determining region having a sufficient framework sequence to specifically bind the epitope). Antigen-binding fragments can be generated by recombinant techniques or by enzymatic or chemical cleavage of the antibody.

[0185] As used herein, "chromatographic support" refers to a solid or porous matrix having a specific chemical composition or a specific three-dimensional structure, or wherein specific chemical groups or macromolecules can be immobilized for 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 cylinders, or other containers.

[0186] As used herein, the term "clarified harvest" refers to a liquid material containing the protein of interest (e.g., anti-α4β7 antibody) extracted from a cell culture (e.g., a fermentation bioreactor) after undergoing one or more process steps to remove solid particles (such as cell debris and particulate impurities) from the material. Following cell culture, the harvest is typically purified using separation techniques such as centrifugation and filtration to remove cells and cell debris. The initial clarification and particulate removal steps produce a "clarified harvest" that can be used, for example, in subsequent chromatographic steps (downstream processing). The clarified harvest is often the starting material for downstream processing, as described in the downstream processing steps herein.

[0187] As used herein, the terms “culture” and “cell culture” generally refer to the (upstream) process of growing cells under controlled conditions, typically outside their natural environment. “Culturing” cells means contacting cells with a cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation. In some embodiments, cell culture refers to methods for generating and maintaining a population of host cells capable of producing a recombinant protein of interest (e.g., an anti-α4β7 antibody), and methods and techniques for generating and collecting the protein of interest. For example, once an expression vector is incorporated into a suitable host (e.g., cultured host cells), the host can be maintained under conditions suitable for expressing the relevant nucleotide-coding sequence and for collecting and purifying the desired recombinant protein. “Cell culture” can also refer to a solution containing cells.

[0188] As used herein, the term "downstream process" refers to one or more techniques used after an upstream process to purify a protein of interest (e.g., an antibody). Downstream process techniques include, for example, using 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 to purify protein products.

[0189] As used herein, the terms "elution solution" or "elution buffer" refer to an aqueous liquid prepared for displacing an antibody, such as a protein of interest, from a chromatographic support (e.g., a resin or membrane). In one embodiment, the elution solution has different biochemical properties than the equilibration and / or washing solutions, such that the protein of interest (e.g., an antibody) preferably associates with the elution solution rather than the chromatographic support (e.g., a resin or membrane).

[0190] As used herein, the term "equilibration solution" refers to an aqueous liquid prepared to establish initial operating conditions for a processing step or chromatographic support (such as a chromatographic operation). Equilibration solutions are used to prepare, for example, a solid phase, such as a chromatographic support (e.g., a resin or membrane), which is used to load the protein of interest (e.g., an antibody).

[0191] As used herein, for example, “flow-through operation” in relation to a chromatographic step refers to the process during loading and washing where proteins are eluted while impurities bind and remain associated with the chromatographic support.

[0192] The term "high molecular weight" or "HMW" is used to refer to antibody complexes with a molecular weight greater than that of the 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).

[0193] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing a minimal sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the receptor hypervariable region are replaced by residues from a hypervariable region of a non-human species (donor antibody) with the desired specificity, affinity, and capability. In some cases, the frame region (FR) residues of the human antibody are replaced by corresponding non-human residues. Additionally, humanized antibodies may contain residues not found in the receptor antibody or in the donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain substantially all, and typically both, variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable CDR loops of the non-human antibody, and all or substantially all of the FRs are FRs of the human antibody sequence. Optionally, a humanized antibody will also contain at least a portion of the antibody constant region (Fc), typically at least a portion of the 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).

[0194] As used herein, the term "impurity" in relation 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 impurity" refers to one or more impurities present in a composition (e.g., a solution) containing a protein but not originating 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 buffer solutions, and leachable materials from material handling containers or chromatographic supports. Process-related impurities that can form proteins (e.g., antibodies) during preparation (upstream and / or downstream treatments). As used herein, the term "host cell impurity" refers to any protein, nucleic acid contaminant, lipid contaminant, or byproduct introduced by a host cell line, cell culture medium, or cell culture. The term "host cell protein" refers to a protein byproduct introduced by a host cell line, cell culture medium, or cell culture. Examples of impurities include, but are not limited to, Chinese hamster ovary protein (CHOP), Escherichia coli protein, yeast protein, simian COS protein, or myeloma cell protein (e.g., NSO protein (derived from mouse plasmacytoma cells of BALB / c mice)). Host cell proteins do not include the protein of interest generated in a host cell expression system. For example, when CHO cells are used to generate recombinant antibodies or fragments thereof, the term “host cell protein” covers proteins derived from CHO cells, not 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 of the antibody of interest, types of misfolding, types of oxidation or deamidation, or low molecular weight fragments.

[0195] 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., mammalian cell). In some embodiments, the recombinant protein is an isotype of an antibody belonging to the group consisting of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In some embodiments, the recombinant antibody is IgG1.

[0196] The term “recombinant host cell” (which is used interchangeably with the term “host cell” herein) includes cells in which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the specific subject cell but also to the progeny of such cells. Because certain modifications can occur in progeny due to mutations or environmental influences, such progeny may indeed differ from the parent cells but are still included within the scope of the term “host cell” as used herein. Furthermore, it should be understood that, unless otherwise stated, the use of the term “cell,” such as host cell or mammalian cell or mammalian host cell, is intended to include a population of cells.

[0197] "Substantially purified" in relation to the desired protein means that the purified sample containing 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 and less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5% of impurities.

[0198] As used herein, in the context of protein (e.g., antibody) preparation, the term “upstream process” refers to activities involving the production and collection of proteins (e.g., antibodies) from host cells (e.g., when culturing cells to produce the protein of interest (e.g., antibody)).

[0199] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of guiding the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0200] As used herein, the term "washing fluid" or "washing solution" refers to an aqueous liquid prepared for displacing unbound contaminants from a chromatographic support (such as a resin or membrane). In some embodiments, the washing fluid is passed over a solid support (such as a resin or membrane) after loading of a protein (e.g., an antibody of interest) and before elution of the protein of interest (e.g., an antibody). In one embodiment, the washing fluid has biochemical properties similar to those of an equilibrium solution.

[0201] II. Anti-α4β7 integrin antibody

[0202] The methods disclosed herein can be used to produce large quantities of highly purified compositions containing antibodies against α4β7 integrin. It will be apparent that any method used to produce the 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 known by its trade name... Takeda Pharmaceuticals, Inc. is known to have vedolizumab, a humanized antibody comprising a human IgG1 framework and a constant region, as well as an antigen-binding CDR derived from the mouse antibody Act-1. The vedolizumab CDR, variable region, and mutant Fc region (mutated to eliminate Fc effector function) are described in U.S. Patent No. 7,147,851, which is incorporated herein by reference in its entirety.

[0203] Vedolizumab is a humanized monoclonal antibody that specifically binds to α4β7 integrin (e.g., the α4β7 complex). Vedolizumab blocks the interaction of α4β7 integrin with mucosal addressing cell adhesion molecule-1 (MAdCAM-1) and inhibits the migration of memory T lymphocytes across the endothelium into inflamed gastrointestinal parenchyma. Vedolizumab does not bind to or inhibit the function of integrin α4β1 or αEβ7, nor does it antagonize the interaction of α4 integrin with vascular cell adhesion molecule-1 (VCAM-1).

[0204] α4β7 integrin is expressed on the surface of discrete subsets of memory T lymphocytes that preferentially migrate to the gastrointestinal tract. MAdCAM-1 is primarily expressed on intestinal endothelial cells and plays a crucial role in T lymphocyte homing to intestinal lymphoid tissue. The interaction between α4β7 integrin and MAdCAM-1 is considered an important contributing factor to mucosal inflammation, such as chronic inflammation, a hallmark of ulcerative colitis and Crohn's disease. Vedolizumab is used to treat inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, pouchitis (including chronic pouchitis), graft-versus-host disease, and HIV.

[0205] 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 containing 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 containing 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 containing the amino acid sequence of SEQ ID NO:9 and a light chain containing the amino acid sequence of SEQ ID NO:10. Vedolizumab and its sequences are also described in U.S. Patent Publication No. 2014 / 0341885 and U.S. Patent Publication No. 2014-0377251, the entire contents of which are expressly incorporated herein by reference. The methods disclosed herein can be performed using antibodies containing the binding regions (e.g., CDRs) or variable regions described above.

[0206] The method of the present invention can be used to generate anti-α4β7 antibodies in mammalian cells, particularly vedolizumab or antibodies having a binding region (i.e., CDR) or variable region of vedolizumab.

[0207] Exemplary strategies for antibody production

[0208] In some embodiments, the methods described herein may be combined with one or more additional steps to facilitate the production and / or purification of vedolizumab, including one or more steps described below. For long-term, high-yield production of recombinant proteins such as vedolizumab, mammalian host cells may be engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab). Exemplary cell culture procedures and considerations for producing monoclonal antibodies such as vedolizumab are described below in: 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.

[0209] In some embodiments, the initial recovery can be performed by sequentially employing pH lowering, centrifugation, and filtration steps to remove cells and cell debris (including HCP) from the bioreactor harvest. In some embodiments, the invention relates to subjecting the sample mixture from said initial recovery to one or more of the following purification steps: affinity chromatography, anion exchange (AEX), cation exchange (CEX), hydrophobic interaction chromatography (HIC), ceramic hydroxyapatite chromatography (CHT), and / or mixed mode (MM). In some embodiments, the order of steps can affect the quality of the resulting antibody composition by adjusting the levels of aggregates, impurities, or isotypes.

[0210] In one exemplary embodiment, a composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), mixed mode, cation exchange, and anion exchange.

[0211] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), mixed mode, anion exchange, and cation exchange.

[0212] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), cation exchange, mixed mode, and anion exchange.

[0213] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), anion exchange, mixed mode, and cation exchange.

[0214] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: mixed mode, affinity chromatography (e.g., protein A), anion exchange, and cation exchange.

[0215] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), CHT chromatography, cation exchange, and anion exchange.

[0216] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), CHT chromatography, anion exchange, and cation exchange.

[0217] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), cation exchange, CHT chromatography, and anion exchange.

[0218] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), hydrophobic interaction chromatography, cation exchange, and anion exchange.

[0219] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), hydrophobic interaction chromatography, anion exchange, and cation exchange.

[0220] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), anion exchange, hydrophobic interaction chromatography, and cation exchange.

[0221] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), cation exchange, hydrophobic interaction chromatography, and anion exchange.

[0222] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), cation exchange, anion exchange, and hydrophobic interaction chromatography.

[0223] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: affinity chromatography (e.g., protein A), anion exchange, cation exchange, and hydrophobic interaction chromatography.

[0224] In another exemplary embodiment, the composition containing vedolizumab can be purified using a process comprising the following steps in sequence: hydrophobic interaction chromatography, affinity chromatography (e.g., protein A), anion exchange, and cation exchange.

[0225] It should be understood that purification steps do not necessarily have to be adjacent to each other; various other process steps (such as filtration or virus reduction steps) can be inserted between chromatographic steps without interfering with the effect of the order of chromatographic steps on the charge curve.

[0226] To adjust the levels of basic isoforms present in the vedolizumab composition, an incubation step may be incorporated between any of the aforementioned purification steps as described herein. Alternatively or additionally, as described herein, the purification process may be adapted to minimize the duration of antibody exposure to low pH conditions.

[0227] 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 using anion exchange chromatography with a low conductivity buffer as described herein.

[0228] To regulate the yield of vedolizumab in the purification process including CHT, the CHT conditions described herein can be used at any stage of the vedolizumab purification process using ceramic hydroxyapatite chromatography.

[0229] Some embodiments of the present 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, reversed-phase HPLC, silica gel chromatography, heparin chromatography, and Sepharose chromatography. TM Further anion exchange chromatography and / or further cation exchange chromatography, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (e.g., using protein G, antibody, specific substrate, ligand, or antigen as capture reagent).

[0230] In some implementations, unbound flow-through and washing fractions can be further separated, and combinations of fractions that provide the purity of the target product can be combined.

[0231] In some embodiments, protein concentrations can be adjusted to achieve differential partitioning behavior between the antibody product and product-related substances, thereby further improving purity and / or yield. In some embodiments, loading can be performed at different protein concentrations during the loading operation to improve product quality / yield at any particular purification step.

[0232] In some implementations, the column temperature can be varied independently to improve the separation efficiency and / or yield of any particular purification step.

[0233] In some embodiments, the loading and washing solution matrices can be different or composed of mixtures of chemicals, while achieving similar "resin interaction" behavior, enabling the aforementioned novel separation. For example, but not as a limitation, the loading and washing solutions can differ in ionic strength or pH, while maintaining substantially similar functionality in terms of product rinsing achieved during the washing step. In some embodiments, additives such as amino acids, sugars, PEGs, etc., can be added to the loading or washing step to modulate partitioning behavior, thereby achieving separation efficiency and / or yield.

[0234] In some embodiments, the loading and washing steps can be controlled by measuring the levels of product-related impurities / substances in the column effluent or collection tank, or both, in-line, online, or offline, to achieve a target product quality and / or yield. In some embodiments, the loading concentration can be dynamically controlled by in-line or batch or continuous dilution with a solution or other solution to achieve the desired distribution for improved separation efficiency and / or yield.

[0235] Some embodiments of the present invention employ ultrafiltration and percolation steps to further concentrate and formulate the proteins of interest, such as antibody products. Ultrafiltration is described in detail below: 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 87762-456-9). One filtration process is tangential flow filtration, as described in the Millipore catalogue entitled “Pharmaceutical Process Filtration Catalogue,” pp. 177-202 (Bedford, Mass., 1995 / 96). Ultrafiltration is generally considered to refer to filtration using filters with pore sizes smaller than 0.1 μm. By employing filters with such small pore sizes, the volume of the sample can be reduced by allowing the sample solution to permeate through the pores of the filter membrane, while retaining proteins (such as antibodies) above the membrane surface.

[0236] Percolation is a method of removing and exchanging salts, sugars, and non-aqueous solvents from bound species using a membrane filter, to remove low molecular weight species and / or species that cause rapid changes in ionic and / or pH environments. Microsolutes can be removed most effectively by adding solvent to the percolated solution at a rate approximately equal to the percolation flow rate. This washes away microspecies from the solution at a constant volume, effectively purifying the retained protein of interest. In some embodiments of the invention, a percolation step is optionally employed to exchange the various solutions used in conjunction with the invention and to remove impurities from protein formulations prior to further chromatographic or other purification steps.

[0237] Ultrafiltration / percolation (UF / DF) of vedozimab can be performed using selected devices and membranes, such as polyethersulfone or regenerated cellulose, for example... or Membrane, in The box (MilliporeSigma, Burlington MA). In one embodiment, UF is performed using a cellulose membrane cast on a microporous polyethylene membrane having a molecular weight cutoff of 30 kDa.

[0238] III. Preparation of vedotin compositions containing altered levels of basic isoform species

[0239] In addition to the major (or main) isoform of vedolizumab, formulations of vedolizumab derived from mammalian host cells typically contain small amounts of acidic and / or basic isoforms. The acidic and basic isoforms can be quantified using methods known in the art, including, for example, cation exchange-high performance liquid chromatography (CEX-HPLC). Based on differences in retention time on CEX resin, acidic and basic vedolizumab isoforms can be separated from the major isoform. Typically, as... Figure 1 As described, the acidic vedolizumab isoforms present in the vedolizumab liquid formulation have shorter retention times relative to the major isoform, while the basic vedolizumab isoforms have longer retention times relative to the major isoform. The vedolizumab formulation may contain more than one acidic isoform and / or more than one basic isoform with slightly different charges, thus eluting from CEX resin with different retention times. In this document, multiple basic peaks are mentioned regarding their retention times on CEX resin; the first basic isoform peak eluted from CEX resin after the major isoform peak is referred to herein as "basic peak 1," the second basic isoform peak eluted from CEX resin after the major isoform peak is referred to herein as "basic peak 2," the third basic isoform peak eluted from CEX resin after the major isoform peak is referred to herein as "basic peak 3," and so on.

[0240] In pharmaceutical antibody formulations, it may be necessary to maximize the percentage of the antibody present as the major isotype while minimizing the percentage of basic and / or acidic isotypes. In one aspect, the present invention provides a method for adjusting the percentage of basic vedolizumab isotypes in a composition comprising vedolizumab. This method is based on the surprising discovery that the distribution of vedolizumab isotypes can be modulated by changes in pH. The basic isotypes of vedolizumab are particularly sensitive to pH fluctuations. As described herein, this pH-dependent modulation of the isotype distribution is driven at least in part by fluctuations in the level of basic peak 2 and the accompanying fluctuations in the level of the major vedolizumab isotype.

[0241] Without being bound by theory, and based at least in part on the findings presented herein, it is believed that at least two basic isoform variants exist in some vedolizumab formulations. The first variant, eluted from CEX resin as “basic peak 1,” is attributed to the presence of a lysine residue at the carboxyl terminus of the IgG heavy chain. The second variant, eluted from CEX resin as “basic peak 2,” is attributed to the isomerization of one or more aspartic residues in the antibody to form a succinimide intermediate. In some embodiments, one or more aspartic residues have undergone isomerization to form a succinimide intermediate. A glycine or serine residue adjacent to aspartic acid at the “n+1 position” (an adjacent amino acid close to the carboxyl terminus) can facilitate the isomerization of aspartic acid residues to succinimide. In some embodiments, identifying this variant of vedolizumab containing succinimide instead of aspartic acid at residue 102 of SEQ ID NO:1 allows for the reduction, minimization, or removal of this basic isoform variant in vedolizumab formulations. In some embodiments, the basic isotype variant can be removed by a method comprising, for example, fractionating the formulation containing vedolizumab (e.g., on CEX resin) and removing (or failing to collect) fractions containing succinimide instead of aspartic acid at residue 102 of SEQ ID NO:1, which can also be identified as fractions of vedolizumab eluted from CEX resin as “basic peak 2”. In some embodiments, the level of this basic isotype variant (referred herein to as the “succinimide variant” or optionally “basic isotype peak 2” or “BP2” variant) can be minimized by controlling the pH exposure of the antibody during vedolizumab production and purification. In some embodiments, compositions containing a reduced level of this basic isotype variant may contain a corresponding increase in the relative proportion of the major isotype of vedolizumab. Thus, in some embodiments, compositions containing a reduced level of this basic isotype variant may have increased potency relative to compositions containing an increased level of this basic isotype variant.

[0242] In one embodiment, this document provides a method for purifying vedolizumab with reduced basic allotype levels, wherein vedolizumab is maintained at pH 5.5 or higher, pH 5.6 or higher, pH 5.7 or higher, pH 5.8 or higher, pH 5.9 or higher, pH 6.0 or higher, pH 6.1 or higher, pH 6.2 or higher, pH 6.3 or higher, pH 6.4 or higher, or pH 6.5 or higher. 6.5, 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 the initial recovery of vedolizumab from the cell culture harvest via ultrafiltration / percolation (UF / DF) and the formulation of vedolizumab in a pharmaceutically acceptable carrier. By maintaining vedolizumab at pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or higher than pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 during most of the purification process, vedolizumab can be kept at pH values ​​lower than 6.5. A sustained period at a pH of 5.5–6.5, 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 the initial recovery of vedolizumab from cell culture harvest by ultrafiltration / percolation (UF / DF) and the formulation of vedolizumab in a pharmaceutically acceptable carrier, reduces the number of basic vedolizumab isoforms. In some embodiments, the method is carried out on a commercial manufacturing scale, for example using vedolizumab formulations derived from cell culture harvests produced at a scale of 1000L, 2000L, 3000L, 4000L, or 5000L (e.g., at least 3000L).

[0243] Therefore, in one aspect, the present invention provides a method for producing a low-basic species composition of an anti-α4β7 antibody or its antigen-binding moiety, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing an anti-α4β7 antibody or its antigen-binding moiety, and (ii) purifying the anti-α4β7 antibody or its antigen-binding moiety from the cell culture harvest, wherein the antibody is exposed to pH 3.5 or lower (e.g., pH 2.5-3.5, pH lower than 3.0, or pH lower than 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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 3.5 or lower (e.g., pH 2.5-3.5, pH lower than 3.0, pH 2.5-3.5 ... The duration of pH (2.5-3.5, below 3.0, or below 3.5) is longer, 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-basic composition contains a lower BP2 level compared to the control. In some embodiments, the antibody or its antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding moiety is vedozizumab or its antigen-binding moiety. In some embodiments, the steps of purifying the anti-α4β7 antibody or its antigen-binding moiety include one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and combinations thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding moiety is GS-CHO cells. In other embodiments, the host cell is DHFR-CHO cells. In some embodiments, the low-basic composition contains 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 basic isoform. In some embodiments, the low-basic composition contains less than 4%, less than 3%, less than 2%, or less than 1% of basic isoform peak 2.

[0244] In another aspect, the present invention provides a method for producing a low-basic species composition of an anti-α4β7 antibody or its antigen-binding moiety thereof, the method comprising (i) providing a clear cell culture harvest obtained from a culture of recombinant host cells expressing an anti-α4β7 antibody or its antigen-binding moiety thereof, and (ii) purifying the anti-α4β7 antibody or its antigen-binding moiety from the cell culture harvest, wherein the antibody is exposed to pH 4.0 or a pH below 4.0 (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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 4.0 or a pH below 4.0 (e.g., pH 3.6 to 4.0). 3.6 to 4.0) for a longer duration, 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-basic composition contains a lower BP2 level compared to the control. In some embodiments, the antibody or its antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding moiety is vedozizumab or its antigen-binding moiety. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding moiety includes one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and combinations thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding moiety is GS-CHO cells. In other embodiments, the host cell is DHFR-CHO cells. In some embodiments, the low-basic composition contains 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 basic isoform. In some embodiments, the low-basic composition contains less than 4%, less than 3%, less than 2%, or less than 1% of basic isoform peak 2.

[0245] In another aspect, the present invention provides a method for producing a low-basic species composition of an anti-α4β7 antibody or its antigen-binding moiety, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing an anti-α4β7 antibody or its antigen-binding moiety, and (ii) purifying the anti-α4β7 antibody or its antigen-binding moiety from the cell culture harvest, wherein the antibody is exposed to pH 4.5 or lower (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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 4.5 or lower (e.g., pH 4.1 to 4.5). 4.1 to 4.5) for a longer duration, 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 composition contains a lower BP2 level compared to the control. In some embodiments, the antibody or its antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding moiety is vedozizumab or its antigen-binding moiety. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding moiety includes one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and combinations thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding moiety is GS-CHO cells. In other embodiments, the host cell is DHFR-CHO cells. In some embodiments, the low-basic composition contains 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 basic isoform. In some embodiments, the low-basic composition contains less than 4%, less than 3%, less than 2%, or less than 1% of basic isoform peak 2.

[0246] In another aspect, the present invention provides a method for producing a low-basic species composition of an anti-α4β7 antibody or its antigen-binding moiety, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing an anti-α4β7 antibody or its antigen-binding moiety, and (ii) purifying the anti-α4β7 antibody or its antigen-binding moiety from the cell culture harvest, wherein the antibody is exposed to pH 5.0 or lower (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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 5.0 or lower (e.g., pH 4.6 to 5.0). 4.6 to 5.0) for a longer duration, 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 composition contains a lower BP2 level compared to the control. In some embodiments, the antibody or its antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding moiety is vedozizumab or its antigen-binding moiety. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding moiety includes one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and combinations thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding moiety is GS-CHO cells. In other embodiments, the host cell is DHFR-CHO cells. In some embodiments, the low-basic composition contains 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 basic isoform. In some embodiments, the low-basic composition contains less than 4%, less than 3%, less than 2%, or less than 1% of basic isoform peak 2.

[0247] In another aspect, the present invention provides a method for producing a low-basic species composition of an anti-α4β7 antibody or its antigen-binding moiety, the method comprising (i) providing a clarified cell culture harvest obtained from a culture of recombinant host cells expressing an anti-α4β7 antibody or its antigen-binding moiety, and (ii) purifying the anti-α4β7 antibody or its antigen-binding moiety from the cell culture harvest, wherein the antibody is exposed to pH 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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 5.5 or less (e.g., pH 5.1 to 5.5). 5.1 to 5.5) for a longer duration, 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 composition contains a lower BP2 level compared to the control. In some embodiments, the antibody or its antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the antibody or its antigen-binding moiety is vedozizumab or its antigen-binding moiety. In some embodiments, the step of purifying the anti-α4β7 antibody or its antigen-binding moiety includes one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and combinations thereof. In some embodiments, the host cell expressing the anti-α4β7 antibody or its antigen-binding moiety is GS-CHO cells. In other embodiments, the host cell is DHFR-CHO cells. In some embodiments, the low-basic composition contains 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 basic isoform. In some embodiments, the low-basic composition contains less than 4%, less than 3%, less than 2%, or less than 1% of basic isoform peak 2.

[0248] In some implementations, the methods provided herein are carried out on a commercial manufacturing scale, for example using vedolizumab formulations derived from cell culture harvests produced at a scale of 1000L, 2000L, 3000L, 4000L, or 5000L (e.g., at least 3000L).

[0249] In some aspects, the methods described herein can be used to adjust the level of basic vedolizumab isotype in compositions containing vedolizumab (e.g., liquid compositions). In some embodiments, the methods can be used to produce vedolizumab compositions having low levels of basic vedolizumab isotypes.

[0250] In one aspect, the present invention provides a method for reducing the level of basic vedolizumab isotypes in a composition containing vedolizumab by incubating the composition at a pH greater than 6.5 for a period of time sufficient to reduce the level of basic vedolizumab isotypes. In one embodiment, the method is carried out at an ambient temperature, for example, 20°C–25°C. In other embodiments, the method is carried out at 2°C–8°C.

[0251] In another aspect, the present invention provides a method for producing a vedolizumab-containing composition having a reduced level of basic vedolizumab isotypes. The method may include providing a vedolizumab-containing composition at a pH of 6.5 or higher, and incubating the vedolizumab-containing composition for a period of time sufficient to reduce the level of basic vedolizumab isotypes, thereby producing a vedolizumab-containing composition having a reduced level of basic vedolizumab isotypes.

[0252] To effectively reduce the level of basic vedolizumab isoforms in the vedolizumab composition, incubation is preferably performed at pH 6.5 or a pH higher than pH 6.5.

[0253] 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 may 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. Optionally, the pH of the vedolizumab composition may 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, such as 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 may be in the range of about 7.0-7.5. In other embodiments, the pH of the vedolizumab composition may be in the range of about 7.5-8.0. In other embodiments, the pH of the vedolizumab composition may be in the range of about 8.0-8.5. In exemplary embodiments, the pH of the vedolizumab composition may 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.

[0254] The composition containing vedolizumab can be incubated at the aforementioned pH (e.g., pH 6.5 or higher) for a period sufficient to reduce the percentage of basic vedolizumab isoforms in the vedolizumab composition. In exemplary embodiments, incubation occurs for periods of 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 periods 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, incubation can occur for about 20 minutes to about 1 hour, about 20 minutes to about 1 hour, about 20 minutes to about 2 hours, about 20 minutes to about 3 hours, about 1 hour to about 3 hours, about 1 hour to about 5 hours, or about 5 hours to about 8 hours. In some embodiments, incubation can occur for about 8 hours to about 168 hours (7 days) or longer. In some embodiments, incubation can occur for about 8-168 hours, for example, 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, incubation can occur for approximately 10 hours to approximately 168 hours or longer, such as approximately 10-168 hours, approximately 12-168 hours, approximately 18-168 hours, approximately 24-168 hours, approximately 36-168 hours, approximately 48-168 hours, approximately 72-168 hours, approximately 96-168 hours, approximately 120-168 hours, or approximately 144-168 hours. In some embodiments, incubation can occur for approximately 0.5-7 days, such as approximately 0.5-5 days, approximately 0.5-4 days, approximately 0.5-3 days, approximately 0.5-2 days, or approximately 0.5-1 day. In some embodiments, incubation can occur for approximately 1-5 days, approximately 1-3 days, or approximately 1-2 days. In exemplary embodiments, incubation at pH 6.5 or above 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 ≥25% of the total purification time, for example, the duration from the provision of a clear cell culture harvest to the end with purified antibody UF / DF.

[0255] In other embodiments, the incubation occurs for a period of time sufficient to reduce the percentage of basic vedolizumab isoform in the composition by 1% or more, such as 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. In an exemplary embodiment, the incubation occurs for a period of time sufficient to reduce the percentage of basic vedolizumab isoform in the composition by 1% to 5%. In other embodiments, the incubation period is sufficient to reduce the percentage of basic vedolizumab isotypes in the composition by 1-10%. In other embodiments, the incubation period is sufficient to reduce the percentage of basic vedolizumab isotypes in the composition by 2-10%. In other embodiments, the incubation period is sufficient to reduce the percentage of basic vedolizumab isotypes in the composition by 5-10%. In other embodiments, the incubation period is sufficient to reduce the percentage of basic vedolizumab isotypes in the composition by 2-20%. In other embodiments, the incubation period is sufficient to reduce the percentage of basic vedolizumab isotypes in the composition by 5-20%.

[0256] In other embodiments, the incubation occurs for a period of time sufficient to produce a low-basic 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 isotypes. In some embodiments, the method includes incubating the composition at a pH greater than pH 6.3 for a period of time sufficient to bring the level of basic isotype peak 2 to less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the method includes dispensing the composition containing 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 8.9). Incubate at pH 9.0; and incubate the composition containing the anti-α4β7 antibody (e.g., vedolizumab) for 20 minutes or longer, such as 30 minutes or longer, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, 5 hours or longer, 6 hours or longer, 7 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.

[0257] In some embodiments, the pH of the vedolizumab composition may be maintained at or above pH 6.3 during incubation. In other embodiments, the pH of the vedolizumab composition may be maintained at or above pH 6.5 during incubation. In some embodiments, the vedolizumab composition is maintained at approximately the same pH throughout the duration of the incubation period.

[0258] Incubation can be performed at any suitable temperature. For example, incubation can be performed at a temperature ranging from about 0°C to 40°C. In another embodiment, incubation can be performed at a temperature ranging from about 1°C to 37°C. In some embodiments, incubation is performed at a temperature ranging from about 0°C to 4°C or from 4°C to 8°C. In other embodiments, incubation is performed at ambient temperature. For example, incubation can be performed at a temperature ranging from about 20°C to 25°C. In other embodiments, incubation is performed at 37°C or about 37°C. In some embodiments, incubation is performed at a temperature ranging from about 1°C to 25°C. In some embodiments, incubation is performed at a temperature ranging from about 5°C to 18°C. In some embodiments, incubation is performed at a temperature ranging from about 15°C to 30°C. In some embodiments, incubation is performed at a temperature ranging from about 33°C to 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.

[0259] The aforementioned method may optionally include an additional step of adjusting the pH of the vedolizumab composition. For example, if it is necessary to reduce the percentage of basic vedolizumab species in the vedolizumab-containing composition, the method may include a step of raising the pH of the composition before incubation. For example, if the vedolizumab-containing composition is at a pH below 6.5, the method may include a step of raising the pH of the composition to 6.5 or above 6.5. Conversely, if it is necessary to increase the percentage of basic vedolizumab species in the vedolizumab-containing composition, the method may include a step of lowering the pH of the composition before incubation. For example, if the pH of the vedolizumab-containing composition is at or above 6.5, the method may include a step of lowering the pH of the composition to below 6.5.

[0260] Compositions containing vedolizumab can be liquid solutions. In some embodiments, the composition containing vedolizumab is derived from host cells used to produce vedolizumab or its antigen-binding moiety. In some embodiments, the host cells are mammalian cells. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells, such as CHO cells lacking dihydrofolate reductase (DHFR) expression or CHO cells lacking glutamine synthase (GS) expression.

[0261] After isolating antibodies from cell cultures, the aforementioned methods can be incorporated into small-scale or large-scale vedolizumab purification processes. In some embodiments, the initial recovery of vedolizumab from host cell cultures (e.g., bioreactor harvests) can be performed using centrifugation and filtration steps. The methods described herein can be performed on the vedolizumab composition before or after centrifugation and / or before or after filtration to reduce the level of basic isoforms in the composition at the corresponding purification stage. After the initial recovery, downstream process steps that can be used to purify vedolizumab from process-related impurities and / or product-related impurities include, but are not limited to, affinity chromatography (e.g., protein A chromatography), deep filtration, cation exchange (CEX), anion exchange (AEX), mixed-mode chromatography (MM), ceramic hydroxyapatite chromatography (CHT), hydrophobic interaction chromatography (HIC), ultrafiltration, and / or percolation. The methods described herein can be performed on the vedolizumab composition before or after any downstream process step to reduce the level of basic isoforms in the composition at the corresponding purification stage.

[0262] For example, the vedolizumab composition may be incubated before or after affinity chromatography as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., affinity chromatography loading material or affinity chromatography eluent) to pH 6.5 or a pH higher than pH 6.5.

[0263] In another embodiment, the vedolizumab composition may be incubated before or after deep filtration as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition to pH 6.5 or a pH higher than pH 6.5 before or after deep filtration.

[0264] In another embodiment, the vedolizumab composition may be incubated before or after cation exchange (CEX) as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., CEX loading material or CEX eluent) to pH 6.5 or a pH higher than pH 6.5.

[0265] In another embodiment, the vedolizumab composition may be incubated before or after anion exchange (AEX) as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., AEX loading material or AEX flow product) to pH 6.5 or a pH higher than pH 6.5.

[0266] In another embodiment, the vedolizumab composition may be incubated before or after mixed-mode chromatography (MM) as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., MM loading material or MM eluent) to pH 6.5 or a pH higher than pH 6.5.

[0267] In another embodiment, the vedolizumab composition may be incubated before or after ceramic hydroxyapatite chromatography (CHT) as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., CHT loading material or CHT eluent) to pH 6.5 or a pH higher than pH 6.5.

[0268] In another embodiment, the vedolizumab composition may be incubated before or after hydrophobic interaction chromatography (HIC) as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition (e.g., HIC loading material or HIC eluent) to pH 6.5 or a pH higher than pH 6.5.

[0269] In another embodiment, the vedolizumab composition may be incubated before or after ultrafiltration and / or percolation (UF / DF) as described herein. In one embodiment, the method may include adjusting the pH of the vedolizumab composition to pH 6.5 or higher before or after UF / DF.

[0270] IV. Compositions containing reduced basic isoforms of vedolizumab

[0271] In some aspects, the present invention provides vedolizumab compositions comprising reduced levels of basic vedolizumab isotypes. In some embodiments, compositions having reduced levels of basic isotypes can be obtained by methods provided herein (e.g., see Section III and Examples). In some embodiments, compositions having reduced levels of basic isotypes are produced by methods provided herein (e.g., see Section III). Thus, in one aspect, compositions comprising vedolizumab are provided herein, wherein the compositions are produced by methods comprising, in particular, incubating the vedolizumab-containing composition at a pH greater than pH 6.5 for a time sufficient to reduce the level of basic isotypes in the composition. After incubation, the vedolizumab-containing composition may optionally be subjected to a further purification step, said purification step being designed, for example, to reduce the levels of process-derived impurities and / or product-derived impurities in the composition.

[0272] On the other hand, this document provides compositions comprising vedolizumab or its antigen-binding moiety, wherein the compositions are obtained by the methods described above, for example by limiting the duration of exposure of the antibody to low pH conditions during purification.

[0273] In some embodiments, this document provides compositions comprising vedolizumab or its antigen-binding moiety, wherein the compositions are obtained, in particular, by methods comprising: (i) providing a clear cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab or its antigen-binding moiety; and (ii) purifying vedolizumab or its antigen-binding moiety from the cell culture harvest, wherein the antibody or its antigen-binding moiety is exposed to pH 3.5 or lower (e.g., pH 2.5-3.5, pH lower than 3.0, or pH lower than 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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 3.5 or lower (e.g., pH 2.5-3.5, pH lower than 3.0, pH 2.5-3.5 ... (pH 2.5-3.5, pH below 3.0 or pH below 3.5) for longer periods, i.e., more than 20 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, more than 3 hours, more than 5 hours, more than 7 hours, more than 10 hours or more than 12 hours.

[0274] In some embodiments, this document provides compositions comprising vedolizumab or its antigen-binding moiety, wherein the compositions are obtained, in particular, by methods comprising: (i) providing a clear cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab or its antigen-binding moiety; and (ii) purifying vedolizumab or its antigen-binding moiety from the cell culture harvest, wherein the antibody or its antigen-binding moiety is exposed to pH 4.0 or lower (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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 4.0 or lower (e.g., pH 3.6 to 4.0). (3.6 to 4.0) lasting longer, i.e., more than 20 minutes, more than 30 minutes, more than 45 minutes, more than 1 hour, more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 15 hours, more than 18 hours or more than 24 hours.

[0275] On the other hand, this document provides compositions comprising vedolizumab or its antigen-binding moiety, wherein the compositions are obtained by means of, in particular, a method comprising: (i) providing a clear cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab or its antigen-binding moiety, and (ii) purifying vedolizumab or its antigen-binding moiety from the cell culture harvest, wherein the antibody or its antigen-binding moiety is exposed to pH 4.5 or less (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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 4.5 or less (e.g., pH 4.1 to 4.5). 4.1 to 4.5) lasting longer, i.e., more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, more than 36 hours, more than 48 hours, more than 72 hours or more than 96 hours.

[0276] On the other hand, this document provides compositions comprising vedolizumab or its antigen-binding moiety, wherein the compositions are obtained by means of, in particular, a method comprising: (i) providing a clear cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab or its antigen-binding moiety, and (ii) purifying vedolizumab or its antigen-binding moiety from the cell culture harvest, wherein the antibody or its antigen-binding moiety is exposed to pH 5.0 or lower (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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 5.0 or lower (e.g., pH 4.6 to 5.0). (4.6 to 5.0) lasting longer, 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.

[0277] On the other hand, this document provides compositions comprising vedolizumab or its antigen-binding moiety, wherein the compositions are obtained by means of, in particular, a method comprising: (i) providing a clear cell culture harvest obtained from a culture of recombinant host cells expressing vedolizumab or its antigen-binding moiety, and (ii) purifying vedolizumab or its antigen-binding moiety from the cell culture harvest, wherein the antibody or its antigen-binding moiety is exposed to pH 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 its antigen-binding moiety has a reduced basic isotype level (determined by CEX) compared to a control, wherein the control is a composition comprising an anti-α4β7 antibody or its antigen-binding moiety produced by the same method, wherein the antibody is exposed to pH 5.5 or less (e.g., pH 5.1 to 5.5). 5.1 to 5.5) lasting longer, i.e., more than 3 hours, more than 5 hours, more than 10 hours, more than 12 hours, more than 18 hours, more than 24 hours, more than 36 hours, more than 48 hours, more than 72 hours or more than 96 hours.

[0278] In some embodiments of the foregoing aspects, the antibody or its antigen-binding portion comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:5.

[0279] In some embodiments of the foregoing aspects, the steps of purifying the anti-α4β7 antibody or its antigen-binding portion include one or more of protein A chromatography, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and combinations thereof.

[0280] In some embodiments of the foregoing aspects, the host cell expressing the anti-α4β7 antibody or its antigen-binding moiety is a GS-CHO cell. In other embodiments, the host cell is a DHFR-CHO cell.

[0281] In some embodiments of the foregoing, the composition contains 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 basic isoforms.

[0282] In some embodiments of the foregoing, the composition contains less than 4%, less than 3%, less than 2%, or less than 1% of basic isotype peak 2.

[0283] In some embodiments, this document provides a vedolizumab composition wherein the basic type of vedolizumab accounts for 15% or less of the vedolizumab isotype in the composition. For example, in some embodiments, this document provides 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 isotype.

[0284] In some embodiments, the level of the basic isoform 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 the basic isoform 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 the basic isoform 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 the basic isoform 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 the basic isoform 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 the basic isoform 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 the basic isoform 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 the basic isoform 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 the basic isoform in the vedolizumab composition is about 5% or less.

[0285] In some embodiments, the percentage of basic isoforms in the vedolizumab composition is reduced by about 1% or more relative to the percentage of basic isoforms in a vedolizumab composition produced by the same method as described herein without incubation at a pH greater than pH 6.5, 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.

[0286] The percentage of basic isotypes in a composition containing vedolizumab can be determined by any suitable method, including but not limited to CEX-HPLC. In some embodiments, this document provides low-basic-type compositions comprising an anti-α4β7 antibody or its antigen-binding moiety (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 isotypes of the anti-α4β7 antibody or its antigen-binding moiety, wherein, as determined by CEX, the basic isotypes have a net positive charge relative to the major isotype of the anti-α4β7 antibody or its antigen-binding moiety, wherein the anti-α4β7 antibody or its antigen-binding moiety comprises a heavy chain variable region containing SEQ ID NO:1 and a light chain variable region containing SEQ ID NO:5. In some embodiments, the composition comprises a first basic isotype peak (BP1) and a second basic isotype peak (BP2). In some such embodiments, the composition contains less than 2% BP2, less than 1.5% BP2, less than 1% BP2, or less than 0.7% BP2. In some embodiments, the composition contains 1.5% to 2.5% BP2, 1.2% to 2.2% 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, or 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.

[0287] 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 2:12, 2:10, 2:8, 2:6, 2:4, 3:12, 3:11, 3:9, 3:6, 3:5, 3:4, 4:12, 4:11, 4:10, 4:8, 4:6, 4:5, 5:12, 5:11, 5:10, 5:9, 5:8, 5:6, 6:12, 6:11, 6:10, 6:8, 6:7, 7:12, 7:11, 7:10, 7:9, 7:8, 8:12, 8:11, 8:10, 8:9, 9:12, 9:11, or 9:10.

[0288] In some embodiments, the low-basic composition comprises less than 8% (e.g., less than 7%, less than 6%, or less than 5%) of a total basic isotype of anti-α4β7 antibody (e.g., vedolizumab). In some embodiments, the low-basic 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 a total basic isotype of anti-α4β7 antibody (e.g., vedolizumab).

[0289] In some embodiments, the aforementioned compositions may be incorporated into pharmaceutical compositions comprising an anti-α4β7 antibody or its antigen-binding moiety (e.g., vedolizumab) and a pharmaceutically acceptable carrier or excipient. Therefore, in some embodiments, this document provides pharmaceutical compositions comprising a low-basic type of anti-α4β7 antibody or its antigen-binding moiety (e.g., vedolizumab) and a pharmaceutically acceptable carrier. Antibody formulations may be maintained as liquids or lyophilized into dry antibody formulations. On one hand, dry, lyophilized antibody formulations are provided in single-dose vials 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, anti-α4β7 antibodies (e.g., vedolizumab) are in stable liquid pharmaceutical compositions stored at about 2°C–8°C in containers (e.g., vials, syringes, or cartridges) until administered to a subject in need. In some embodiments, the syringe or cartridge may provide a single dose of antibody in the form of 54 mg, 108 mg, 160 mg, or 216 mg. 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 comprises L-arginine, L-histidine, L-histidine monohydrochloride, sucrose, and / or polysorbate 80. In another embodiment, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody comprises citrate, arginine, histidine, and / or polysorbate 80. Other formulations and uses of anti-α4β7 antibodies are described, for example, in U.S. Patent Nos. 9,764,033 and 10,040,855. The entire contents of each of the aforementioned patents are incorporated herein by reference.

[0290] V. Preparation of Vidozazumab Compositions Containing Reduced Host Cell Protein Levels

[0291] In some aspects, the present invention provides a method for producing a composition containing vedolizumab with reduced levels of host cell protein. This method is based on the surprising discovery that using an AEX buffer (e.g., a loading buffer) with reduced conductivity relative to standard operating conditions produces a vedolizumab composition with reduced levels of host cell protein (HCP) compared to a vedolizumab composition produced under standard operating conditions.

[0292] Therefore, in one aspect, the present invention provides a method for producing a composition containing vedolizumab with a reduced amount of host cell protein (HCP), wherein the method involves contacting a sample containing vedolizumab and HCP with an anion exchange resin in the presence of a loading buffer having reduced conductivity relative to standard buffer conditions, and collecting the flow-through material from the anion exchange resin, wherein the flow-through material contains vedolizumab and a reduced amount of HCP. In an exemplary embodiment, AEX is performed in flow-through mode, wherein vedolizumab is not bound to the AEX resin and is collected in the flow-through material without a separate elution step.

[0293] The standard operating range of buffer conductivity for anion exchange (AEX) (e.g., via anion exchange Q membrane adsorbent) is approximately 11-15 mS / cm (average approximately 13.6 mS / cm). Therefore, in some embodiments, standard AEX buffer conditions include loading buffers having conductivity of approximately 11 mS / cm or greater, approximately 12 mS / cm or greater, approximately 13 mS / cm or greater, approximately 14 mS / cm or greater, or approximately 15 mS / cm or greater. In some embodiments, the standard buffer conductivity is approximately 11 mS / cm to approximately 12 mS / cm, approximately 11 mS / cm to approximately 13 mS / cm, approximately 11 mS / cm to approximately 14 mS / cm, or approximately 11 mS / cm to approximately 15 mS / cm. In some embodiments, the standard buffer solution has a conductivity of 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 solution has a conductivity of about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, or about 15 mS / cm.

[0294] The AEX buffers (e.g., AEX loading buffers) used in the methods described herein have reduced conductivity compared to standard AEX buffer conditions. For example, in some embodiments, loading buffers with reduced conductivity have 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, loading buffers with reduced conductivity have a conductivity of about 11 mS / cm or less.

[0295] In some embodiments, the loading buffer having 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 (inclusive of one or more of the ranges above). 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 (inclusive of one or more of the above ranges). In some embodiments, the loading buffer having 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 (inclusive of one or more of the above ranges).

[0296] In some embodiments, the loading buffer having reduced conductivity has values ​​of approximately 1 mS / cm, approximately 1.5 mS / cm, approximately 2 mS / cm, approximately 2.5 mS / cm, approximately 3 mS / cm, approximately 3.5 mS / cm, approximately 4 mS / cm, approximately 4.5 mS / cm, approximately 5 mS / cm, approximately 5.5 mS / cm, approximately 6 mS / cm, approximately 6.5 mS / cm, approximately 7 mS / cm, and approximately 7 mS / cm. Conductivity of 5 mS / cm, approximately 8 mS / cm, approximately 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.

[0297] 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 increased conductivity relative to the loading buffer. In other embodiments, the wash buffer has decreased conductivity relative to the loading buffer.

[0298] In some embodiments, the loading buffer comprises sodium chloride and / or sodium phosphate. In exemplary embodiments, the loading buffer contains 40-70 mM NaCl (e.g., 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, or 70 mM NaCl). In some embodiments, the loading buffer contains 55-65 mM NaCl. Alternatively or additionally, the loading buffer may contain sodium phosphate. In exemplary embodiments, the loading buffer contains 20-50 mM sodium phosphate (e.g., 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate). In some embodiments, the loading buffer contains 35-45 mM sodium phosphate. In some embodiments, the loading buffer has a pH of pH 6.5 or higher, e.g., pH 6.5-8.5, pH 7.0-7.5, pH 6.8-7.4, etc. In some implementations, 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.

[0299] In some embodiments, this document provides a method for purifying α4β7 antibodies (e.g., vedolizumab), the method comprising contacting a solution containing the antibody with AEX resin equilibrated in a low conductivity buffer in flow-through mode and collecting the flow-through material. In one embodiment, the low conductivity solution comprises a mixture of NaCl and a buffer solution with 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.

[0300] In some embodiments, the anion exchange resin is formatted as anion exchange membrane. In some embodiments, the anion exchange resin is formatted as anion exchange column. In some embodiments, the anion exchange resin contains quaternary amine functional groups. Exemplary AEX resins include, but are not limited to, Mustang Q (Pall Corporation, PortWashington, NY) and 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).

[0301] HCP can be derived from host cells used to produce vedolizumab or its antigen-binding moiety. For example, in some embodiments, vedolizumab is produced in Chinese hamster ovary (CHO) cells, and HCP is a CHO cell protein. In some embodiments, HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression. In some embodiments, HCP is derived from CHO cells lacking glutamine synthase (GS) expression.

[0302] In some embodiments, the amount of HCP in the flow material 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.

[0303] In some embodiments, the amount of HCP in the flow material is 1 ppm to 8 ppm, 2 ppm to 8 ppm, 3 ppm to 8 ppm, 4 ppm to 8 ppm, 5 ppm to 8 ppm, 6 ppm to 8 ppm, or 7 ppm to 8 ppm, including one or more of the ranges mentioned above. In some embodiments, the amount of HCP in the flow material is 1 ppm to 2 ppm, 1 ppm to 3 ppm, 1 ppm to 4 ppm, 1 ppm to 5 ppm, 1 ppm to 6 ppm, 1 ppm to 7 ppm, or 1 ppm to 8 ppm, including one or more of the ranges mentioned above. In some embodiments, the amount of HCP in the flow material is 1 ppm to 3 ppm, 3 ppm to 5 ppm, or 5 ppm to 7 ppm.

[0304] In some 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 produced when the same sample is used with a loading buffer having a standard conductivity (e.g., conductivity of 11-15 mS / cm or higher).

[0305] In some embodiments, the amount of HCP in the flow stream 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 some embodiments, the amount of HCP in the flowstream 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 flowstream produced when the same sample is used with a loading buffer having a standard conductivity (e.g., about 11-15 mS / cm).

[0306] In some embodiments, the amount of HCP in the flowstream 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 some embodiments, relative to the amount of HCP in the flowstream produced when the same sample is used with a loading buffer having a standard conductivity (e.g., about 11-15 mS / cm) for the same method, the amount of HCP in the flowstream 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%, about 2 5% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 98%.

[0307] Samples containing vedolizumab and HCP are derived from mammalian cell cultures, optionally following 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 combinations thereof). Additionally, following the AEX method described herein, the HCP level in vedolizumab-containing samples may optionally be 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 combinations thereof).

[0308] Therefore, in some embodiments, the methods described herein may include one or more additional purification steps to further reduce HCP levels in samples containing vedolizumab. In one embodiment, the present invention provides a method for reducing HCP levels in compositions containing vedolizumab using the AEX method described herein, and the method further includes one or more additional purification steps. These 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 exemplary embodiments, 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 combinations thereof.

[0309] In an exemplary embodiment, the present invention provides a method for reducing the HCP level in a composition comprising vedolizumab, the method comprising providing a composition comprising vedolizumab and HCP, purifying vedolizumab from the HCP by affinity chromatography, mixed-mode chromatography, and / or cation exchange chromatography, and further purifying vedolizumab from the HCP by performing the AEX method described herein. In one embodiment, the loading material for the AEX method described herein comprises a cation exchange eluent. The foregoing method can be used to generate a composition comprising vedolizumab using an anion exchange buffer with a conductivity of 11-15 mS / cm or higher, which has a reduced HCP level relative to the HCP level present in a composition generated by performing the same method.

[0310] In another exemplary embodiment, the present invention provides a method for reducing the HCP level in a composition containing vedolizumab, the method comprising providing a composition containing vedolizumab and HCP, purifying vedolizumab from the HCP by affinity chromatography, cation exchange chromatography, and / or hydroxyapatite chromatography (e.g., ceramic hydroxyapatite (CHT) chromatography), and further purifying vedolizumab from the HCP by performing the AEX method described herein. In one embodiment, the loading material for the AEX method described herein comprises hydroxyapatite chromatographic eluent. The foregoing method can be used to generate a composition containing vedolizumab using an anion exchange buffer with a conductivity of 11-15 mS / cm or higher, which has a reduced HCP level relative to the HCP level present in a composition generated by performing the same method.

[0311] The content of vedolizumab and / or the content of host cell protein 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 measurements (such as nanoparticle trajectory analysis NTA, NanoSight Ltd, Wiltshire, UK).

[0312] In some embodiments, the method further includes treating the AEX flow material by a process including ultrafiltration and / or percolation to exchange the elution buffer for a buffer containing one or more pharmaceutically acceptable carriers or excipients.

[0313] VI. Compositions containing vedotinib with reduced host cell proteins

[0314] In some aspects, the present invention provides vedolizumab compositions comprising reduced host cell proteins. In some embodiments, compositions having reduced host cell proteins are produced by methods provided herein (e.g., see section V). Thus, in one aspect, compositions comprising vedolizumab are provided herein, wherein the compositions are produced by contacting a sample containing vedolizumab and HCP with an anion exchange resin in the presence of a loading buffer having reduced conductivity relative to standard buffer conditions; and collecting the flow-through material from the anion exchange resin, wherein the flow-through material comprises vedolizumab and a reduced amount of HCP. After elution from the AEX resin, the flow-through material may optionally be treated to exchange the elution buffer for a buffer comprising one or more pharmaceutically acceptable carriers or excipients, thereby forming a pharmaceutical composition having a reduced amount of HCP. This buffer exchange step may be performed using standard methods, including, for example, ultrafiltration and / or percolation.

[0315] HCP can be derived from host cells used to produce vedolizumab or its antigen-binding moiety. For example, in some embodiments, vedolizumab is produced in Chinese hamster ovary (CHO) cells, and HCP is a CHO cell protein. In some embodiments, HCP is derived from CHO cells lacking dihydrofolate reductase (DHFR) expression. In some embodiments, HCP is derived from CHO cells lacking glutamine synthase (GS) expression.

[0316] In some 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.

[0317] In some embodiments, the amount of HCP in the composition is 1 ppm to 8 ppm, 2 ppm to 8 ppm, 3 ppm to 8 ppm, 4 ppm to 8 ppm, 5 ppm to 8 ppm, 6 ppm to 8 ppm, or 7 ppm to 8 ppm, including one or more of the ranges mentioned above. In some embodiments, the amount of HCP in the composition is 1 ppm to 2 ppm, 1 ppm to 3 ppm, 1 ppm to 4 ppm, 1 ppm to 5 ppm, 1 ppm to 6 ppm, 1 ppm to 7 ppm, or 1 ppm to 8 ppm, including one or more of the ranges mentioned above. In some embodiments, the amount of HCP in the composition is 1 ppm to 3 ppm, 3 ppm to 5 ppm, or 5 ppm to 7 ppm.

[0318] In some 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 the composition produced by using the same sample with a loading buffer having a standard conductivity (e.g., conductivity of 11-15 mS / cm or higher).

[0319] In some 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 some embodiments, the amount of HCP flowing through the 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 the composition produced by using the same sample with a loading buffer having a standard conductivity (e.g., conductivity of 11-15 mS / cm or higher).

[0320] In some 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 some embodiments, relative to the amount of HCP in the composition produced when the same sample is used with a loading buffer having a standard conductivity (e.g., 11-15 mS / cm or a conductivity greater than 11-15 mS / cm), the amount of HCP flowing through the 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%, or about 20%. % to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 98%.

[0321] The content of vedolizumab and / or the content of host cell protein 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 measurements (such as nanoparticle trajectory analysis NTA, NanoSight Ltd, Wiltshire, UK).

[0322] VII. Preparation of vedolizumab using mixed-mode chromatography

[0323] This article describes a buffer for maximizing vedolizumab yield after elution from a mixed-mode chromatographic resin. Under high loading conditions (e.g., loading concentrations of at least 14 g / L, 15 g / L, 17 g / L, 10 g / L, 25 g / L, 30 g / L, 35 g / L, or more), protein loss occurs during washing in mixed-mode purification using, for example, ceramic hydroxyapatite resin. To increase the loading capacity of mixed-mode resins (e.g., ceramic hydroxyapatite resin) and improve vedolizumab yield, the equilibration buffer, loading buffer, and washing buffer can be optimized to reduce antibody loss during column washing. Surprisingly, the method described herein allows for applying higher loading concentrations to mixed-mode resins and increases the yield by 2%–3% relative to standard mixed-mode buffers without altering the product quality (e.g., percentage of aggregates) in the final eluate.

[0324] Therefore, in one aspect, the present invention provides a method for increasing the yield of vedolizumab recovered after elution from a mixed-mode chromatographic resin, the method comprising equilibrating the mixed-mode chromatographic resin with an equilibration buffer, loading a solution containing vedolizumab and a loading buffer onto the mixed-mode chromatographic resin such that the vedolizumab binds to the mixed-mode chromatographic resin, washing the mixed-mode chromatographic resin with a washing buffer, and eluting the vedolizumab from the mixed-mode chromatographic resin with an elution buffer, wherein the equilibration buffer, loading buffer, and / or washing buffer have a pH of 7.0 or lower. The pH of the standard buffer used for mixed-mode chromatography can be higher, i.e., pH 7.2 or higher. In some embodiments of the invention, an equilibration buffer with a pH of 7.0 or lower is used. In some embodiments, a loading buffer with a pH of 7.0 or lower is used. In some embodiments, a washing buffer with a pH of 7.0 or lower is used.

[0325] A buffer pH range below 7.0 can be used to prevent loss of vedolizumab during the washing step of mixed-mode purification. For example, in some embodiments, the equilibration 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, the buffer may have a pH of about 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or 5.5. In some embodiments, the buffer pH is in the range of about 7.0–5.5. In other embodiments, the buffer pH is in the range of about 7.0–6.0. In other embodiments, the pH of the buffer solution is in the range of about 7.0-6.5. In other embodiments, the pH of the buffer solution is in the range of about 6.8-5.8. In other embodiments, the pH of the buffer solution is in the range of about 6.8-6.0. In other embodiments, the pH of the buffer solution is in the range of about 6.8-6.5. In other embodiments, the pH of the buffer solution is about 6.6-6.8.

[0326] Alternatively or additionally, the buffer used for equilibrating, loading, and / or washing the mixed-mode chromatographic resin used to purify vedolizumab may have a total salt concentration of less than 70 mM. For example, the buffer may have a salt concentration of less than 70 mM, less than 65 mM, less than 60 mM, less than 55 mM, less than 50 mM, less than 45 mM, less than 40 mM, less than 35 mM, or less than 30 mM. In some embodiments, the buffer has a salt concentration of about 70 mM, about 65 mM, about 60 mM, about 55 mM, about 50 mM, about 45 mM, about 40 mM, about 35 mM, or about 30 mM. In other embodiments, the buffer has a salt concentration in the range of 30-70 mM. In some embodiments, the buffer has a salt concentration in the range of 40-65 mM. In some embodiments, the buffer has a salt concentration in the range of 45-65 mM. In some embodiments, the buffer has a salt concentration in the range of 50-60 mM. 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 salts present in the buffer used for equilibrating, loading, and / or washing the CHT chromatography resin used to purify vedolizumab include sodium chloride and / or sodium phosphate.

[0327] In some embodiments, the buffer used for equilibrating, loading, and / or washing the mixed-mode chromatographic resin used to purify vedolizumab may have a sodium chloride concentration of less than 70 mM. For example, the buffer may have a sodium chloride concentration of less than 70 mM, less than 65 mM, less than 60 mM, less than 55 mM, less than 50 mM, less than 45 mM, less than 40 mM, less than 35 mM, or less than 30 mM. In some embodiments, the buffer has a sodium chloride concentration of about 70 mM, about 65 mM, about 60 mM, about 55 mM, about 50 mM, about 45 mM, about 40 mM, about 35 mM, or about 30 mM. In other embodiments, the buffer has a sodium chloride concentration in the range of 30-70 mM. In some embodiments, the buffer has a sodium chloride concentration in the range of 40-65 mM. In some embodiments, the buffer has a sodium chloride concentration in the range of 45-65 mM. In some embodiments, the buffer has a sodium chloride concentration in the range of 50-60 mM. In some embodiments, the buffer solution has a sodium chloride concentration in the range of 40-50 mM. In some embodiments, the buffer solution has a sodium chloride concentration in the range of 45-55 mM. In some embodiments, the aforementioned sodium chloride buffer solution may further contain sodium phosphate. In some embodiments, the aforementioned sodium chloride buffer solution may further contain 1-30 mM sodium phosphate, for example, 5-20 mM sodium phosphate, 10-20 mM sodium phosphate, etc. In exemplary embodiments, the sodium chloride buffer solution may further contain sodium phosphate at concentrations of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM.

[0328] The buffer solution described above can be used to equilibrate, load, and / or wash the mixed-mode chromatographic resin during the vedolizumab purification process. In an exemplary embodiment, the mixed-mode resin may be a ceramic hydroxyapatite resin.

[0329] In some embodiments, two of the equilibration buffer, loading buffer, and wash buffer have the same pH. In some embodiments, two of the equilibration buffer, loading buffer, and wash buffer have the same salt concentration. In some embodiments, two of the equilibration buffer, loading buffer, and wash buffer have the same pH and the same salt concentration. In some embodiments, the equilibration buffer, loading buffer, and wash buffer all have the same pH. In some embodiments, the equilibration buffer, loading buffer, and wash buffer all have the same salt concentration. In some embodiments, the equilibration buffer, loading buffer, and wash buffer all have the same pH and the same salt concentration.

[0330] In some embodiments, methods for increasing the yield of vedolizumab recovered after loading a mixed-mode chromatographic resin include elution at a pH less than 7, i.e., pH 5.5 to 6.9 or pH 6.5 to 6.8, and at a salt concentration of 40 to 60 mM or 45 to 55 mM (e.g., NaCl). The method may further include washing the column at a pH less than 7, i.e., pH 5.5 to 6.9 or pH 6.5 to 6.8.

[0331] The buffers and methods described herein improve the yield of vedolizumab eluted from mixed-mode columns compared to yields obtained when using buffers with a pH greater than 7.0 and / or a salt concentration greater than 70 mM and / or a sodium chloride concentration greater than 70 mM for equilibration, loading, and / or washing steps. In some embodiments, the yield is improved by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, or more. In some embodiments, the yield is improved by more than 2% relative to buffers with a pH greater than 7.0. In some embodiments, the yield is improved by more than 3% relative to buffers with a pH greater than 7.0. In some embodiments, the yield is improved by more than 4% relative to buffers with a pH greater than 7.0. In some embodiments, the yield is improved by more than 5% relative to buffers with a pH greater than 7.0.

[0332] VIII. Methods for assessing the purity of compositions containing anti-α4β7 antibodies

[0333] The purity of a composition containing an antibody (e.g., vedolizumab) can be assessed by any suitable method, including but not limited to the methods described herein.

[0334] (a) Assessing the types of alkaline isoforms

[0335] This invention provides methods for adjusting (e.g., reducing) the level of basic isotypes in a composition containing vedolizumab or its antigen-binding moiety, and methods for adjusting (e.g., increasing) the level of the major isotype in a composition containing vedolizumab. The relative amounts of basic vedolizumab isotypes and the relative amounts of the major vedolizumab isotype 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 based on their overall surface charge. After dilution to a low ionic strength using a mobile phase, the test sample can be injected onto a CEX column, such as the Dionex Pro-Pac. TMA WCX-10 column (Thermo Fisher Scientific, Waltham, MA (USA)) is equilibrated 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 can be designated as acidic, basic, or major isotype. Acidic peaks elute from the column with shorter retention times than the major isotype peaks, and basic peaks elute with longer retention times than the major isotype peaks. The percentages of major isotypes, total percentages of acidic species, and total percentages of basic species are reported. The retention times of the major isotypes in the sample are compared to those of a reference standard to determine concordance. In some embodiments, a CEX-HPLC method is used. For example, compositions containing vedolizumab and its acidic and / or basic species can be separated using cation exchange chromatography as described above, followed by HPLC analysis of the eluted peaks. In one implementation, 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 The CEX-HPLC profiles of exemplary vedozizumab formulations are provided in the document.

[0336] In one embodiment, the CEX assay method includes diluting the test sample to a low ionic strength, injecting it 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 peak at 280 nm and designating the peak as an acidic peak, main peak, or basic peak, wherein the acidic peak is eluted first with the shortest retention time, the main peak is eluted second, and the basic peak is eluted with the longest retention time, and the peak area is quantified and its amount is calculated as a percentage of the total peak area.

[0337] (b) Assess host cell protein levels

[0338] This invention provides a method for regulating (e.g., reducing) the level of residual host cell proteins in a composition containing vedolizumab or its antigen-binding moiety. 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)). Host cell proteins in a test sample can be captured using a fixed polyclonal anti-CHO HCP antibody. The captured proteins can then be detected using a suitable detection reagent, for example, a horseradish peroxidase-labeled form of the same antibody. In this exemplary embodiment, the amount of captured peroxidase can be colorimetrically measured at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is proportional to the concentration of CHO HCP. The HCP concentration can be determined by comparison with a CHO HCP standard curve (as included in the test kit) and reported as a percentage of the total protein level in the antibody preparation. In another embodiment, the rabbit-rabbit (“RaRa”) method illustrated herein can be used to determine HCP. The polyclonal anti-CHO HCP antibody is produced by immunizing rabbits with harvested material made from null cells under manufacturing conditions similar to those for vedolizumab, and the antibody is affinity purified. Host CHO cell proteins in the vedolizumab sample are captured using a fixed polyclonal anti-CHO HCP antibody, then detected using a biotinylated form of the same antibody, followed by detection with horseradish peroxidase-conjugated streptavidin. The amount of captured peroxidase is measured colorimetrically at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is proportional to the concentration of CHO HCP. The HCP concentration is determined by comparison with a CHO HCP standard curve included in the test kit and reported as a percentage of total protein. In another embodiment, the rabbit-goat (“RaGo”) method illustrated herein can be used to determine HCP. Polyclonal anti-CHO HCP antibodies were produced by immunizing rabbits and goats with harvested material made from naked cells using a manufacturing process similar to that of vedotin. The antibody aggregates 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 sequentially adding goat anti-CHO affinity-purified antibody and donkey anti-goat IgG reagent (labeled with horseradish peroxidase). The amount of captured peroxidase was colorimetrically measured at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB), and was proportional to the concentration of CHO HCP.HCP concentration was determined by comparison with the CHO HCP standard curve contained in the test kit and reported as a concentration (ng / mg) relative to total vedozimab.

[0339] A suitable CHO HCP assay for use in conjunction with the various embodiments provided herein involves capturing HCP using a polyclonal anti-CHOHCP antibody, detecting HCP after binding with a horseradish peroxidase-labeled polyclonal anti-CHO HCP antibody that converts the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a substrate that is colorimetrically quantified at 450 nm. In one embodiment, a suitable HCP ELISA for use in conjunction with the various embodiments provided herein is an ELISA method for capturing HCP using a fixed polyclonal anti-CHO HCP antibody, preferably the CHO HCP ELISA Kit 3G provided by Cygnus Technologies (Southport, NC (USA)), whereby the captured protein is detected by the same antibody in horseradish peroxidase-labeled form, and the amount of captured peroxidase is colorimetrically measured at 450 nm using 3,3',5,5'-tetramethylbenzidine (TMB), subsequently determining the HCP concentration by comparison with a CHO HCP standard curve.

[0340] (c) Evaluate size variants

[0341] In some embodiments, the levels of aggregates, monomers, and fragments in chromatographic samples produced using the techniques described herein are analyzed. In the various embodiments set forth herein, size exclusion chromatography (SEC) can be used to determine the relative levels of monomers, high molecular weight (HMW) aggregates, and low molecular weight (LMW) degradation products present in a population of antibodies or their antigen-binding moieties (e.g., vedozizumab). The SEC method provides size-based separation of antibody monomers from HMW species and LMW degradation products. Commercially available SEC columns can be used, along with appropriate buffers to analyze the test samples and reference standards. 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 tandem with an isocratic phosphate-sodium chloride buffer system (pH 6.8). Elution of protein species is monitored at 280 nm. The main peak (monomer) and total peak area are evaluated to determine purity. Sample purity (%) (calculated as monomer %), HMW aggregate %, and / or LMW degradation product % are reported.

[0342] In one implementation, the SEC analysis involves injecting the sample onto two G3000SWxl columns connected in series and running it in an isocratic phosphate-sodium chloride buffer system (pH 6.8), where the elution of protein species is monitored at 280 nm and the main peak (monomer) and total peak area are measured.

[0343] IX. Pharmaceutical Compositions and Their Uses

[0344] The vedolizumab-containing compositions provided herein, for example, compositions containing vedolizumab with reduced levels of basic isoforms and / or reduced levels of host cell proteins, may be incorporated into pharmaceutical formulations for therapeutic use. Pharmaceutical formulations containing vedolizumab can be prepared by any suitable method.

[0345] In one aspect, the pharmaceutical formulation comprising the composition described herein is a lyophilized pharmaceutical formulation. In another aspect, the lyophilized formulation may be stored as a single dose in a container (e.g., a vial). The container (e.g., the vial) is refrigerated, for example at about 2°C–8°C or at room temperature, for example at about 20°C to 35°C, about 25°C, or about 30°C, until it is administered to a subject in need. The vial may be, for example, 10, 20, or 50 cc vials. The container (e.g., a vial) may 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 anti-α4β7 antibody. In one instance, the vial contains about 200 mg of anti-α4β7 antibody. The vial may contain sufficient anti-α4β7 antibody (e.g., vedolizumab) to allow delivery (e.g., manufactured for delivery) of approximately 100 mg, approximately 108 mg, approximately 150 mg, approximately 200 mg, approximately 300 mg, approximately 400 mg, or approximately 600 mg of anti-α4β7 antibody. For example, the vial may contain approximately 15%, approximately 12%, approximately 10%, or approximately 8% more anti-α4β7 antibody than the stated dosage.

[0346] In some embodiments, the compositions described herein are formulated as dried, lyophilized pharmaceutical preparations that can be reconstituted in a liquid (such as sterile water) for administration. Administration of the reconstituted preparation can be performed via parenteral injection through one of the routes described above. Intravenous injection can be performed by infusion, such as by further dilution with sterile isotonic saline, a buffer (e.g., phosphate-buffered saline), or Ringer's solution (lactated or glucose).

[0347] In some embodiments, the compositions described herein are formulated as liquid pharmaceutical preparations 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, 165 mg, or 216 mg every two, three, or four weeks after the start of therapy or after the third follow-up dose.

[0348] On the other hand, the compositions comprising anti-α4β7 antibodies (e.g., vedolizumab) described herein are in the form of stable liquid pharmaceutical compositions stored in containers (e.g., vials, syringes, or cartridges) at about 2°C–8°C until administered to the subject in need. 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 may be a 1 mL or 2 mL container (e.g., for a 160 mg / mL dose) or more than 2 mL (e.g., for higher doses (at least 320 mg or 400 mg or higher)). 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) may 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 may be manufactured to deliver about 90 to 120 mg, about 95 to 115 mg, about 100 to 112 mg, or about 108 mg of anti-α4β7 antibody (e.g., vedolizumab). In other embodiments, the syringe or cartridge may 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 anti-α4β7 antibody (e.g., vedolizumab).

[0349] Containers suitable for storing and freezing the purified compositions described herein include polycarbonate bottles (for IV preparations) or PETG bottles (for subcutaneous preparations). After aliquoting the preparations into the bottles, freezing can be performed (e.g., at -60 degrees Celsius or lower).

[0350] The pharmaceutical composition may comprise any vedolizumab composition provided herein, such as a vedolizumab-containing composition having a reduced level of basic isotype, and / or a vedolizumab-containing composition and a pharmaceutically acceptable carrier or excipient having a reduced level of host cell protein. In some embodiments, the pH of the pharmaceutical composition is between 6.0 and 7.0, for example, 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-pH 6.8, pH 6.6-7.0, pH 6.7-6.9, or pH 6.8-7.0. 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.

[0351] 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 for anti-α4β7 antibodies provided herein comprises arginine, histidine, and / or polysorbate 80. In some embodiments, the pharmaceutical composition for anti-α4β7 antibodies provided herein comprises citrate, arginine, histidine, and / or polysorbate 80.

[0352] The vedolizumab-containing compositions provided herein (e.g., vedolizumab-containing compositions having reduced levels of basic isotypes) and / or vedolizumab-containing compositions having reduced levels of host cell proteins can be used in methods for inhibiting integrin α4β7 activity in vitro or in vivo.

[0353] In one respect, the compositions described herein can be used to treat a disease or condition in a subject, including administering to the subject an amount of the composition containing an anti-α4β7 antibody that is effective in treating a human disease or condition. The human subject may be an adult (e.g., 18 years of age or older), an adolescent, or a child. The human subject may be a person 65 years of age or older.

[0354] In one embodiment, the composition described herein is used to treat subjects who may have previously lacked an adequate response, lost a response, or been intolerant 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 had an inadequate response to, been intolerant of, or exhibited corticosteroid dependence to corticosteroid treatment (e.g., inflammatory bowel disease). An inadequate response to corticosteroids is defined as a history of at least one four-week induction regimen comprising a dose equivalent to 30 mg of prednisone orally daily for two weeks or intravenously for one week, with persistent signs and symptoms of active disease. Loss of response to corticosteroids is defined as two failed attempts to gradually reduce the corticosteroid dose to below the equivalent of 10 mg of prednisone orally daily. Intolerance to corticosteroids includes a history of Cushing's syndrome, osteopenia / osteoporosis, hyperglycemia, insomnia, and / or infection.

[0355] Immunomodulatory agents can be, for example, oral azathioprine, 6-mercaptopurine, or methotrexate. Inadequate response to immunomodulatory agents is defined as the presence of persistent signs and symptoms of active disease despite a history of at least one 8-week regimen or oral azathioprine (≥1.5 mg / kg), 6-mercaptopurine (≥0.75 mg / kg), or methotrexate (≥12.5 mg / week). Intolerance to immunomodulatory agents includes, but is not limited to, nausea / vomiting, abdominal pain, pancreatitis, abnormal LFT, lymphopenia, TPMT gene mutations, and / or infection.

[0356] 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 (tertuzumab), SIMPONI (golimab); or circulating receptor fusion proteins, such as ENBREL (etanercept). Inadequate response to a TNF-α antagonist is defined as a history of at least one four-week induction regimen, including infliximab 5 mg / kg IV at least two weeks apart; adalimumab 80 mg subcutaneously followed by 40 mg at least two weeks apart; or 400 mg subcutaneously tertuzumab at least two weeks apart, with the presence of signs and symptoms of persistent active disease. Loss of response to a TNF-α antagonist is defined as the recurrence of symptoms during maintenance administration following prior clinical benefit. Intolerance to TNFα antagonists includes, but is not limited to, infusion-related reactions, demyelination, congestive heart failure, and / or infection.

[0357] As used in this study for subjects with ulcerative colitis, loss of maintenance remission was defined as an increase of at least 3 points in the Mayo score and at least 2 points in the modified Baron score.

[0358] In one implementation, treatable diseases include, but are not limited to, inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis, celiac disease, nontropical stomatitis, enteropathy associated with seronegative arthropathy, microscopic colitis or collagen-producing colitis, eosinophilic gastroenteritis, or pouchitis resulting from rectocele and ileoanal anastomosis. In some implementations, the inflammatory bowel disease is Crohn's disease or ulcerative colitis. Other treatable diseases include, for example, primary sclerosing cholangitis (PSC) and graft-versus-host disease (GVHD).

[0359] Ulcerative colitis can be moderate to severe active ulcerative colitis (e.g., Mayo score of 6 to 12, endoscopic subscale score of 2 or 3). In patients with moderate to severe active ulcerative colitis, treatment can lead to the induction and maintenance of clinical response, the induction and maintenance of clinical remission, or mucosal healing. Treatment can also lead to a reduction, elimination, or reduction and elimination of corticosteroid use (e.g., remission without corticosteroids).

[0360] Crohn's disease can be moderate to severe active Crohn's disease (e.g., a Crohn's Disease Activity Index (CDAI) score of 220 to 450). Treatment can achieve a clinical response or clinical remission in patients with moderate to severe active Crohn's disease. Treatment can also lead to a reduction, elimination, or reduction and elimination of corticosteroid use (e.g., remission in the absence of corticosteroids).

[0361] Pancreatitis and insulin-dependent diabetes mellitus are other diseases that can be treated with the compositions of the present invention. MAdCAM (e.g., MAdCAM-1) has been reported to be expressed by some blood vessels in the exocrine pancreas of NOD (non-obese diabetic) mice and BALB / c and SJL mice. The expression of MAdCAM (e.g., MAdCAM-1) has been reported to be induced on the endothelium of inflamed islets of the pancreas in NOD mice, and MAdCAM (e.g., MAdCAM-1) is a major addressin expressed by the endothelium of NOD islets in the early stages of pancreatitis (Hanninen, A. et al., J. Clin. Invest., 92:2509-2515 (1993)). Treatment of NOD mice with anti-MAdCAM or anti-β7 antibodies has prevented the development of diabetes (Yang et al., Diabetes, 46:1542-1547 (1997)). Furthermore, lymphocytes expressing α4β7 were observed to accumulate within the islets, and MAdCAM-1 was associated with lymphoma cells via α4β7 binding to blood vessels from inflamed islets (Hanninen, A. et al., J. Clin. Invest., 92:2509-2515 (1993)) or the gastrointestinal tract in mantle cell lymphoma (Geissmann et al., Am. J. Pathol., 153:1701-1705 (1998)).

[0362] Examples of mucosal tissue-related inflammatory diseases that can be treated with the compositions of the present 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; Behçet's disease, such as Behçet's disease of the intestines or pericholangiitis (bibitis and tissues surrounding the liver); and graft-versus-host disease (e.g., graft-versus-host disease in the gastrointestinal tract (e.g., after bone marrow transplantation) (Petrovic et al. Blood 103:1542-1547 (2004)). As seen in Crohn's disease, inflammation often extends beyond the mucosal surface, thus 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.

[0363] The present invention also relates to a method for inhibiting leukocyte infiltration into mucosal tissues. The present invention also relates to a method for treating cancers (e.g., α4β7-positive tumors such as lymphoma). Other examples of mucosal tissue-related inflammatory diseases that can be treated using formulations of the present invention include mastitis (breast) and irritable bowel syndrome.

[0364] Diseases or pathogens whose etiology utilizes the interaction between MAdCAM (e.g., MAdCAM-1) and 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, for example, WO2008140602).

[0365] The compositions of the present invention are administered with an effective amount of anti-α4β7 antibody to inhibit the binding of α4β7 integrin to its ligand. For therapeutic purposes, the effective amount will be sufficient to achieve the desired therapeutic (including prophylactic) effect (e.g., sufficient to reduce or prevent α4β7 integrin-mediated binding and / or signaling, thereby inhibiting leukocyte adhesion and infiltration and / or associated cellular responses). An effective amount of anti-α4β7 antibody, for example sufficient to maintain an effective titer of saturation (e.g., neutralization) of α4β7 integrin, can induce a clinical response or remission in inflammatory bowel disease. An effective amount of anti-α4β7 antibody can lead to mucosal healing in ulcerative colitis or Crohn's disease. The formulations of the present invention can be administered in single doses or multiple doses. The dosage can be determined by methods known in the art and can depend on, for example, an individual's age, sensitivity, tolerance, and overall health. Examples of administration methods include local routes, such as nasal or inhalation or transdermal administration; enteral routes, such as via feeding tube or suppository; and parenteral routes, such as intravenous, intramuscular, subcutaneous, intra-arterial, 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.

[0366] In some respects, dosing regimens for treating the diseases described herein (e.g., UC or Crohn's disease) have two phases: an induction phase and a maintenance phase. In the induction phase, an antibody or its antigen-binding fragment is administered in a manner that rapidly 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 a clinical response and improving symptoms of inflammatory bowel disease. Induction phase treatment may be administered to patients during the maintenance phase of anti-α4β7 antibody therapy when the patient is receiving anti-α4β7 antibody therapy for the first time, when treatment is initiated after a long period without 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 anti-α4β7 antibody therapy), or in cases of recurrence of inflammatory bowel disease symptoms (e.g., relapse after disease remission). In some embodiments, the induction phase regimen results in a higher mean trough serum concentration (e.g., the concentration maintained just before the next dose) than the mean steady-state trough serum concentration maintained during the maintenance phase.

[0367] During the maintenance phase, the antibody or its antigen-binding fragment is administered in a manner consistent with a response achieved through induction therapy with stable levels of the antibody or its antigen-binding fragment. Maintenance regimens can prevent the recurrence or relapse of symptoms of inflammatory bowel disease. Maintenance regimens may offer convenience to patients (e.g., a simple dosing regimen) or require infrequent treatment. In some embodiments, the maintenance regimen may include administration of the anti-α4β7 antibody or its antigen-binding fragment (e.g., in the formulations described herein) via a strategy selected from the group consisting of: low dose, infrequent administration, self-administration, and a combination of any of the foregoing.

[0368] In one implementation, for example, during the induction phase of therapy, the dosing regimen provides an effective amount of the anti-α4β7 antibody or antigen-binding fragment in the formulation described herein for inducing remission of inflammatory bowel disease in a human patient. The duration of the induction period may be approximately four, five, six, seven, or eight weeks of treatment. In some implementations, the induction regimen may utilize a strategy selected from the group consisting of: high-dose anti-α4β7 antibody or its antigen-binding fragment, frequent administration of anti-α4β7 antibody or its antigen-binding fragment, and a combination of high-dose anti-α4β7 antibody or its antigen-binding fragment and frequent administration of anti-α4β7 antibody or its antigen-binding fragment (e.g., in the formulation described herein). Induction administration may be once or more than one dose, such as at least two doses. During the induction phase, a dose may be administered once daily, every other day, twice weekly, once weekly, once every ten days, once every two weeks, or once every three weeks. In some implementations, the induction dose is administered within the first two weeks of therapy with the anti-α4β7 antibody. In one embodiment, induction administration may be performed once at the start of treatment (day 0) and again approximately two weeks after the start of treatment. In another embodiment, the induction phase lasts for six weeks. In yet another embodiment, the induction phase lasts for six weeks, and multiple induction doses are administered during the first two weeks.

[0369] In some embodiments, for example, when initiating treatment of patients with severe inflammatory bowel disease (e.g., in patients who have failed anti-TNFα therapy), the induction period needs to be longer than that for 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 (treatment start), 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 (treatment start), a dose at week 2, a dose at week 6, and a dose at week 10.

[0370] The dosage can be administered once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, or once every ten weeks. Higher or more frequent dosages, such as every other day, once weekly, once every two weeks, once every three weeks, or once every four weeks, can be used to induce remission of active disease or to treat new patients, such as to induce tolerance to anti-α4β7 antibodies. Doses every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, or once every ten weeks can be used for prophylactic therapy, such as to maintain remission in patients with chronic disease. In one aspect, the treatment regimen is administered on day 0, approximately week 2, approximately week 6, and thereafter every one or two weeks. In some aspects, the treatment regimen is administered on day 0, approximately week 2, approximately week 6, and thereafter every eight weeks. In another aspect, the induction treatment regimen is administered every other day for a total of six treatments.

[0371] In some respects, optimized dosing regimens can achieve sustained clinical remission, for example, clinical remission that persists for at least two, three, or four nursing care visits within a six-month or one-year period after the start of treatment.

[0372] In some respects, effective dosing regimens can achieve sustained clinical responses, such as clinical responses that last for at least 6 months, at least 9 months, or at least 1 year after the start of treatment.

[0373] The present disclosure is further illustrated by the following examples. The examples provided are for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way.

[0374] Example

[0375] The following examples describe the steps in an exemplary process for purifying vedolizumab from CHO cell cultures.

[0376] Example 1. Controlling the charged isoform of vedolizumab

[0377] Vedolizumab has three charged isoforms: acidic, major, and basic. Based on the relative areas of the chromatograms representing the acidic, major, and basic isoforms, cation exchange (CEX)-HPLC can be used to quantify the isoform distribution of vedolizumab. Figure 1 The figure shows exemplary CEX-HPLC curves depicting these three types of vedolizumab.

[0378] The distribution of charged isoforms of vedolizumab was evaluated using CEX-HPLC after storage under various conditions. As summarized in Table 1, the retention conditions during the manufacturing process of vedolizumab affected the distribution of charged isoforms, with the basic isoforms being most significantly affected by the retention conditions.

[0379] Table 1. Qualitative changes in basic isoforms in intermediates during the process.

[0380]

[0381]

[0382] These results demonstrate that the proportions of different isoforms of vedolizumab can be predictably modulated by maintaining the antibody under certain conditions. The basic isoforms tend to increase with duration of retention at pH less than approximately 6.5. In contrast, the basic isoforms decrease with duration of retention at pH greater than approximately 6.5. These changes were observed in vedolizumab produced at pilot-scale (Table 2) and manufacturing scale (Table 3).

[0383] Table 2. Vedolizumab isotypes during retention period - pilot-scale purification

[0384]

[0385] Table 3. Vedolizumab isotype-manufacturing-scale purification during retention period

[0386]

[0387]

[0388] Further experiments were conducted to evaluate the charged isotype distribution of vedolizumab derived from GS-CHO cells. The percentages of major, acidic, and basic species of vedolizumab were assessed after storage for 0–7 days at various pH values ​​(i.e., pH 4.7, 5.1, 5.3, 5.7, 5.9, 6.1, 6.5, or 6.9) at 5°C or room temperature. As shown in Tables 4–11, improved stability was observed at pH values ​​greater than or equal to 5.9. Following protein A capture and elution, the typical neutralized pH range (approximately pH 4.9 to 5.2) was associated with increased formation of basic species. The increase in basic species was slowed when vedolizumab was stored at pH 5.9 or 6.1 compared to the basic species observed during storage at pH < 5.9. As shown in Tables 10 and 11, the increase in basic species was stopped or even reversed at pH values ​​greater than or equal to 6.5.

[0389] Table 4. Vedolizumab isotype distribution during retention at pH 4.7

[0390]

[0391]

[0392] Table 5. Vedolizumab isotype distribution during retention in the process at pH 5.1

[0393] Storage temperature (°C) Days retained Acidity % Main % Alkalinity % [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

[0394] Table 6. Vedolizumab isotype distribution during retention in the process at pH 5.3

[0395] Storage temperature (°C) Days retained Acidity % Main % Alkalinity % [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

[0396] Table 7. Vedolizumab isotype distribution during retention in the process at pH 5.7

[0397]

[0398]

[0399] Table 8. Vedolizumab isotype distribution during retention in the process at pH 5.9

[0400] Storage temperature (°C) Days retained Acidity % Main % Alkalinity % [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

[0401] Table 9. Vedolizumab isotype distribution during retention in the process at pH 6.1

[0402] Storage temperature (°C) Days retained Acidity % Main % Alkalinity % [T0] 0 14.52 71.73 13.75 5℃ 1 14.54 71.51 13.95 5℃ 2 14.39 71.57 14.04 5℃ 3 14.42 71.69 13.89 5℃ 5 14.38 71.80 13.82 5℃ 7 14.58 71.21 14.21 Room temperature 1 14.35 71.61 14.04 Room temperature 2 14.33 71.18 14.49 Room temperature 3 14.55 70.64 14.81 Room temperature 5 14.38 69.88 15.74 Room temperature 7 14.27 69.40 16.33

[0403] Table 10. Vedolizumab isotype distribution during retention in the process at pH 6.5

[0404] Storage temperature (°C) Days retained Acidity % Main % Alkalinity % [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

[0405] Table 11. Vedolizumab isotype distribution during retention in the process at pH 6.9

[0406] Storage temperature (°C) Days retained Acidity % Main % Alkalinity % [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

[0407] 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.

[0408] Example 2. Reduction of the basic isotype of vedolizumab

[0409] To further evaluate the effect of pH on the formation of basic isoforms of vedolizumab, 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, major isoform, and basic isoform.

[0410] As shown in Table 12, the three peaks corresponding to the basic type of vedolizumab appeared at pH 6.3 (control) (i.e., "basic peak 1", "basic peak 2", and "basic peak 3"). At higher pH levels, basic peak 2 was observed to decrease significantly, as shown in Table 12.

[0411] Table 12. Sensitivity of alkaline peak 2 to increased pH

[0412] Control After exposure to 200 mM Tris HCI (pH 9) Name Area % Area % Alkaline peak-1 6.85 6.8 Alkaline peak-2 3.09 0.85 Alkaline peak-3 0.83 0.56

[0413] Material eluted from CEX resin with a characteristic retention time of basic peak 2 was collected, enzymatically digested, and subsequently analyzed by mass spectrometry (MS). A characteristic MS peak for succinimide was present in the basic peak 2 CEX fraction from the control formulation, but not in the formulation analyzed after exposure to pH 9. Analysis of the primary amino acid sequence of vedolizumab identified aspartic residues in vedolizumab's CDR-H3 located near residues favorable for the isomerization of aspartic acid to succinimide, specifically glycine and serine at position n+1 (CDR-H3:GGY). D GWDYAIDY (SEQ ID NO:4)). This finding suggests that the increase in the number of basic species observed in vedozimab at low pH may be attributed to the isomerization of this aspartic acid residue to succinimide. Maintaining the antibody at neutral or near-neutral pH can slow down 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).

[0414] To further evaluate the effect of pH on basic peak 2, the relative peak area was determined by CEX-HPLC after the antibody was 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, consistent with the findings reported in Example 1.

[0415] Table 13. Loss of alkaline peak 2 at elevated pH

[0416] pH Peak area % Loss % 6.5 3.3 0.0 7 3.26 1.2 8 2.13 35.5 8.5 1.36 58.8 9 0.69 79.1

[0417] The levels of vedolizumab isotypes corresponding to basic peak 2 were then assessed 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 the DHFR-CHO and GS-CHO cell lines.

[0418] Table 14. Loss of basic peak 2 in antibody preparations derived from two different CHO cell lines

[0419]

[0420] Example 3. Effect of anion exchange loading conductivity on host cell protein clearance rate

[0421] Since it is generally necessary to reduce the amount of host cell protein contaminants in therapeutic protein compositions, methods for producing vedolizumab compositions with reduced host cell protein content were examined.

[0422] The standard operating range for the buffer conductivity of anion exchange (AEX) (e.g., via anion exchange Q membrane adsorbent) is approximately 11–15 mS / cm (average approximately 13.6 mS / cm). To evaluate impurity clearance under conditions outside the standard operating range, impurity clearance was tested in compositions obtained using lower conductivity AEX conditions. Two independent starting formulations (harvest 1 and harvest 2) of vedolizumab clarified harvests were tested. All samples were AEX purified after the conductivity of the loaded material was adjusted to either standard conductivity (approximately 13.6 mS / cm) or low conductivity (approximately 11 mS / cm). As shown in Table 15, the HCP level was reduced in the low conductivity AEX loaded material compared to the standard conductivity AEX loaded material.

[0423] Table 15. HCP clearance rate obtained using rabbit-goat (RaGo) process-specific ELISA

[0424]

[0425]

[0426] Next, AEX performance (flow-through mode) was tested using loading buffers with conductivity ranges below standard operating conditions (i.e., below 13-15 mS / cm). Table 16 summarizes the loading conditions, conductivity, host cell protein content, and percentages of acidic and basic isoforms. The 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.

[0427] Table 16. Conductivity Variation Study – Q-film Performance

[0428]

[0429] The effect of reduced AEX conductivity on HCP clearance was then evaluated using two different HCP ELISA methods. In the first method, rabbit primary antibody and anti-rabbit secondary antibody were used in HCP ELISA (RaRa). The second method utilized rabbit primary antibody and goat secondary antibody 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 demonstrated that reducing the conductivity of the AEX-loaded material improved HCP clearance.

[0430] Table 17. Standard conductivity AEX loading conditions (approximately 13.3 mS / cm)

[0431]

[0432]

[0433] Table 18. Low conductivity AEX loading conditions (approximately 10.0 mS / cm)

[0434]

[0435] The amount of HCP in recombinant protein formulations is variable. As shown in this paper, HCP clearance in vedolizumab formulations can be improved by reducing conductivity during purification using AEX. Regardless of the HCP concentration in the starting material, lower conductivity AEX conditions achieved a greater reduction in HCP compared to standard higher conductivity AEX conditions.

[0436] Example 4. Effects of equilibration and washing conditions on the loading capacity of mixed-mode resins

[0437] To minimize vedolizumab loss during purification, various purification conditions were examined to identify steps that reduced antibody product yield. Protein loss was observed during the washing step of ceramic hydroxyapatite (CHT) purification under high-concentration loading conditions. For example, Figure 2 The elution profiles of vedolizumab from CHT columns were compared using standard wash and equilibration buffers (75 mM NaCl, 10 mM sodium phosphate, pH 7.2) after loading 27 mg / ml or 35 mg / ml. Figure 2 As shown, under high-concentration loading conditions (35 mg / ml), proteins begin to be expelled from the column during the washing step.

[0438] To increase the loading capacity of vedolizumab on CHT columns and improve vedolizumab yield by reducing protein loss during the CHT washing step, alternative CHT equilibration buffers, loading buffers, and washing buffers were evaluated. The vedolizumab loading (g / L) and yield achieved on the CHT column were determined by using a lower pH buffer (50 mM NaCl, 10 mM sodium phosphate, pH 7.7) for CHT equilibration and washing, compared to CHT column operations using a standard CHT buffer at elevated pH (75 mM NaCl, 10 mM sodium phosphate, pH 7.2). Figure 3 As shown, significant protein loss occurred during the wash step using standard CHT buffer conditions (dashed line), but no protein loss was observed using the low pH buffer (solid line). Furthermore, as summarized in Table 19, using the low pH buffer in the equilibration and wash steps allows for a larger loading volume on the CHT column and results in a 2%–3% higher yield in the final eluent compared to that achieved using standard CHT buffer, without altering product quality (e.g., aggregate percentage).

[0439] Table 19. Comparative Analysis: Standard CHT vs. CHT with Low pH Buffer

[0440]

[0441] Equivalent Case

[0442] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0443] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods, and embodiments are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described herein.

[0444] sequence list

[0445]

[0446]

Claims

1. A method of producing a composition comprising vedolizumab, the method comprising: providing a composition comprising vedolizumab at a pH of pH 6.5-9; and incubating the composition comprising vedolizumab for a period of at least 10 hours; thereby producing a composition comprising vedolizumab, wherein the incubation is performed during purification of vedolizumab, and wherein the incubation is performed prior to formulating vedolizumab in a pharmaceutically acceptable buffer, and wherein the composition comprising vedolizumab is derived from a mammalian cell culture expressing vedolizumab.

2. The method of claim 1, wherein the method is a method of producing a composition comprising vedolizumab having reduced levels of basic vedolizumab isoform species, and wherein the method produces a composition comprising vedolizumab having reduced levels of basic vedolizumab isoform species.

3. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 16% basic vedolizumab isoform species.

4. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 15% basic vedolizumab isoform species.

5. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 14% basic vedolizumab isoform species.

6. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 13% basic vedolizumab isoform species.

7. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 12% basic vedolizumab isoform species.

8. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 11% basic vedolizumab isoform species.

9. The method of claim 1 or 2, wherein the method produces a composition comprising vedolizumab having < 10% basic vedolizumab isoform species.

10. The method of claim 1, wherein the incubation is performed during purification of vedolizumab, and wherein the incubation is performed prior to ultrafiltration / diafiltration of vedolizumab.

11. The method of claim 1, wherein the incubation is performed at ambient temperature.

12. The method of claim 1, wherein the incubation is performed at 15°C-30°C.

13. The method of claim 1, wherein the incubation is performed at 20°C-25°C.

14. The method of claim 1, wherein the composition comprising vedolizumab is provided at a pH of 6.5-8.

5.

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

0.

16. The method of claim 1, wherein the composition comprising vedolizumab is provided at a pH of 7.0-7.

5.

17. The method of claim 1, wherein the composition comprising vedolizumab is provided at a pH of 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.

18. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 10-120 hours.

19. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 12-120 hours.

20. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 12-96 hours.

21. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 12-72 hours.

22. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 12-48 hours.

23. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of at least 12 hours.

24. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 24-120 hours.

25. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 24-96 hours.

26. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 24-72 hours.

27. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of 24-48 hours.

28. The method of claim 1, wherein the composition comprising vedolizumab is incubated for a period of time of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, or about 120 hours.

29. The method of claim 1, wherein the composition comprising vedolizumab comprises a first basic isoform peak and a second basic isoform peak, and wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having reduced levels of the second basic isoform peak.

30. The method of claim 29, wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having less than 2% of the second basic isoform peak.

31. The method of claim 29, wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having less than 1.5% of the second basic isoform peak.

32. The method of claim 29, wherein the method produces a composition comprising vedolizumab or an antigen-binding portion thereof having less than 1% of the second basic isoform peak.

33. The method of claim 29, wherein the method produces a composition comprising vedolizumab, or an antigen binding portion thereof, having less than 0.5% of a second basic isoform peak.

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

35. The method of claim 34, wherein the CHO cell culture comprises CHO cells lacking dihydrofolate reductase (DHFR) expression.

36. The method of claim 34, wherein the CHO cell culture comprises CHO cells lacking glutamine synthetase (GS) expression.

37. The method of claim 1, wherein the method further comprises purifying the composition comprising vedolizumab from mammalian host cell proteins (HCPs) using one or more chromatography separation steps selected from the group consisting of affinity chromatography, cation exchange chromatography, anion exchange chromatography, and ceramic hydroxyapatite (CHT) chromatography.

38. The method of claim 37, wherein the composition comprising vedolizumab is purified using an affinity chromatography resin comprising protein A.

39. The method of claim 38, wherein the composition comprising vedolizumab is purified using affinity chromatography prior to the incubation.

40. The method of claim 38, wherein the composition comprising vedolizumab is purified using affinity chromatography after the incubation.

41. The method of claim 37, wherein the composition comprising vedolizumab is purified using cation exchange chromatography.

42. The method of claim 41, wherein the composition comprising vedolizumab is purified using cation exchange chromatography prior to the incubation.

43. The method of claim 41, wherein the composition comprising vedolizumab is purified using cation exchange chromatography after the incubation.

44. The method of claim 37, wherein the composition comprising vedolizumab is purified using anion exchange chromatography.

45. The method of claim 44, wherein the composition comprising vedolizumab is purified using anion exchange chromatography prior to the incubation.

46. The method of claim 44, wherein the composition comprising vedolizumab is purified using anion exchange chromatography after the incubation.

47. The method of claim 37, wherein the composition comprising vedolizumab is purified using CHT chromatography.

48. The method of claim 47, wherein the composition comprising vedolizumab is purified using CHT chromatography prior to the incubation.

49. The method of claim 47, wherein the composition comprising vedolizumab is purified using CHT chromatography after the incubation.

50. The method of claim 1, wherein the method comprises incorporating the composition into a pharmaceutical formulation.

51. The method of claim 50, wherein the pharmaceutical formulation is a lyophilized pharmaceutical formulation.

52. The method of claim 51, wherein the lyophilized pharmaceutical formulation is a dry lyophilized pharmaceutical formulation.

53. The method of claim 52, further comprising the step of reconstituting the dry lyophilized pharmaceutical formulation with a liquid to make it suitable for administration.

54. The method of claim 50, wherein the pharmaceutical formulation is a liquid pharmaceutical formulation.

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