Antibody purification methods and compositions thereof

Abstract

To provide a method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, and a composition obtainable thereby.SOLUTION: This method comprises obtaining a composition comprising an anti-α4β7 antibody by contacting a solution comprising the anti-α4β7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow flow through of the anti-α4β7 antibody through the HIC resin, wherein the HIC resin is characterized as a high hydrophobic HIC resin, and the anti-α4β7 antibody is a humanized antibody, is an IgG1 antibody, comprises a heavy chain variable region comprising a CDR3 domain, a CDR2 domain, and a CDR1 domain, each having a specific sequence, and comprises a light chain variable region comprising a CDR3 domain, a CDR2 domain, and a CDR1 domain, each having a specific sequence.SELECTED DRAWING: None

Description

Technical Field

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

[0002] Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 859,580, filed on June 10, 2019. The entire contents of the above application are incorporated herein by reference.

[0003] Sequence Listing This application includes a sequence listing that is electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on June 5, 2020 is named T103022_1120WO_SL.txt and has a size of 10,015 bytes.

Background Art

[0004] Large-scale and economical purification of proteins is becoming an increasingly important issue in the biotechnology industry. Generally, biopharmaceuticals are produced by cell culture using prokaryotic cell lines such as bacterial cells, or eukaryotic cell lines such as mammalian or fungal cells, which are engineered to produce large amounts of the desired therapeutic protein. Since the cell lines used are biological, they must be supplied with a complex cell culture medium containing sugars, amino acids, and, optionally, growth factors supplied from formulations of animal serum. The desired recombinant therapeutic protein is contaminated with process-related impurities such as cell culture medium components, host cell proteins (HCPs), host nucleic acids, and / or chromatography media materials, as well as aggregates, misfolded species, and so on. ​​​​​​​​​or from product-related impurities such as fragments of the target protein for use as a human therapeutic agent It has been a difficult problem to separate it to a sufficient purity for this purpose.

[0005] Product-related and process-related impurities including aggregates interfere with the purification process and may affect the protein during storage and / or may cause adverse reactions when the antibody is administered as a pharmaceutical to a subject (Shukla et al., J. C hromatogr. B. Analyt. Technol. Biomed. Life S ci., 848(1), 28 - 39).

[0006] Therefore, there is a continuing need in the art for improved methods for purifying therapeutic proteins such as antibodies while effectively removing impurities, improving the protein recovery rate, and maintaining the therapeutic requirements conditions, for example, for purifying therapeutic proteins such as antibodies.

Summary of the Invention

[0007] The present invention particularly provides a method for purifying an anti-α4β7 antibody such as vedolizumab from, for example, a liquid solution.

[0008] In one aspect, the present invention is a method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising contacting a matrix comprising protein A with the liquid solution comprising the anti-α4β7 antibody and one or more impurities to bind the anti-α4β7 antibody to the protein A and washing the matrix comprising protein A with a washing solution and then contacting the matrix with an elution solution having a pH of 3.2 to 4 to elute the anti-α4β7 antibody from the matrix comprising protein A to obtain a composition comprising the anti-α4β7 antibody ​​​​​ and wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and is represented by SEQ ID NO: 4. the CDR3 domain described in SEQ ID NO:3, the CDR2 domain described in SEQ ID NO:2 a heavy chain variable region comprising a CDR1 domain of the sequence A light chain comprising the CDR2 domain set forth in SEQ ID NO:7 and the CDR1 domain set forth in SEQ ID NO:6. The method further features a method comprising:

[0009] In one embodiment, the method comprises administering a composition containing less than 1% high molecular weight (HMW) aggregates to an anti-alpha The method is used to obtain a 4β7 antibody from a liquid solution containing the antibody and one or more impurities, the method comprising the steps of: A matrix containing tein A is contacted with a liquid solution containing anti-α4β7 antibody and one or more impurities. and binding the anti-α4β7 antibody to Protein A by contacting the antibody with Protein A. The washing of the matrix with a washing solution containing 1,2-dichlorophenyl ether, and the dissolution of the matrix with an elution solution having a pH of 3.2 to 4 were performed. The release of anti-α4β7 antibodies from a matrix comprising protein A by contacting the matrix with a liquid. Elution results in a composition containing less than 1% HMW aggregates.

[0010] In one embodiment, Protein A is immobilized on a solid phase. In one embodiment, the solid phase is The material may include one or more of beads, gels, and resins.

[0011] In one embodiment, the wash solution has a pH of about 7. In one embodiment, the elution solution is Contains enoic acid.

[0012] In one embodiment, the elution solution has a pH of 3.2 to 3.7.

[0013] In another aspect, the present invention provides a method for extracting an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities. A method for obtaining a composition comprising an α4β7 antibody, the method comprising contacting a solution containing an anti-α4β7 antibody and at least 1 impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow a flow-through of the anti-α4β7 antibody through the HIC resin, whereby a composition comprising the anti-α4β7 antibody is obtained, the HIC resin being characterized as a highly hydrophobic HIC resin, and the anti-α4β7 antibody being a humanized antibody, an IgG1 antibody, comprising a heavy chain variable region containing the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and comprising a light chain variable region containing the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6. In one embodiment, the method is for obtaining a composition comprising the anti-α4β7 antibody and less than 0.6% HMW aggregates from a liquid solution containing the anti-α4β7 antibody and one or more impurities, the method comprising contacting the solution containing the anti-α4β7 antibody and at least 1 impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow a flow-through of the

[0014] anti-α4β7 antibody through the HIC resin, whereby a composition comprising the anti-α4β7 antibody and less than 0.6% HMW aggregates is obtained, the anti-α4β7 antibody being a humanized antibody, an IgG1 antibody, comprising a heavy chain variable region containing the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and comprising a light chain variable region containing the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6. ​​​​​

[0015] In one embodiment, the HIC resin is equilibrated with a phosphate buffer having a pH of less than about 7.2. In one embodiment, the phosphate buffer contains from about 0.35 mM to about 0.15 mM potassium phosphate.

[0016] In one embodiment, the resin load is from about 55 to 75 mg / ml.

[0017] In one embodiment, the composition contains less than about 0.22 ppm of residual Protein A.

[0018] In one embodiment, the composition contains less than about 0.3 ppm of host cell protein (HCP).

[0019] In one embodiment, the highly hydrophobic HIC resin has an average pore size of from about 50 to 150 μm.

[0020] In one embodiment, the highly hydrophobic HIC resin has an average pore size of about 100 nm and / or a pore size of about 10 0 μm.

[0021] In another aspect, the present invention provides a method for producing a formulation comprising an anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising binding the anti-α4β7 antibody to a mixed-mode chromatography resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin; washing the mixed-mode chromatography resin with a washing solution; and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 3.9 or higher to obtain a purified formulation comprising the anti-α4β7 antibody, wherein the anti-α4β7 antibody has the heavy chain set forth in SEQ ID NO: 1. A method comprising a variable heavy chain region and a variable light chain region as set forth in SEQ ID NO: 2.

[0022] In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates. In one embodiment of the above aspect, the method is for obtaining a formulation containing less than 1% high molecular weight (HMW) aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method comprises binding the anti-α4β7 antibody to a resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the resin by contacting the resin with an elution solution having a pH of 3.9 or higher, thereby obtaining a formulation containing less than 1% HMW aggregates.

[0023] In one embodiment, the elution solution has a pH of 4.1 or higher. In another embodiment, the elution solution has a pH of about 3.9 to about 4.4. In one embodiment, the elution solution has a pH of 4.1 or higher. In another embodiment, the elution solution has a pH of about 3.9 to about 4.4.

[0024] In some embodiments, the elution solution has a conductivity of 30 mS / cm or less. In certain embodiments, the elution solution has a conductivity of about 20 mS / cm to about 30 mS / cm. In some embodiments, the elution solution has a conductivity of 30 mS / cm or less. In certain embodiments, the elution solution has a conductivity of about 20 mS / cm to about 30 mS / cm.

[0025] In some embodiments, the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM. In some embodiments, the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM.

[0026] In certain embodiments, the mixed-mode chromatography resin is Capto Adhere ImpRes. In certain embodiments, the mixed-mode chromatography resin is Capto Adhere ImpRes.

[0027] In some embodiments of the above aspect, the method uses a cation exchange (CEX) resin. Further comprising purifying the anti-α4β7 antibody. In some such embodiments, the CEX resin is operated in a bind / elute mode.

[0028] In another aspect, the present invention provides a method for producing a formulation comprising an anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, the method comprising binding the anti-α4β7 antibody to a mixed-mode chromatography resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising the purified anti-α4β7 antibody, and the anti-α4β7 antibody comprising a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2. α4β7 antibody, and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising the purified anti-α4β7 antibody, and the anti-α4β7 antibody comprising a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2. impurities, binding the anti-α4β7 antibody to the resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising the purified anti-α4β7 antibody, and the anti-α4β7 antibody comprising a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2. impurities, binding the anti-α4β7 antibody to the resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising the purified anti-α4β7 antibody, and the anti-α4β7 antibody comprising a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 2. characterized.

[0029] In some embodiments of the above aspects, the method is for obtaining a formulation comprising a high yield of anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, the method comprising binding the anti-α4β7 antibody to a mixed-mode chromatography resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising a high yield of anti-α4β7 antibody. impurities with a mixed-mode chromatography resin, binding the anti-α4β7 antibody to the resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising a high yield of anti-α4β7 antibody. impurities with a mixed-mode chromatography resin, binding the anti-α4β7 antibody to the resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising a high yield of anti-α4β7 antibody. impurities with a mixed-mode chromatography resin, binding the anti-α4β7 antibody to the resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin, washing the mixed-mode chromatography resin with a washing solution, and eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or less and a conductivity of 28 mS / cm or less to obtain a formulation comprising a high yield of anti-α4β7 antibody.

[0030] In some embodiments, the elution solution has a pH of 4.0 or less. In other embodiments , the elution solution has a pH of from about pH 4.2 to about pH 3.8.

[0031] In some embodiments, the elution solution has a conductivity of from about 18 mS / cm to about 28 mS / cm and has.

[0032] In some embodiments, the elution solution contains NaCl at a concentration of from about 160 mM to about 240 mM and contains.

[0033] In some embodiments of the above aspects, the mixed-mode chromatography resin is resin 1 and is contacted with at least 55 g of anti-α4β7 antibody per liter of resin. In certain embodiments, the mixed mode chromatography resin is contacted with from about 55 g to about 80 g of anti-α4β7 antibody per liter of resin and is contacted with the body.

[0034] In some embodiments of the above aspects, the mixed-mode chromatography resin is Cap to Adhere ImpRes.

[0035] In some embodiments of the above aspects, the method further comprises purifying the anti-α4β7 antibody using a cation exchange (CEX) resin . In some such embodiments, the CEX resin is operated in a bind / elute mode.

[0036] In another aspect, the present invention is a method for producing a formulation comprising an anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising contacting a liquid solution comprising an anti-α4β7 antibody and one or more impurities with a cation exchange (CEX) resin to obtain an anti-α4β 7 antibody, by Binding the 7 antibody to the resin, washing the CEX resin with a washing solution, and contacting the resin with an elution solution having a conductivity of 16 mS / cm or less to elute the anti-α4 β7 antibody from the CEX resin to obtain a preparation containing the purified anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises the heavy chain variable region set forth in SEQ ID NO: 1 and the light chain variable region set forth in SEQ ID NO: 2.

[0037] In one embodiment of the above aspect, the method is for obtaining a preparation containing a reduced level of HMW aggregates from a liquid solution containing the anti-α4β7 antibody and one or more impurities, and the method comprises binding the anti-α4β7 antibody to the resin by contacting a liquid solution containing the anti-α4β7 antibody and one or more impurities with the CEX resin, washing the CEX resin with the washing solution, and contacting the resin with an elution solution having a conductivity of 16 mS / cm or less to elute the anti-α4β7 antibody from the CEX resin, whereby a preparation containing a reduced level of HMW aggregates is obtained. In some embodiments of the above aspect, the elution solution has a conductivity of 14 mS / cm or less. In other embodiments, the elution solution has a conductivity of about 11-16 mS / cm. In still other embodiments, the elution solution has a conductivity of about 12-14 mS / cm. In some embodiments of the above aspect, the elution solution contains about 70 mM to about 110 mM of NaCl.

[0038] In some embodiments of the above aspect, the elution solution has a pH of about pH 5 to about pH 6.

[0039]

[0040] ​​​​​​​It is. In certain embodiments, the elution solution has a pH of from about pH 5.1 to about pH 5.8.

[0041] In some embodiments of the above aspects, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 to 7 0 g of antibody per liter of resin. In certain embodiments, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 to 60 g of antibody per liter of resin.

[0042] In some embodiments of the above aspects, the CEX resin is Nuvia HR-S.

[0043] In some embodiments of the above aspects, the method further comprises purifying the anti-α4β7 antibody using a mixed-mode chromatography resin. In some such embodiments, the mixed-mode chromatography resin is operated in a binding / elution mode.

[0044] In another aspect, the present invention provides a method for generating a formulation comprising an anti-α4β7 antibody from a liquid solution comprising the major isoform of the anti-α4β7 antibody and one or more basic isoform species, the method comprising binding the anti-α4β7 antibody to a cation exchange (CEX) resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more basic isoform species with the CEX resin, washing the CEX resin with a wash solution, and eluting the anti-α4β7 antibody from the CEX resin by contacting the resin with an elution solution having a conductivity of 11 mS / cm or more, thereby obtaining a formulation comprising the purified anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises the heavy chain variable region set forth in SEQ ID NO: 1 and the light chain variable region set forth in SEQ ID NO: 2.

[0045] .

[0045] In one embodiment of the above aspect, the method is for obtaining a formulation with a reduced level of the basic isoform species of an anti-α4β7 antibody from a liquid solution containing the major isoform of the anti-α4β7 antibody and one or more basic isoform species. The method comprises contacting a liquid solution containing the anti-α4β7 antibody and one or more basic isoform species with a CEX resin to bind the anti-α4β7 antibody to the resin, washing the CEX resin with a washing solution, and contacting the resin with an elution solution having a conductivity of 11 mS / cm or more to elute the anti-α4β7 antibody from the CEX resin, thereby obtaining a formulation containing a reduced level of the basic isoform species. In one embodiment, the purified composition comprises from about 4% to about 20% basic isoform. In some embodiments of the above aspect, the elution solution has a conductivity of 12 mS / cm or more. In other embodiments, the elution solution has a conductivity of about 11 - 16 mS / cm. In yet other embodiments, the elution solution has a conductivity of about 12 - 14 mS / cm. In some embodiments of the above aspect, the elution solution has a pH of about pH 5 to about pH 6. In certain embodiments, the elution solution has a pH of about pH 5.1 to about pH 5.8.

[0046] In some embodiments of the above aspect, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 - 70 g of antibody per liter of resin. In certain embodiments, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 - 60 g of antibody per liter of resin.

[0047] In some embodiments of the above aspect, the elution solution has a conductivity of 12 mS / cm or more. In other embodiments, the elution solution has a conductivity of about 11 - 16 mS / cm. In yet other embodiments, the elution solution has a conductivity of about 12 - 14 mS / cm. In some embodiments of the above aspect, the elution solution has a pH of about pH 5 to about pH 6. In certain embodiments, the elution solution has a pH of about pH 5.1 to about pH 5.8.

[0048] In some embodiments of the above aspect, the elution solution has a pH of about pH 5 to about pH 6. In certain embodiments, the elution solution has a pH of about pH 5.1 to about pH 5.8. In some embodiments of the above aspect, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 - 70 g of antibody per liter of resin. In certain embodiments, the anti-α4β7 antibody

[0049] In some embodiments of the above aspect, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 - 70 g of antibody per liter of resin. In certain embodiments, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 - 60 g of antibody per liter of resin. In some embodiments of the above aspect, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 - 70 g of antibody per liter of resin. In certain embodiments, the anti-α4β7 antibody

[0050] In some embodiments, the elution solution contains sodium chloride, for example, 70 to 110 mM of sodium chloride.

[0051] In some embodiments of the above aspect, the CEX resin is Nuvia HR-S.

[0052] In some embodiments of the above aspect, the method further comprises purifying the anti-α4β7 antibody using a mixed-mode chromatography resin. In some such embodiments, the mixed-mode chromatography resin is operated in a binding / elution mode. using to purify the anti-α4β7 antibody. In some such embodiments, the mixed mode chromatography resin is operated in a binding / elution mode.

[0053] In any one embodiment of the above aspect, the antibody is produced in Chinese hamster ovary (CHO ) host cells.

[0054] In one embodiment, the host cells are GS-CHO cells.

[0055] In any one embodiment of the above aspect, the anti-α4β7 antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO: 5. variable region sequence and the light chain variable region sequence set forth in SEQ ID NO: 5.

[0056] In any one embodiment of the above aspect, the anti-α4β7 antibody is vedolizumab.

[0057] Furthermore, the present invention also includes the following embodiments.

[0058] 1. A method for obtaining a composition containing less than 1% HMW aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities, comprising: contacting the liquid solution containing the anti-α4β7 antibody and the one or more impurities with a matrix containing protein A to bind the anti-α4β7 antibody to the protein A, and performing; washing the matrix containing the Protein A with a washing solution; contacting the matrix with an elution solution having a pH of 3.2 to 4 to elute the anti-α4β7 antibody from the matrix containing the Protein A, and obtaining a composition containing less than 1% of HMW aggregates; wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and contains a heavy chain variable region containing the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and contains a light chain variable region containing the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6; the method comprising: 2. The method according to item 1, wherein the Protein A is immobilized on a solid phase. 3. The method according to item 2, wherein the solid phase comprises one or more of beads, gels, and resins. 4. The method according to any one of items 1 to 3, wherein the pH of the washing solution is about 7. 5. The method according to any one of items 1 to 4, wherein the elution solution contains citric acid. 6. The method according to any one of items 1 to 5, wherein the pH of the elution solution is 3.2 to 3.7.

[0059] 7. A method for obtaining a composition containing an anti-α4β7 antibody and less than 0.6% of HMW aggregates from a liquid solution containing the anti-α4β7 antibody and one or more impurities,

[0060]

[0061]

[0062]

[0063]

[0064] ​​​​​​​​A solution containing an anti-α4β7 antibody and at least one impurity is subjected to hydrophobic interaction chromatography (HIC) resin, and the flow-through of the anti-α4β7 antibody through the HIC resin is contacted under conditions that allow it, to obtain a composition containing the anti-α4β7 antibody and less than 0.6% H MW aggregates, wherein the HIC resin is characterized as a highly hydrophobic resin, the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and has the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and contains a heavy chain variable region, and has the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6, and contains a light chain variable region, the method.

[0065] 8. The method according to item 7, wherein the HIC resin is equilibrated with a phosphate buffer having a pH of less than about 7.2.

[0066] 9. The method according to item 8, wherein the phosphate buffer contains from about 0.35 mM to about 0.15 mM potassium phosphate.

[0067] 10. The method according to any one of items 7 to 9, wherein the resin load is from about 55 to 75 mg / ml.

[0068] 11. The method according to any one of items 7 to 10, wherein the composition contains less than about 0.22 ppm of residual Protein A.

[0069] 12. The method according to any one of items 7 to 11, wherein the composition contains less than about 0.3 ppm of host cell protein (HCP). ​​​​​​​

[0070] 13. The method according to any one of items 7, wherein the highly hydrophobic HIC resin has an average pore size of about 50 to 150 μm. ~12.

[0071] 14. The method according to any one of items 7 to 12, wherein the highly hydrophobic HIC resin has an average pore size of about 100 nm and / or a pore size of about 100 μm.

[0072] 15. The method according to any one of items 1 to 14, wherein the antibody is produced in Chinese hamster ovary (CHO) cells.

[0073] 16. The method according to item 15, wherein the host cell is a GS-CHO cell.

[0074] 17. The method according to any one of items 1 to 16, wherein the anti-α4β7 antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO: 5.

[0075] 18. The method according to any one of items 1 to 16, wherein the anti-α4β7 antibody is vedolizumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0076]

Figure 1

Figure 2-1

Figure 2-2

Figure 2-3

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Mode for Carrying Out the Invention

[0077] The present invention particularly relates to a purification method for controlling the amounts of product-related substances (such as aggregates such as high molecular weight (HMW) aggregates, misfolded species, or protein fragments) and / or process-related impurities (such as host cell proteins (HCPs), host cell nucleic acids, viruses, chromatography materials, and / or medium components) present in a preparation of an anti-α4β7 antibody or an antigen-binding fragment thereof, such as vedolizumab.

[0078] I. Definitions To make the present invention more readily understandable, certain terms are first defined.

[0079] The "α4β7 integrin", or "α4β7" (used interchangeably throughout this specification), which is a cell surface molecule, is a heterodimer of an α4 chain (CD49D, ITGA4) and a β7 chain (ITGB7). The human α4 integrin and β7 integrin genes (GenBank (National Center for Biotechnology Information, Bethesda, Md.) RefSeq accession numbers NM_000885 and NM_000889, respectively) are expressed by B and T lymphocytes, particularly memory CD4+ lymphocytes. As is typical for many integrins, α4β7 can exist in a resting or activated state. Ligands for α4β7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM (e.g., MAdCAM-1)). An antibody that binds to α4β7 integrin is referred to herein as an "anti-α4β7 antibody".

[0080] As used herein, an antibody or antigen-binding fragment having "binding specificity for the α4β7 complex" binds to α4β7 but does not bind to α4β1 or α β7. Vedolizumab is an example of an antibody having binding specificity for the α4β7 complex. E mab. The term "antibody" as used herein refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.

[0081] Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. 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 divided into hypervariable regions called complementarity determining regions (CDRs) with more conserved regions in between called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CD R2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. In some embodiments, the antibody has a "fragment crystallizable" (Fc) region. In certain embodiments, the antibody is of the IgG1 isotype and has a κ light chain. A "CDR" or "complementarity determining region" is a region of hypervariability intervening within a more conserved region called the framework region (FR).

[0082]

[0083] ​​​​​​​ As used herein, the terms "antigen-binding fragment" or "antigen-binding portion" of an antibody refer to Fab, Fab’, F(ab’) 2 , and Fv fragments, single-chain antibodies, functional heavy-chain antibodies (nanobodies), and any portion of an antibody that has specificity for at least one desired epitope and competes with the intact antibody for specific binding (e.g., an isolated portion of a complementarity-determining region having a framework sequence sufficient to specifically bind to an epitope). Antigen-binding fragments can be produced by recombinant techniques or by enzymatic or chemical cleavage of an antibody. The "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from the non-human antibody. In many cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable regions of the recipient are replaced with residues from the hypervariable regions of a non-human species such as mouse, rat, rabbit, or non-human primate (donor antibody) having the desired specificity, affinity, and functionality. Optionally, framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These alterations are made to further improve the performance of the antibody. Generally, a humanized antibody contains at least one, and typically two, variable domains substantially all of whose hypervariable CDR loops correspond to those of the non-human antibody and substantially all of whose FRs correspond to those of the human antibody sequence. A humanized antibody may optionally contain at least a portion of the antibody constant region (Fc).

[0084] ​​​​​​​​​​​​Further details include those of some, typically human antibodies. Jones et al., Nature 321:522-525 (1986), Ri echmann et al., Nature 332:323-329 (1988), and Presta, Curr. Struct. Biol. 2:593-596 (1992 ) for more information.

[0085] As used herein, a "recombinant antibody" refers to an antibody that is introduced into a host cell, e.g., a mammalian cell. Transcription and translation of gene(s) incorporated into recombinant expression vector(s) In certain embodiments, the recombinant protein is an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, The antibody is of an isotype selected from the group consisting of IgD, IgE, or IgD. In the present invention, the recombinant antibody is an IgG1.

[0086] The term "recombinant host cell" (used interchangeably herein with the term "host cell") The term includes cells into which a recombinant expression vector has been introduced. It is understood that the term is intended to refer not only to the cell but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, Although such progeny may not actually be identical to the parent cell, as used herein, In addition, unless otherwise specified, the term "host cell" includes, for example, The term "cell" is used, such as host cell or mammalian cell or mammalian host cell. It should be understood that when used, the term is intended to include a population of cells.

[0087] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of the introduced host cell. A particular vector can induce the expression of a nucleic acid to which the vector is operably linked. Such a vector is referred to herein as an "expression vector". As used herein, the term "upstream process" in relation to a protein refers to activities that generate and recover a protein (e.g., an antibody) from a host cell (e.g., in cell culture for generating a protein of interest such as an antibody). As used herein, the term "downstream process" refers to one or more techniques used after an upstream process for purifying a protein such as an antibody of interest. For example, techniques for downstream processes include, for example, affinity chromatography including protein A affinity chromatography, ion exchange chromatography such as anion or cation exchange ion exchange chromatography, size exclusion chromatography, mixed mode chromatography, hydrophobic interaction chromatography (HIC), or purification of the protein product using displacement chromatography. As used herein, the terms "culture" and "cell culture" generally refer to the process by which cells are grown under controlled conditions, generally outside of their natural environment. To "culture" cells

[0088]

[0089]

[0090] "Culturing" refers to contacting cells with a cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation. In certain embodiments, cell culture refers to methods for generating and maintaining a population of host cells capable of producing a recombinant protein of interest, e.g., an anti-α4β7 antibody, as well as methods and techniques for generating and recovering the protein of interest. For example, after an expression vector has been incorporated into a suitable host, e.g., a host cell in culture, the host can be maintained under conditions suitable for expression of the relevant nucleotide coding sequence and for recovery and purification of the desired recombinant protein. "Cell culture" may also refer to a solution containing cells. As used herein, "clarified harvest" refers to a liquid material containing a protein of interest, e.g., an anti-α4β7 antibody, extracted from a cell culture, e.g., a fermentation bioreactor, after one or more steps have been performed to remove solid particles such as cell debris and particulate impurities from the material. After cell culture, the harvest is typically purified using separation techniques such as centrifugation and filtration to remove cells and cell debris. The initial clarification, particle removal step yields a "clarified harvest" that can be used, for example, in subsequent chromatography steps (downstream processes). The clarified harvest is generally the starting material in downstream processes such as those described herein. As used herein, "chromatography support" refers to a material having a specific chemical composition or specific three-dimensional structure, or to chromatography including affinity chromatography, gel filtration (size exclusion chromatography), or ion exchange chromatography.

[0091]

[0092] ​ A solid or porous matrix to which specific chemical groups or macromolecules can be immobilized for performing chromatography. Examples of chromatography supports include, but are not limited to, resins (e.g., agarose) or membranes. As used herein, the "chromatography housing" refers to a structure that houses a chromatography support. Examples of chromatography housings include columns or cartridges, or other containers. As used herein, the "buffer" refers to an aqueous solution that resists changes in pH due to the action of its acid-base conjugate components. The "buffer" is used to establish a specific set of conditions for regulating the control of a process step or a chromatography support such as a chromatography resin or membrane.

[0093] As used herein, the term "equilibration solution" refers to an aqueous solution formulated to establish the initial operating conditions of a process step or a chromatography support, e.g., for a chromatography operation. The equilibration solution is used to prepare a solid phase, e.g., a chromatography support such as a resin or membrane, for loading a target protein, e.g., an antibody.

[0094] As used herein, the term "washing solution" or "washing liquid" refers to an aqueous solution formulated to displace unbound contaminants from a chromatography support such as a resin or membrane. In some embodiments, the washing solution is used after loading a target protein, e.g., an antibody, and before eluting the target protein, e.g., an antibody, from a solid support such as a resin or membrane.

[0095] or membrane. or passed through the membrane. In one embodiment, the wash solution has biochemical properties similar to the equilibration solution. It is.

[0096] As used herein, the term "flow-through operation" means that during washing, the protein does not substantially bind to the matrix, e.g., a hydrophobic chromatography resin, and / or elutes, while impurities remain bound to the chromatography support. refers to a process such that. It refers to.

[0097] As used herein, the terms "elution solution" or "eluate" refer to an aqueous solution formulated to displace a target protein, e.g., an antibody, from a chromatography support, e.g., a resin or a membrane. In one embodiment, the elution solution has biochemical properties different from those of the equilibration solution and / or the wash solution such that the target protein, e.g., an antibody, associates more readily with the elution solution than with the chromatography support, e.g., a resin or a membrane.

[0098] As used herein, the term "impurities" with respect to impurities contained in a solution containing an antibody to be purified includes both process-related impurities and product-related impurities.

[0099] As used herein, the term "process-related impurities" refers to impurities (or impurities) that are present in a composition containing a protein, e.g., a solution, but are not derived from the protein itself. For example, process-related impurities include, but are not limited to, cell culture components, host cell components e.g., host cell proteins (HCP), host cell nucleic acids, or also lipid-containing intracellular structures or fragments thereof, viruses, trace metals from buffers, or Examples include leachable substances from ions, containers for handling materials, or chromatographic supports. Process-related impurities can be generated during the preparation of proteins, such as antibodies (upstream and / or downstream processes). The term "host cell impurity" as used herein refers to host cell lines, cell culture media, or any proteinaceous, nucleic acid contaminants, lipid contaminants, or by-products introduced by cell culture. Examples of impurities include, but are not limited to, Chinese

[0100] Hamster Ovary Protein (CHOP), E. coli proteins, enzyme proteins, simian COS S protein, or myeloma cell proteins (e.g., NS0 protein (derived from BALB / c mouse-derived mouse plasmacytoma)).

[0101]

[0101] The term "product-related impurity" as used herein includes impurities derived from the protein of interest, such as the antibody itself. For example, product-related impurities include, but are not limited to, aggregates (e.g., HMW), misfolded species, oxidized or deamidated species, or low molecular weight fragments of the antibody of interest.

[0102] The term "aggregate" or "aggregates" as used herein refers to the association of multiple antibodies or antibody fragments. For example, an aggregate can be a dimer, trimer, tetramer, or multimer larger than a tetramer of an antibody and / or antibody fragment. Antibody aggregates can be soluble or insoluble. The bonds between aggregated molecules can be covalent or non-covalent, regardless of the mechanism by which the molecules associate. The bond can be direct between the aggregated molecules, or It may also be indirect through other molecules that link the molecules to each other. Examples of the latter include, but are not limited to, disulfide bonds with other proteins, hydrophobic bonds with lipids, charge bonds with DNA, affinity bonds with leached protein A, or mixed-mode bonds with multiple components. Aggregates can form irreversibly during protein expression in cell culture, during protein purification in downstream processes, or during storage of pharmaceuticals. The presence of aggregates in solution can be determined, for example, using size exclusion chromatography (SEC) (e.g., SEC by UV detection, SEC by light scattering detection (SEC-LSD)), field flow fractionation, sedimentation velocity method of ultracentrifugal analysis, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced). Among these are, but not limited to, disulfide bonds with other proteins, hydrophobic bonds with lipids charge bonds with DNA, affinity bonds with leached protein A, or mixed-mode bonds with multiple components. Aggregates can form irreversibly during protein expression in cell culture, during protein purification in downstream processes, or during storage of pharmaceuticals. The presence of aggregates in solution can be determined, for example, using size exclusion chromatography (SEC) (e.g., SEC by UV detection, SEC by light scattering detection (SEC-LSD)), field flow fractionation, sedimentation velocity method of ultracentrifugal analysis, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced). formed during protein expression in cell culture, during protein purification in downstream processes, or during storage of pharmaceuticals The presence of aggregates in solution can be determined, for example, using size exclusion chromatography (SEC) (e.g., SEC by UV detection, SEC by light scattering detection (SEC-LSD)), field flow fractionation, sedimentation velocity method of ultracentrifugal analysis, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced). field flow fractionation, sedimentation velocity method of ultracentrifugal analysis, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced). capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced). can be determined.

[0103] The term "high molecular weight" or "HMW" is used to refer to antibody complexes having a molecular weight greater than that of monomeric antibodies. In one embodiment, the HMW aggregates have a molecular weight greater than about 147 kDa. The presence of high molecular weight aggregates can be determined, for example, by standard methods well known in the art such as size exclusion chromatography (SEC). The term "high molecular weight" or "HMW" is used to refer to antibody complexes having a molecular weight greater than that of monomeric antibodies. In one embodiment, the HMW aggregates have a molecular weight greater than about 147 kDa. The presence of high molecular weight aggregates can be determined, for example, by standard methods well known in the art such as size exclusion chromatography (SEC). The presence of high molecular weight aggregates can be determined, for example, by standard methods well known in the art such as size exclusion chromatography (SEC). can be determined by standard methods well known in the art such as size exclusion chromatography (SEC). can be determined.

[0104] "Substantially purified" with respect to a desired protein means that a purified sample containing the protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% pure. Comprising the recombinant protein of interest, with impurities less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5%. This means that it is less than the specified percentage.

[0105] The term "about" means that the subsequent value is not an exact value but is at the center point of a range that is ±5% of that value. When the value is a relative value given as a percentage, the term "about" means that the subsequent value is not an exact value but is at the center point of a range that is ±5% of that value, meaning that the upper limit of the range cannot exceed a value of 100%.

[0106] II. Methods and Compositions Related to Antibody Purification Disclosed herein are methods for purifying anti-α4β7 antibodies, such as vedolizumab, from, for example, a liquid solution, such as a clarified harvest from a mammalian cell culture. The present invention provides a specific embodiment of an antibody purification process that reduces the level of impurities present in the antibody solution, such as process-related impurities, including cell culture medium components, host cell proteins (HCPs), host cell nucleic acids, viruses, and chromatography materials, as well as product-related impurities, including aggregates (including HMW aggregates), misfolded species, or fragments of the protein of interest. The methods of the present invention are useful for purifying anti-α4β7 antibodies, particularly antibodies having the binding region of vedolizumab (i.e., CDR or variable region), thereby enabling the formulation of the antibody for use in human patients. Specifically, the methods disclosed herein are useful for obtaining low levels of antibody aggregation, such as HMW antibody aggregates. In certain embodiments, the methods disclosed herein provide for about 0% to about 1% HMW antibody aggregates. The methods of the present invention are based at least in part on purifying antibodies having the binding region of vedolizumab (i.e., CDR or variable region), thereby enabling the formulation of the antibody for use in human patients. This can be done.

[0107] Specifically, the methods disclosed herein are useful for obtaining low levels of antibody aggregation, such as HMW antibody aggregates. In certain embodiments, the methods disclosed herein provide for about 0% ~5.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1. 5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3% , 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3 .2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4 %, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8 %, 4.9%, or 5%) of aggregates, e.g., compositions having HMW aggregates are provided . In certain embodiments, the methods disclosed herein are from about 0% to 2%, 2% or less, 1.9 % or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1 .3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0 .7% or less, 0.6% or less, or 0.5% or less of aggregates, e.g., compositions having HMW aggregates are provided . The present invention also includes compositions comprising an anti-α4β7 antibody and said low level of HMW aggregates . Specifically, using the methods disclosed herein, anti-α4β7 antibody vedolizumab, or an antibody having the antigen-binding region of vedolizumab can be produced. Vedolizumab is also , known by the trade name ENTYVIO® (Takeda Pharmaceutical Company). Vedolizumab is a humanized antibody comprising the framework and constant regions of human IgG1 and the antigen-binding CDRs derived from the murine antibody Act-1 . The CDRs, variable regions, and mutated Fc regions (mutated to abolish Fc effector function) of vedolizumab are incorporated herein by reference . It is described in U.S. Patent No. 7,147,851, the entire contents of which are incorporated herein by reference.

[0108] Vedolizumab specifically binds to α4β7 integrin, such as the α4β7 complex, and blocks the interaction between α4β7 integrin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1), inhibiting the migration of memory T lymphocytes through the endothelium into the inflamed gastrointestinal parenchymal tissue. Vedolizumab is a humanized monoclonal antibody that does not bind to α4β1 and αEβ7 integrins, does not inhibit their function, and does not antagonize the interaction between α4 integrin and vascular cell adhesion molecule 1 (VCAM-1).

[0109] α4β7 integrin is expressed on the surface of a distinct subset of memory T lymphocytes that selectively migrate to the gastrointestinal tract. MAdCAM-1 is mainly expressed on endothelial cells of the intestine and plays an important role in the homing of T lymphocytes to intestinal lymphoid tissue. The interaction between α4β7 integrin and MAdCAM-1 has been shown to be an important contributing factor to mucosal inflammation, such as the chronic inflammation characteristic of ulcerative colitis and Crohn's disease. The use of vedolizumab can treat inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, ileitis including chronic ileitis, graft-versus-host disease, and HIV.

[0110] 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 contains a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4. Vedolizumab contains a light chain variable region It includes a light chain variable region containing a CDR3 of 8. In one embodiment, the antibody includes 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. The sequences of vedolizumab and of vedolizumab are both incorporated herein by reference in their entirety in U.S. Patent Application Publication No. 2014 / 0341885 and U.S. Patent Application Publication No. 2014-0377251. The methods disclosed herein can be carried out using an antibody comprising a binding region (e.g., a CDR or variable region) described above and in the accompanying Sequence Listing. The methods of making antibodies are well known in the art. Mammalian hosts are engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab). The overall cell culture process and considerations in the production of any monoclonal antibody, such as vedolizumab, are described in Li et al. (2010) mAbs 2:5,466-477, and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685, which are incorporated herein by reference. When using cell culture methods, the anti-α4β7 antibody may be produced intracellularly, in the periplasmic space, or secreted directly into the medium. In embodiments where the anti-α4β7 antibody is produced intracellularly, particulate debris of the host cells or lysed cells (e.g., resulting from homogenization) can be removed by various means including, but not limited to, centrifugation or filtration. When the anti-α4β7 antibody is secreted into the medium, the supernatant from such an expression system can be concentrated using a commercially available protein concentration filter. U.S. Patent Application Publication No. 2014 / 0341885 and U.S. Patent Application Publication No. 2014-0377251 are also incorporated herein by reference in their entirety. The methods disclosed herein can be carried out using an antibody comprising a binding region (e.g., a CDR or variable region) described above and in the accompanying Sequence Listing. It includes a light chain variable region containing a CDR3 of 8. In one embodiment, the antibody includes 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. The sequences of vedolizumab and of vedolizumab are both incorporated herein by reference in their entirety in U.S. Patent Application Publication No. 2014 / 0341885 and U.S. Patent Application Publication No. 2014-0377251. The methods disclosed herein can be carried out using an antibody comprising a binding region (e.g., a CDR or variable region) described above and in the accompanying Sequence Listing.

[0111] The methods of making antibodies are well known in the art. Mammalian hosts are engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab). The overall cell culture process and considerations in the production of any monoclonal antibody, such as vedolizumab, are described in Li et al. (2010) mAbs 2:5,466-477, and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685, which are incorporated herein by reference. When using cell culture methods, the anti-α4β7 antibody may be produced intracellularly, in the periplasmic space, or secreted directly into the medium. In embodiments where the anti-α4β7 antibody is produced intracellularly, particulate debris of the host cells or lysed cells (e.g., resulting from homogenization) can be removed by various means including, but not limited to, centrifugation or filtration. When the anti-α4β7 antibody is secreted into the medium, the supernatant from such an expression system can be concentrated using a commercially available protein concentration filter. The methods of making antibodies are well known in the art. Mammalian hosts are engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab).

[0112] The overall cell culture process and considerations in the production of any monoclonal antibody, such as vedolizumab, are described in Li et al. (2010) mAbs 2:5,466-477, and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685, which are incorporated herein by reference. When using cell culture methods, the anti-α4β7 antibody may be produced intracellularly, in the periplasmic space, or secreted directly into the medium. In embodiments where the anti-α4β7 antibody is produced intracellularly, particulate debris of the host cells or lysed cells (e.g., resulting from homogenization) can be removed by various means including, but not limited to, centrifugation or filtration. When the anti-α4β7 antibody is secreted into the medium, the supernatant from such an expression system can be concentrated using a commercially available protein concentration filter. The methods of making antibodies are well known in the art. Mammalian hosts are engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab). The overall cell culture process and considerations in the production of any monoclonal antibody, such as vedolizumab, are described in Li et al. (2010) mAbs 2:5,466-477, and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685, which are incorporated herein by reference. The solution is first concentrated.

[0113] The medium or lysate may be subjected to one or more of the following: precipitation, flocculation, centrifugation, and / or filtration. Clarified cell culture supernatant that has been subjected to a processing step to remove particulate cell debris, or clarified A yield is then formed. The antibody, e.g., an anti-α4β7 antibody (e.g., vedolizumab or The antibody having a binding region corresponding to Dolizumab was purified as described in detail below. , impurities (e.g., cell culture medium components, host cell proteins (HCPs), host cell nucleic acids, viruses Process-related impurities such as eluents, and chromatographic materials, as well as aggregates (HMW generation of proteins that are not specifically targeted (including aggregates), misfolded species, or fragments of the protein of interest (substance-related impurities) are removed.

[0114] The purification process may be carried out using the upstream processes described above and / or by other conventional processes. Therefore, the antibody can be produced after the antibody has been purified. After the compound is obtained, process-related impurities such as other proteins produced by the cells, and separating the antibody of interest from product-related impurities. Such separations are performed using CEX, AEX, and / or MM chromatography. In certain embodiments, the affinity separation step(s), the ion exchange separation step (multiple possibilities), mixed mode step(s), and / or hydrophobic interaction separation step(s). A combination of one or more different purification methods may also be used, including The purification process separates the mixture of antibodies into groups based on their charge, hydrophobicity, and / or size. In one aspect of the invention, such further separation step is to separate hydrophobic, anionic Chromatography involving hydrophobic, hydrophilic, or cationic interactions (or combinations thereof) is used. Many chromatography resins are commercially available for each of these methods, and the purification scheme can be precisely adjusted according to the specific antibody involved. Each of these separation methods involves flowing the antibody through the column at different rates to achieve increasing physical separation as the antibody further passes through the column, or enabling selective attachment to the separation resin (or medium). Then, different eluents are used to differentially elute the antibody. In some cases, the antibody of interest is separated from impurities when the impurities specifically bind to the resin of the column and the antibody of interest does not (i.e., the antibody of interest is included in the flow-through), and in other cases, the antibody of interest attaches to the resin of the column and the impurities and / or product-related substances are eluted from the resin of the column during the washing cycle, and then the antibody is released by changing the liquid around the resin, and the antibody of interest is eluted from the column. In certain embodiments, in the "capture step", a solution containing the antibody is subjected to affinity chromatography to purify the antibody from impurities. In certain embodiments, the chromatography material is capable of selectively or specifically binding to the antibody of interest ("capture"). Non-limiting examples of such chromatography materials include Protein A, Protein G, for example, a chromatography material containing an antigen to which the antibody of interest binds, and a chromatography material containing an Fc-binding protein. In certain embodiments, the affinity chromatography steps described herein are performed as described herein.

[0115] In a particular embodiment, in the "capture step", a solution containing the antibody is subjected to affinity chromatography to purify the antibody from impurities. In certain embodiments, the chromatography material is capable of selectively or specifically binding to the antibody of interest ("capture"). Non-limiting examples of such chromatography materials include Protein A, Protein G, for example, a chromatography material containing an antigen to which the antibody of interest binds, and a chromatography material containing an Fc-binding protein.

[0116] In certain embodiments, the affinity chromatography steps described herein , subjecting the clarified harvest containing the anti-α4β7 antibody to a protein A matrix (e.g., a chromatography column containing protein A resin). In certain embodiments , protein A is useful for the affinity purification and isolation of various antibody isotypes, particularly IgG1, IgG2, and IgG 4. Protein A is a bacterial cell wall protein that binds to mammalian IgG mainly through its Fc region . Protein A has, in its native state, five IgG-binding domains and domains of other unknown functions .

[0117] Purification of anti-α4β7 antibody using protein A resin In one aspect, the methods described herein involve the use of protein A to purify an anti-α4β7 antibody (e.g., vedolizumab) from a liquid solution containing the antibody and one or more impurities, e.g., a clarified harvest. The method involves binding the anti-α4β7 antibody to an affinity chromatography matrix such as protein A. In certain embodiments , the antibody solution can be loaded onto the affinity chromatography matrix at greater than 10 g / L, e.g., 10 - 50 g / L, 20 - 45 g / L, or 30 - 40 g / L. For example, the antibody solution can be, the antibody solution can be about 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 2 0 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L , 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 4 0 g / L. 0 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L , 47 g / L, 48 g / L, 49 g / L, or 50 g / L can be loaded onto the matrix of Protein A affinity -chromatography.

[0118] Protein A resins are available from multiple vendors. One suitable resin is MabSelect™ sold by GE Healthcare. Suitable resins include, but are not limited to, Mab Select SuRe™, MabSelect SuRe LX, MabSel ect, MabSelect Xtra, rProtein A Sepharose, MabSelect™ ProA resin, ProSep HC, ProSep Ultra, and ProSep Ultra Pl us sold by EMD Millipore, and MabCapture sold by Life Technologies.

[0119] The Protein A column can be equilibrated with a suitable equilibration solution prior to loading the sample. After loading the column, the column can be washed one or more times with a set of suitable solutions to reduce one or more impurities, and the anti-α4β7 antibody remains bound to Protein A.

[0120] In some embodiments, the Protein A matrix is washed multiple times. In some embodiments, the Protein A matrix is washed three times. In one embodiment, one or more of the wash solutions contain phosphate. In one embodiment, the affinity column contains PBS ​​​​​​ After washing with the first washing solution, wash with a second washing solution containing NaCl and PBS, and then it can be washed with a third washing solution containing PBS. In one embodiment, the first and the third washing solutions are the same. In one embodiment, the second washing solution contains NaCl (e.g., 1 M NaCl) and PBS and has a pH of 7.2. In another embodiment, the pH of one or more of the washing solutions is about 7.0 to 7.4. In one embodiment, the pH of one or more of the washing solutions is about 7.2.

[0121] In other embodiments, the affinity column is washed with a first washing solution containing PBS and then washed with second and third solutions containing a buffer such as citrate, acetate, or phosphate. In one embodiment, the second and third solutions contain sodium citrate buffer. In one embodiment, the sodium citrate buffers in the second and third washing solutions are the same. In one embodiment, the sodium citrate buffers in the second and third washing solutions are different. In one embodiment, the sodium citrate buffer in the second washing solution has a higher molar concentration than the sodium citrate buffer in the third washing solution. In one embodiment, the sodium citrate buffer in the second washing solution has a molar concentration of 75 mM to 125 mM or 100 mM, and the sodium citrate buffer in the third washing solution has a molar concentration of 15 mM to 40 mM or 25 mM. In one embodiment, the pH of the final washing solution is 5.6 to 6.2. In one embodiment, the elution solution has approximately the same conductivity as the final washing solution.

[0122] Next, the Protein A column can be eluted using a suitable elution solution. For example, glycine-HCL, acetic acid, or citric acid can be used as the elution solution. In one embodiment, the elution solution is citric acid, for example, a sodium citrate elution solution. In some embodiments, the elution solution can have a pH of about 3.0 to 4.0 (e.g., about 3.1 to 4.0, 3.2 to 4.0, 3.3 to 4.0, 3.4 to 4.0, 3.5 to 4.0, 3.6 to 4.0, 3.7 to 4.0, 3.8 to 4.0, or 3.9 to 4.0). . In certain embodiments, the elution solution has a pH of about 3.0 to 3.4. In some embodiments, the elution solution can have a pH of about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5 , about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0. In some embodiments, the elution solution has a pH of 3.3 or higher. In some embodiments, the elution solution has a pH of 3.4 or higher. In some embodiments, the elution solution has a pH of 3.5 or higher. In some embodiments, the elution solution has a pH of 3.6 or higher. In some embodiments, the elution solution has a pH of 3.7 or higher. In some embodiments, the elution solution has a pH of 3.8 or higher. In some embodiments, the elution solution has a pH of 3.9 or higher. The elution solution can be monitored using techniques well known in the art. After collecting the desired eluate fraction, it can be prepared for further processes.

[0123] As shown in the examples, the elution buffer pH of the Protein A affinity column Compared with embodiments having a lower pH (e.g., 2.9 - 3.3), the elution buffer of the Protein A affinity column has a higher pH (e.g., 3.3 - 4.0). In such embodiments, the eluate containing the anti-α4β7 antibody has fewer impurities such as HMW aggregates. In some embodiments, the eluate contains the anti-α4β7 antibody and contains about 0% - 5.0% (e.g., 0 - 0.1%, 0 - 0.2%, 0 - 0.3%, 0 - 0.4%, 0 - 0.5%, 0 - 0.6% , 0 - 0.7%, 0 - 0.8%, 0 - 0.9%, 0 - 1%, 0 - 1.1%, 0 - 1.2% , 0 - 1.3%, 0 - 1.4%, 0 - 1.5%, 0 - 1.6%, 0 - 1.7%, 0 - 1. 8%, 0 - 1.9%, 0 - 2%, 0 - 2.5%, 0 - 3%, 0 - 3.5%, 0 - 4%, 0 - 4.5%, or 0 - 5%) of HMW aggregates. In some embodiments, the eluate contains the anti-α4β7 antibody and contains about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less , 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less , 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In certain embodiments, the eluate of the Protein A resin contains the anti-α4β7 antibody and contains about 0% - 2%, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less , 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less of aggregates, e.g., such as HMW aggregates. It contains HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody and contains about 1.2 % or less of HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody and contains about 1.1% or less of HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody, and contains about 1% or less (e.g., about 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In one embodiment, the eluate contains an anti-α4β7 antibody and contains about 0.9 % or less of HMW aggregates.

[0124] The buffers and methods described herein can reduce the level of host cell protein (HCP) in a composition such as a composition containing an anti-α4β 7 antibody eluted from Protein A, compared to the level of HCP when an elution buffer having none of the one or more parameters described herein is used. In some embodiments, the eluate of the Protein A resin contains an anti-α4β7 antibody and less than about 250 ppm (e.g., about 240 ppm, 23 0 ppm, 220 ppm, 210 ppm, 200 ppm, 190 ppm, 180 ppm, 170 ppm, 160 ppm, 150 ppm, 140 ppm, 130 ppm, 120 ppm, 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 4 0 ppm, 30 ppm, 20 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2 ppm, or less than 1 ppm) of HCP and contains a composition. In some embodiments, the eluate of the Protein A resin contains an anti-α4β7 antibody and about 1 - 250 ppm (e.g., about 1 - 240 ppm, 1 - 230 ppm, 1 - 220 ppm, 1 - 210 ppm, 1 - 200 ppm, 1 - 190 ppm, 1 - 180 ppm, 1 - 170 ppm, 1 - 160 ppm, 1 - 150 ppm, 1 - 140 ppm, 1 - 130 ppm, 1 - 120 ppm, 1 - 110 ppm, 1 - 100 ppm, 1 - 90 ppm, 1 - 80 ppm, 1 - 70 ppm, 1 - 60 ppm, 1 - 50 ppm, 1 - 40 ppm, 1 - 30 ppm, 1 - 20 ppm, 1 - 10 ppm, 1 - 9 ppm, 1 - 8 ppm, 1 - 7 ppm, 1 - 6 ppm, 1 - 5 ppm, 1 - 4 ppm, 1 - 3 ppm, 1 - 2 ppm, or 1 - 1 ppm) of HCP and contains a composition. ppm, 1 - 210 ppm, 1 - 200 ppm, 1 - 190 ppm, 1 - 180 ppm, 1 - 170 ppm, 1 - 160 ppm, 1 - 150 ppm, 1 - 140 ppm, 1 - 130 ppm, 1 - 120 ppm, 1 - 110 ppm, 1 - 100 ppm, 1 - 90 ppm, 1 - 80 ppm, 1 - 70 ppm, 1 - 60 ppm, 1 - 50 ppm, 1 - 40 ppm, 1 - 30 ppm, 1 - 20 ppm, 1 - 10 ppm, 1 - 9 ppm, 1 - 8 ppm, 1 - 7 ppm, 1 - 6 ppm, 1 - 5 ppm, 1 - 4 ppm, 1 - 3 ppm, 1 - 2 ppm, or 1 - 1 ppm) of HCP and contains a composition. In some embodiments, the eluate of the Protein A resin contains an anti-α4β7 antibody and about 1 - 250 ppm (e.g., about 1 - 240 ppm, 1 - 230 ppm, 1 - 220 ppm, 1 - 210 ppm, 1 - 200 ppm, 1 - 190 ppm, 1 - 180 ppm, 1 - 170 ppm, 1 - 160 ppm, 1 - 150 ppm, 1 - 140 ppm, 1 - 130 ppm, 1 - 120 ppm, 1 - 110 ppm, 1 - 100 ppm, 1 - 90 ppm, 1 - 80 ppm, 1 - 70 ppm, 1 - 60 ppm, 1 - 50 ppm, 1 - 40 ppm, 1 - 30 ppm, 1 - 20 ppm, 1 - 10 ppm, 1 - 9 ppm, 1 - 8 ppm, 1 - 7 ppm, 1 - 6 ppm, 1 - 5 ppm, 1 - 4 ppm, 1 - 3 ppm, 1 - 2 ppm, or 1 - 1 ppm) of HCP and contains a composition. ppm, 1 to 210 ppm, 1 to 200 ppm, 1 to 190 ppm, 1 to 180 ppm, 1 to 170 ppm, 1 to 160 ppm, 1 to 150 ppm, 1 to 140 ppm, 1 to 13 0 ppm, 1 to 120 ppm, 1 to 100 ppm, 1 to 90 ppm, 1 to 80 ppm, 1 to 70 ppm, 1 to 60 ppm, 1 to 50 ppm, 1 to 40 ppm, 1 to 30 ppm, 1 to 20 ppm, 1 to 10 ppm, 1 to 9 ppm, 1 to 8 ppm, 1 to 7 ppm, 1 to 6 p pm, 1 to 5 ppm, 1 to 4 ppm, 1 to 3 ppm, or 1 to 2 ppm) of HCP and contains a composition containing the same.

[0125] In one embodiment, the anti-α4β7 antibody bound to Protein A is eluted with a (for example, pH 3.3 to 4.0, pH 3.4 to 4.0, pH 3.5 to 4.0, pH 3.6 to 4.0, pH 3.7 to 4.0, pH 3.8 to 4.0, or pH 3.9 to 4.0) elution buffer to obtain an eluate containing the anti-α4β7 antibody and a reduced level of HMW aggregates and / or an eluate containing the anti-α4β7 antibody and / or a reduced level of HCP. In one embodiment, the elution solution has a pH of 3.3 to 3.9. In one embodiment the elution buffer has a pH of 3.3 to 3.8. In one embodiment, the pH of the elution buffer is 3.4 to 3.6. In one embodiment, the pH of the elution buffer is 3.4 to 4.0. In one embodiment, the elution buffer contains citric acid, such as sodium citrate e.g., 100 mM citric acid or 25 mM citric acid. In one embodiment, the Protein A affinity chromatography column is washed and eluted with a buffer containing 25 mM sodium citrate, and the washing buffer is 5.6 to 6.2, 5.7 to 5.9 or a pH of 5.8, and the elution buffer has a pH of 3.3 - 3.9, 3.4 - 3.6 or 3.5.

[0126] In certain embodiments, the material loaded onto the Protein A resin is, for example, a clarified cell culture harvest obtained from a recombinant cell line expressing an anti-α4β7 antibody. In some embodiments, the recombinant cell line (i.e., the host cell line) can be Chinese hamster ovary (CHO) cells. In some embodiments, the CHO cells can be GS-CHO cells deficient in the gene encoding glutamine synthetase. In some embodiments, the CHO cells can be DHFR-CHO cells deficient in the gene encoding dihydrofolate reductase. In some embodiments, the CHO cells can be DHFR-CHO cells deficient in the gene encoding dihydrofolate reductase.

[0127] The Protein A eluate can have its pH and / or conductivity adjusted for subsequent purification steps. The Protein A eluate can be filtered through a depth filter prior to a further chromatography polishing step to remove turbidity and / or various impurities from the antibody of interest.

[0128] Purification of anti-α4β7 antibody using HIC Antibodies, such as anti-α4β7 antibodies (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab), are described in detail below and, as described in Example 2, can also be purified using downstream process technology after purification with Protein A. The purification steps performed in the latter half of the downstream process are often referred to as "polishing" steps, and although the level of impurities can be relatively low, even lower levels are desired considering the properties of the antibody intended for human use. low, even lower levels are desired considering the properties of the antibody intended for human use. ​​​​​Presents unique challenges in terms of desirability.

[0129] In one aspect, the method described herein is a liquid solution containing an antibody and one or more impurities, such as a clarified harvest, using hydrophobic interaction chromatography (HI C) resin, including the purification of anti-α4β7 antibody from the product. Including.

[0130] In one embodiment, the present invention is a method for reducing high molecular weight (HMW) aggregates from an anti-α4β7 antibody solution, the method comprising contacting the antibody solution with hydrophobic interaction chromatography (HIC) resin And provides a method. Hydrophobic interaction chromatography (HIC) Separates proteins based on differences in their surface hydrophobicity by utilizing reversible interactions between proteins and the hydrophobic surface of HIC resin (e.g., a polymer matrix modified with a hydrophobic ligand). Considering the hydrophobicity of anti-α4β7 antibodies such as vedolizumab, using highly hydrophobic HIC resin in the purification process can remove HMW aggregates, residual protein A, and / or host cell protein (HCP) contaminants, and the anti-α4 β7 antibody flows through the HIC resin and does not bind. In some embodiments, highly hydrophobic HIC resins suitable for use in the methods described herein include polymethacrylate-based materials bonded to C6 groups, such as Toyopearl H exyl-650C (Tosoh Biosciences). β7 antibody passes through the HIC resin and does not bind. In some embodiments, HIC is used in "flow-through mode". Thus, as used herein, the "flow-through fraction" refers to the resin provided herein Including. Bonded to the C6 group Including polymethacrylate-based materials.

[0131] In some embodiments, HIC is used in "flow-through mode". Therefore, as used herein, the "flow-through fraction" refers to the resin provided herein Thus, as used herein, the "flow-through fraction" refers to the fraction containing the resin provided herein Refers to the protein in the mobile phase buffer that is recovered in the fraction passing through the column.

[0132] In some embodiments, a solution containing an anti-α4β7 antibody and at least one impurity is contacted with a hydrophobic interaction (HIC) resin under conditions that allow flow-through of the anti-α4β7 antibody through the HIC resin. In one embodiment, the HIC resin has an average pore size of about 100 nm and / or a pore size of about 100 μm. In one embodiment, the HIC resin is equilibrated with a buffer having a pH of less than about 7.2 In one embodiment, the HIC resin is equilibrated with a buffer having a pH of about 5.5 to about 7.2. In one embodiment, the HIC resin is equilibrated with a buffer having a pH of about 5.5 to about 7. In one embodiment, the buffer is a phosphate buffer. In one embodiment, the phosphate buffer contains about 0.35 M to about 0.15 M potassium phosphate. In one embodiment, the resin load is about 55 to 75 mg / ml .

[0133] In one embodiment, a method for purifying an anti-α4β7 antibody using an HIC column involves flowing an anti-α4β7 antibody-containing solution through the column. That is, the purification involves recovering the anti-α4β7 antibody in the flow-through of the column, with the contaminants remaining bound to the column, and the anti-α 4β7 antibody and the column being in a solution containing phosphate, e.g., potassium phosphate, at a concentration of 15 0 to 300 mM, 175 to 250 mM, or about 200 mM, at a pH of 5.2 to 6.5, 5.7 to 6.2, or about 5.9 .

[0134] In some embodiments, such a method using a highly hydrophobic HIC resin is used to obtain an anti-α4 A composition comprising an anti-β7 antibody and HMW aggregates of about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0. 5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3% , less than 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or less than 2% ) can be obtained. In some embodiments, such a method using a highly hydrophobic HIC resin is used to obtain a composition comprising an anti-α4β7 antibody and HMW aggregates of about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less , 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less , 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less , or 0.1% or less). In certain embodiments, such a method using a highly hydrophobic HIC resin is used to obtain a composition comprising an anti-α4β7 antibody and about 0% to 2%, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less , 0.3% or less, 0.2% or less, or 0.1% or less of HMW aggregates. In certain embodiments, such a method using a highly hydrophobic HIC resin is used to obtain a composition comprising an anti-α4β7 antibody and less than 0.6% of HMW aggregates . In one embodiment, a composition comprising an anti-α4β7 antibody and less than 0.5% of HMW aggregates is obtained . In one embodiment, a composition comprising an anti-α4β7 antibody and less than 0.4% of HMW aggregates is obtained . Further, the composition comprises less than about 0.3 ppm of host cell protein (HCP) ​​ is capable, and the host cell is a Chinese hamster ovary ( CHO) cell such as a GS-CHO cell. In one embodiment, the composition contains less than about 0.22 ppm of residual protein A.

[0135] Purification of anti-α4β7 antibody using mixed-mode resin In one aspect, provided herein is a method for purifying an anti-α4β7 antibody, such as vedolizumab, from a liquid solution containing the antibody and one or more impurities, e.g., clarified cell culture harvest, using a mixed-mode chromatography resin in a binding / elution mode In some embodiments, the mixed-mode chromatography resin has properties suitable for high impurity removal rate and high capacity. In one embodiment, the mixed-mode chromatography resin for purifying the anti-α4β7 antibody has strong anion exchange, hydrogen bonding, and hydrophobic binding properties . In another embodiment, the mixed-mode chromatography resin for purifying the anti-α4β7 antibody has strong anion exchange on smaller beads, e.g., beads with a diameter of about 35 - 45 μm, hydrogen bonding, and hydrophobic binding capabilities. In certain embodiments, the mixed-mode chromatography resin used in the methods and compositions described herein is CAPTO™ Adhere ImpRes (GE Healthcare Life Science . In certain embodiments, the mixed-mode chromatography resin used in the methods and compositions described herein is CAPTO™ Adhere (GE Health care Life Sciences, now Global Life Sciences Solutions, LLC) . In certain embodiments, the mixed-mode chromatography resin used in the methods and compositions described herein is CAPTO™ Adhere (GE Health care Life Sciences, now Global Life Scienc s, now Global Life Sciences Solutions, LLC) s, now Global Life Sciences Solutions, LLC) is. In certain embodiments, the mixed-mode chromatography resin used in the methods and compositions described herein is CAPTO™ Adhere (GE Health care Life Sciences, now Global Life Sciences Solutions, LLC) care Life Sciences, now Global Life Sciences Solutions, LLC) es Solutions, LLC). The clarified cell culture harvest can be obtained from host cells recombinantly expressing the anti-α4β 7 antibody. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells such as GS-CHO cells or DHFR-CHO cells.

[0136] The mixed-mode chromatography methods provided herein include binding the anti-α4β7 antibody to a mixed-mode chromatography resin. Further purification steps, including, but not limited to, affinity chromatography (e.g., protein A chromatography), anion exchange (AEX) chromatography, cation exchange (CEX) chromatography, and hydrophobic interaction chromatography (HIC), can be used before and / or after the mixed-mode chromatography methods described herein. Thus, in some embodiments, the load material used in the mixed-mode chromatography can include a protein A eluate, an AEX eluate, a CEX eluate, or a HIC eluate or a recovered HIC flow-through material. The mixed-mode chromatography methods provided herein can further include, in some embodiments, washing the mixed-mode resin with a washing solution and eluting the antibody from the resin.

[0137] In certain embodiments, at least 25 g / L (e.g., at least 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 6 5 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or an antibody solution of 100 g / L) is loaded onto the mixed-mode chromatography resin. This can be done. For example, an antibody solution of at least 55 g / L can be loaded onto the mixed-mode chromatography resin. In certain embodiments, from about 25 g / L to about 100 g / L, for example, from about 25 g / L to about 95 g / L, from about 25 g / L to about 90 g / L, from about 25 g / L to about 85 g / L, from about 25 g / L to about 80 g / L (e.g., from about 30 g / L to about 80 g / L, from about 3 5 g / L to about 80 g / L, from about 40 g / L to about 80 g / L, from about 45 g / L to about 80 g / L, from about 50 g / L to about 80 g / L, from about 55 g / L to about 80 g / L, from about 60 g / L to about 80 g / L, from about 65 g / L to about 80 g / L, from about 70 g / L to about 80 g / L, or from about 75 g / L to about 80 g / L) of the antibody solution can be loaded onto the mixed-mode chromatography resin. For example, an antibody solution of from about 55 g / L to about 80 g / L can be loaded onto the mixed-mode chromatography resin.

[0138] The resin can be washed, if necessary, with a suitable wash buffer that does not elute the antibody bound to the resin. In one embodiment, the resin can be washed, if necessary, with a sodium phosphate wash buffer. In some embodiments, the wash buffer can contain 10 mM sodium phosphate, 25 mM sodium phosphate, 50 mM sodium phosphate, or 75 mM sodium phosphate at a pH that is neutral or near neutral (e.g., pH 6 - 8). Other suitable wash buffers compatible with mixed-mode chromatography are also widely available.

[0139] As used herein, the production of the anti-α4β7 antibody after elution from the mixed-mode chromatography resin Increase the yield of the anti-α4β7 antibody in the agent and / or reduce the level of aggregates (e.g., HMW species ( %)). A buffer is described that increases the yield of the anti-α4β7 antibody and / or reduces the level of aggregates (e.g., HMW species (%)). To increase the yield of the anti-α4β7 antibody and / or reduce the level of aggregates (e.g., HMW species (%)), the pH and / or conductivity of the mixed-mode elution buffer can be adjusted. Suitable elution solutions compatible with mixed-mode chromatography are widely available. In some embodiments, the mixed-mode chromatography elution solution contains a buffer such as citrate, acetate, or phosphate .

[0140] In some embodiments, the elution buffer for use with the mixed-mode chromatography resin by the methods described herein has a pH of pH 3.5 or higher (e.g., pH 3.6 or higher, pH 3.7 or higher, pH 3.8 or higher , pH 3.9 or higher, pH 4.0 or higher , pH 4.1 or higher, pH 4.2 or higher, pH 4.3 or higher , pH 4.4 or higher, or pH 4.5 or higher). For example, the elution buffer for use with the mixed-mode chromatography resin can have a pH of pH 3.9 or higher. In certain embodiments, the elution buffer for use with the mixed-mode chromatography resin by the methods described herein has a pH of about pH 3.9 to about pH 4.5 (e.g., about pH 3.9 to about pH 4.5, about pH 3.9 to about pH 4.4, about pH 3.9 to about pH 4.3, about pH 3 .9 to about pH 4.2, about pH 3.9 to about pH 4.1, or about pH 3.9 to about pH 4.0 . ​​has a pH). In some embodiments, the elution buffer for use in the mixed-mode chromatography method can have a pH of from about pH 3.9 to about pH 4.4.

[0141] In embodiments in addition to or instead of the above, the elution buffer for use with the mixed-mode chromatography resin described herein can have a pH of pH 4.5 or lower (e.g., pH 4.4 or lower, pH 4.3 or lower, pH 4.2 or lower, pH 4.1 or lower, pH 4.0 or lower, pH 3.9 or lower, pH 3.8 or lower, pH 3.7 or lower, pH 3.6 or lower, or pH 3.5 or lower). For example, the elution buffer for use with the mixed-mode chromatography resin can have a pH of pH 4.2 or lower. In certain embodiments, the elution buffer for use with the mixed-mode chromatography resin in the methods described herein can have a pH of from about pH 4.2 to about pH 3.5 (e.g., from about pH 4.2 to about pH 3.6, from about pH 4.2 to about pH 3.7, from about pH 4.2 to about pH 3.8, from about pH 4.2 to about pH 3.9, from about pH 4.2 to about pH 4.0, or from about pH 4 .2 to about pH 4.1). For example, the elution buffer for use with the mixed-mode chromatography resin can have a pH of from about pH 4.2 to about pH 3.8.

[0142] In some embodiments, in the methods described herein, the mixed-mode chromatography ​​​​The elution buffer for use with the phi resin has a conductivity of about 40 mS / cm or less (e.g., about 39 mS / cm, 38 mS / cm, 37 mS / cm, 36 mS / cm, 35 mS / cm, 34 mS / cm, 33 mS / cm, 32 mS / cm, 31 mS / cm, 30 mS / cm, 29 mS / cm, 28 mS / cm, 27 mS / cm, 26 mS / cm, 25 mS / cm, 24 mS / cm, 23 mS / cm, 22 mS / cm, 21 mS / cm, 20 mS / cm, 19 mS / cm, 18 mS / cm, 17 mS / cm, 16 mS / cm, 15 mS / cm, 14 mS / cm, 13 mS / cm, 12 mS / cm, 11 mS / cm, or 10 mS / cm or less). For example, the elution buffer for use with a mixed-mode chromatography resin can have a conductivity of about 30 mS / cm or less. In certain embodiments the elution buffer for use with a mixed-mode chromatography resin in the methods described herein can have a conductivity of from about 10 mS / cm to about 40 mS / cm, such as from about 15 mS / cm to about 35 mS / cm or from about 20 mS / cm to about 30 mS / cm. For example, the elution buffer for use with a mixed-mode chromatography resin can have a conductivity of from about 20 mS / cm to about 30 mS / cm.

[0143] In embodiments in addition to or in place of the above, the elution buffer for use with a mixed-mode chromatography resin in the methods described herein can have a conductivity of about 30 mS / cm or less (e.g., 29 mS / cm, 28 mS / cm, 27 mS / cm, 26 mS / cm 25 mS / cm, 24 mS / cm, 23 mS / cm, 22 mS / cm, 21 mS / cm , 20 mS / cm, 19 mS / cm, 18 mS / cm, 17 mS / cm, 16 mS / cm , 15 mS / cm, 14 mS / cm, 13 mS / cm, 12 mS / cm, 11 mS / cm , or has a conductivity of 10 mS / cm or less). For example, mixed-mode chromatography - The elution buffer for use with the resin can have a conductivity of 28 mS / cm or less . In certain embodiments, in the methods described herein, the elution buffer for use with mixed-mode chromatography resin is from about 10 mS / cm to about 40 mS / cm (e.g., from about 15 mS / cm to about 35 mS / cm, from about 18 mS / cm to about 35 mS / cm , from about 11 mS / cm to about 30 mS / cm, from about 12 mS / cm to about 30 mS / cm, from about 13 mS / cm to about 30 mS / cm, from about 14 mS / cm to about 30 mS / cm, from about 15 mS / c m to about 30 mS / cm, from about 16 mS / cm to about 30 mS / cm, from about 17 mS / cm to about 3 0 mS / cm, from about 18 mS / cm to about 30 mS / cm, from about 19 mS / cm to about 30 mS / cm, from about 20 mS / cm to about 30 mS / cm, from about 21 mS / cm to about 30 mS / cm, from about 22 mS / cm to about 30 mS / cm, from about 23 mS / cm to about 30 mS / cm, from about 24 mS / cm to about 30 mS / cm, from about 25 mS / cm to about 30 mS / cm, from about 26 mS / cm to about 30 mS / cm, or from about 27 mS / cm to about 30 mS / cm). For example , in some embodiments, the elution buffer for use with mixed-mode chromatography resin can have a conductivity of from about 18 mS / cm to about 28 mS / cm

[0144] In some embodiments, in the methods described herein, mixed-mode chromatography The elution buffer for use with the FF resin can contain an ionic salt, such as NaCl, at a concentration of about 10 0 - 300 mM (e.g., about 110 - 290 mM, 120 - 280 mM, 130 - 270 mM, 140 - 260 mM, 150 - 250 mM, 160 - 240 mM, 170 - 230 mM, 180 - 220 mM, or 190 - 210 mM). For example, the elution buffer for use with a mixed - mode chromatography resin can have NaCl at a concentration of about 160 mM to about 240 mM. In certain embodiments, the elution buffer for use with a mixed - mode chromatography resin in the methods described in this specification can have NaCl at a concentration of about 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 15 0 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 m M, 290 mM, or 300 mM. In some embodiments, a method for purifying an anti - α4β7 antibody, such as vedolizumab, from a liquid solution, e.g., a clarified cell culture harvest, using a mixed - mode chromatography resin, includes loading the anti - α4β7 antibody onto a column containing the mixed - mode resin at a concentration of 40 - 90, 50 - 80, or about 65 g of protein per liter of resin, washing the column, and eluting the column with an elution buffer having a pH of 3.5 - 4.5, 3.9 - 4.4, or about 4.1, e.g., sodium citrate buffer. In some embodiments, the method includes that the conductivity of the elution buffer is 15 - 35, 20 - 30, or ~ 25 mS / cm.

[0145] In some embodiments, a method for purifying an anti - α4β7 antibody, such as vedolizumab, from a liquid solution, e.g., a clarified cell culture harvest, using a mixed - mode chromatography resin, includes loading the anti - α4β7 antibody onto a column containing the mixed - mode resin at a concentration of 40 - 90, 50 - 80, or about 65 g of protein per liter of resin, washing the column, and eluting the column with an elution buffer having a pH of 3.5 - 4.5, 3.9 - 4.4, or about 4.1, e.g., sodium citrate buffer. In some embodiments, the method includes that the conductivity of the elution buffer is 15 - 35, 20 - 30, or ~ 25 mS / cm. In some embodiments, a method for purifying an anti - α4β7 antibody, such as vedolizumab, from a liquid solution, e.g., a clarified cell culture harvest, using a mixed - mode chromatography resin, includes loading the anti - α4β7 antibody onto a column containing the mixed - mode resin at a concentration of 40 - 90, 50 - 80, or about 65 g of protein per liter of resin, washing the column, and eluting the column with an elution buffer having a pH of 3.5 - 4.5, 3.9 - 4.4, or about 4.1, e.g., sodium citrate buffer. In some embodiments, the method includes that the conductivity of the elution buffer is 15 - 35, 20 - 30, or ~ 25 mS / cm. In some embodiments, the method includes that the conductivity of the elution buffer is 15 - 35, 20 - 30, or Further comprising including an ionic salt, e.g., NaCl, to provide a pH of about 24 mS / cm. In some embodiments, the method comprises eluting the antibody on a column containing a mixed mode resin, resin 1 In some embodiments, the loading concentration is between 53 and 77 g of protein per liter. The method includes the step of: The method includes eluting the antibody from the column with an elution buffer.

[0146] In some embodiments, purification of anti-α4β7 antibodies is performed using a mixed-mode purification method as described herein. Chromatographic techniques were used in combination with cation exchange (CEX) chromatography. This can be done by:

[0147] In some embodiments, the methods described herein include mixed-mode chromatography. The yield of anti-α4β7 antibodies eluted from the column was determined by one or more of the parameters described herein. The yield is compared to that of a suitable control process using an elution buffer without a meter, e.g. For example, dissolution at pH ≤ 3.7, ≤ 3.6, ≤ 3.5, ≤ 3.3, or ≤ 3.0 This can be improved compared to processes that use buffers. In terms of form, the yield is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% , 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% In some embodiments, the buffers described herein are and the method can provide a recovery rate of 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the anti-α4β7 antibody eluted from the mixed-mode chromatography column. In some embodiments, the buffers and methods described herein can provide a recovery rate of 50% to 95% (e.g., 55 - 95%, 60 - 95%, 65 - 95%, 70 - 95%, 75 - 95%, 80 - 95%, 85 - 95%, 90 - 95%, or more) of the anti-α4β7 antibody eluted from the mixed-mode chromatography column. In some embodiments, such compositions and methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In certain embodiments, a mixed-mode chromatography resin In some embodiments, the buffers and methods described herein can provide a recovery rate of 50% to 95% (e.g., 55 - 95%, 60 - 95%, 65 - 95%, 70 - 95%, 75 - 95%, 80 - 95%, 85 - 95%, 90 - 95%, or more) of the anti-α4β7 antibody eluted from the mixed-mode chromatography column. In some embodiments, the buffers and methods described herein can provide a recovery rate of 50% to 95% (e.g., 55 - 95%, 60 - 95%, 65 - 95%, 70 - 95%, 75 - 95%, 80 - 95%, 85 - 95%, 90 - 95%, or more) of the anti-α4β7 antibody eluted from the mixed-mode chromatography column. In some embodiments, the buffers and methods described herein can provide a recovery rate of 50% to 95% (e.g., 55 - 95%, 60 - 95%, 65 - 95%, 70 - 95%, 75 - 95%, 80 - 95%, 85 - 95%, 90 - 95%, or more) of the anti-α4β7 antibody eluted from the mixed-mode chromatography column. In some embodiments, the buffers and methods described herein can provide a recovery rate of 50% to 95% (e.g., 55 - 95%, 60 - 95%, 65 - 95%, 70 - 95%, 75 - 95%, 80 - 95%, 85 - 95%, 90 - 95%, or more) of the anti-α4β7 antibody eluted from the mixed-mode chromatography column.

[0148] In some embodiments, such compositions and methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. In some embodiments, such methods using a mixed-mode chromatography resin can be used to obtain a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, less than 2%) of HMW aggregates. Using such a method using lipids, an anti-α4β7 antibody and about 0% to 2%, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1. 3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0. 7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or a composition containing aggregates of 0.1% or less can be obtained. In other embodiments, H The level of MW aggregates is at least 1% relative to the level of HMW aggregates in the loaded material , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% , 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, 99%, or more reduced. In some embodiments, using the mixed-mode chromatography method described herein The level of HMW aggregates in a composition containing an anti-α4β7 antibody is described herein Reduced compared to the level of HMW aggregates obtained from a suitable control process using an elution buffer having no one or more of the parameters For example, compared to a process performed using an elution buffer having a pH of 3.7 or less, 3.6 or less, 3.5 or less, 3.3 or less, or 3.0 or less. In some embodiments, the level of HMW aggregates is appropriate At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% compared to a suitable control , 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% ​ 、 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 、 or reduced further.

[0149] Purification of anti-α4β7 antibody using cation exchange (CEX) resin In one aspect, herein, a cation exchange (CEX) resin is used in a bind / elute mode to purify an anti-α4β7 antibody from a liquid solution containing the antibody and one or more impurities, such as a clarified cell culture harvest. A method for purifying an anti-α4β7 antibody, such as vedolizumab, is provided. In some embodiments, the CEX resin for purifying the anti-α4β7 antibody is a strong cation exchange resin. In certain embodiments, the CEX resin compatible for use in the methods and compositions described herein contains a -SO3 functional group. For example, in some embodiments, the CEX resin is Nuvia HR-S. The clarified cell culture harvest can be obtained from host cells that recombinantly express the anti-α4β7 antibody. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. - In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells.

[0150] The CEX method provided herein involves binding the anti-α4β7 antibody to a cation exchange chromatography resin. Further purification steps, including, but not limited to, affinity chromatography (e.g., protein A chromatography), anion exchange (AEX) chromatography, mixed mode chromatography, and hydrophobic interaction chromatography (HIC), can be used before and / or after the CEX method described herein. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. In certain embodiments, the host cells can be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells. Therefore, in some embodiments, the CEX chromatography The loading materials used in chromatography can include protein A eluate, AEX eluate, mixed mode eluate, or HIC eluate. The CEX method provided herein can, in some embodiments, further include washing the CEX resin with a washing solution and eluting the antibody from the resin. Solutions suitable for loading, washing, and eluting proteins compatible with CEX chromatography, such as, for example, an anti-α4β7 antibody, are widely available. In some embodiments, the CEX chromatography solution includes a buffer such as a citrate salt, acetate, or phosphate.

[0151] In certain embodiments, an antibody solution of at least 20 g / L (e.g., at least 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, or 100 g / L) can be loaded onto the CEX resin. For example, an antibody solution of at least 25 g / L can be loaded onto the CEX resin. In certain embodiments, an antibody solution of about 25-100 g / L (e.g., about 25-90 g / L, 25-80 g / L, 25-70 g / L, 25-60 g / L, 25-50 g / L, 25-40 g / L, or 25-30 g / L) can be loaded onto the CEX resin. In certain embodiments, an antibody solution of about 25-70 g / L (e.g., about 25-65 g / L, 30-60 g / L, 35-55 g / L, or 40-50 g / L) can be loaded onto the CEX resin. For example, an antibody solution of about 30-60 g / L can be loaded onto the CEX resin.

[0152] ​​​​​​​​​​​​​​​​The resin can be washed, if necessary, with a suitable wash buffer that does not elute the antibody bound from the resin. In some embodiments, the wash buffer has the same composition as the buffer used to load the antibody onto the resin. In one embodiment, the resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. The resin can be washed, if necessary, with a suitable wash buffer that does not elute the antibody bound from the resin. In some embodiments, the wash buffer has the same composition as the buffer used to load the antibody onto the resin. In one embodiment, the resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. The resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. The resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. The resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. The resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. The resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available. The resin can be washed, if necessary, with sodium citrate buffer, such as 25 mM sodium citrate, 50 mM sodium citrate, 75 mM sodium citrate, or 100 mM sodium citrate. In some embodiments, the wash buffer has a pH in the range of pH 5 - 7, for example, pH 5 - 6, pH 5.5 - 6.5, pH 5.1 - 5.8, pH 5.3 - 5.6, pH 6 - 7 or pH 5.4. Other suitable wash buffers compatible with CEX chromatography are also widely available.

[0153] By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. By adjusting the pH and / or conductivity of the elution buffer, the levels of HMW aggregates, major (main) isoform species, acidic isoform species, and / or basic isoform species in the formulation of the anti-α4β7 antibody eluted from the CEX resin can be adjusted. In some embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of 6.0 or less (for example, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, or 6.0 or less). In certain embodiments, the elution buffer for use with CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6. It has a pH of 0.0 (e.g., from about pH 4.5 to about pH 5.8, from about pH 4.9 to about pH 5.9, from about p H 5.0 to about pH 6.0, from about pH 5.0 to about pH 5.9, from about pH 5.0 to about pH 5.8, from about pH 5.0 to about pH 5.7, from about pH 5.0 to about pH 5.6, or from about pH 5.0 to about p H 5.5). For example, an elution buffer for use with a CEX resin can have a pH of from about pH 5.1 to about pH 5.8. In some embodiments, the pH of the CEX elution buffer is the same as that of the wash buffer.

[0154] In addition to or in place of the above, in embodiments, the elution buffer for use with a CEX resin in the methods described herein has a conductivity of about 20 mS / cm or less (e.g., 19 mS / cm, 18 mS / cm, 17 mS / cm, 16 mS / cm, 15 mS / cm, 14 mS / cm, 13 mS / cm, 12 mS / cm, 11 mS / cm, or 10 mS / cm or less). For example, an elution buffer for use with a CEX resin can have a conductivity of 16 mS / cm or less. In some embodiments, the elution buffer for use with a CEX resin in the methods described herein has a conductivity of from about 10 mS / cm to about 20 mS / cm (e.g., from about 10 mS / cm to about 19 mS / cm, from about 10 mS / cm to about 18 mS / cm, from about 10 mS / cm to about 17 mS / cm, from about 10 mS / cm to about 16 mS / cm, from about 10 mS / cm to about 15 mS / cm, from about 10 mS / cm to about 14 mS / c m, from about 10 mS / cm to about 13 mS / cm, or from about 10 mS / cm to about 12 mS / cm ). In some embodiments, the elution buffer for use with a CEX resin has a conductivity of from about 10 mS / cm to about 20 mS / cm (e.g., from about 10 mS / cm to about 19 mS / cm, from about 10 mS / cm to about 18 mS / cm, from about 10 mS / cm to about 17 mS / cm, from about 10 mS / cm to about 16 The ar can have a conductivity of about 11 mS / cm to about 16 mS / cm. In addition to the above or instead of the above, the elution buffer for use with the CEX resin can have a conductivity of 14 mS / cm or less. In certain embodiments, the elution buffer for use with the CEX resin in the methods described herein can have a conductivity of about 11 mS / cm to about 14 m S / cm, such as about 12 mS / cm to about 14 mS / cm or about 13 mS / cm to about 14 mS / cm. For example, the elution buffer for use with the CEX resin can have a conductivity of about 12 mS / cm to about 14 mS / cm. In addition to the above, or instead of the above, the elution buffer for use with the CEX resin can have a conductivity of 11 mS / cm or more (e.g., 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 m S / cm, 17 mS / cm, 18 mS / cm, 19 mS / cm, or 20 mS / cm or more ). For example, the elution buffer for use with the CEX resin can, in some embodiments, have a conductivity of 12 mS / cm or more. In some embodiments, the elution buffer for use with the CEX resin in the methods described herein can

[0155] have NaCl at a concentration of about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM. For example, the elution buffer for use with the CEX resin can have NaCl at a concentration of about 90 mM to about 120 mM. In certain embodiments , the elution buffer for use with the CEX resin in the methods described herein can be about ​​It has NaCl at a concentration of 50 to 150 mM (for example, about 50 to 140 mM, 60 to 130 mM, 70 to 120 mM, 80 to 110 mM, or 90 to 100 mM). In certain embodiments, the elution buffer for use with a CEX resin in the methods described herein has NaCl at a concentration of about 70 to 120 mM (for example, about 70 to 110 mM, 70 to 100 mM, 70 to 90 mM, or 70 to 80 mM). For example, the elution buffer for use with a CEX resin can have NaCl at a concentration of about 70 mM to about 1100 mM. In some embodiments, a method for purifying an anti-α4β7 antibody, such as vedolizumab, from a liquid solution, such as a clarified cell culture harvest, using a CEX resin involves loading the anti-α4β7 antibody onto a column containing the CEX resin at a concentration of 40 to 90, 50 to 65, or about 57 g of protein per liter of resin, washing the column, and eluting the column with a buffer having a pH of 5 to 6, 5.2 to 5.6, or about 5.4, such as a sodium acetate buffer. In some embodiments, the method further includes including an ionic salt, such as NaCl, so that the conductivity of the elution buffer is 5 to 25, 10 to 17, or about 13 mS / cm. In other embodiments, a method for purifying an anti-α4β7 antibody, such as vedolizumab, from a liquid solution, such as a clarified cell culture harvest, using a CEX resin, such as a strong cation exchange resin, involves eluting the column with a buffer having a pH of 5 to 6, 5.2 to 5.6, or about 5.4 and a conductivity of 5 to 25, 10 to 15, or about 13 mS / cm, such as a sodium acetate buffer. In one embodiment,

[0156] ​​​​​​​​​​​​​​​​​The method involves eluting the capture with an elution buffer having a pH of about 5.4 and a conductivity of about 13 mS / cm. In some embodiments, the CEX resin is loaded with about 57 g of the antibody of the protein per liter of resin.

[0157] In some embodiments, the purification of the anti-α4β7 antibody can be performed by using the CEX resin described herein in combination with mixed-mode chromatography.

[0158] In some embodiments, using the CEX method described herein, a composition comprising the anti-α4β7 antibody and about 0% to 2.0% (e.g., about 0.01%, 0.02%, 0.03%, 0.04%, 0 .05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0 .3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9 %, or 2%) of HMW aggregates can be obtained. In some embodiments, using such a method with the CEX resin, a composition comprising the anti-α4β7 antibody and about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1. 4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0. 8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0. 06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less) of HMW aggregates can be obtained. In certain embodiments Then, using such a method using a CEX resin, the anti-α4β7 antibody is about 0% to 2%, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1. 4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0. 8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0. 06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, and also aggregates of 0.01% or less, such as HMW aggregates, can be obtained. In other embodiments, the level of HMW aggregates is relative to the level of HMW aggregates in the loaded material by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25 %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more reduced. In some embodiments, by using the CEX method provided herein , the level of HMW aggregates in a composition containing an anti-α4β7 antibody is compared to the level of HMW aggregates in a formulation of an anti-α4β7 antibody obtained using a suitable control CEX process using an elution buffer having none of the one or more parameters described herein. For example, a pH of 6.3 or higher, 6.5 or higher, 6.7 or higher, or 6.9 or higher, and / or a conductivity of 18 mS / cm or higher, 19 mS / cm or higher, 20 mS / cm or higher, 22 mS / cm or higher, or 24 mS / cm or higher, is reduced compared to a process performed using an elution buffer and / or a conductivity of 18 mS / cm or higher, 19 mS / cm or higher, 20 mS / cm or higher, 22 mS / cm or higher, or It can be done. In some embodiments, the level of HMW aggregates is compared with an appropriate control and is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0159] In some embodiments of the methods provided herein, the pH and / or conductivity of the elution buffer used to elute the anti-α4β7 antibody from the CEX resin is used to regulate the isoform distribution of the anti-α4β7 antibody present in the eluate. For example, using the pH and / or conductivity of the elution buffer, the percentage of the major (main) antibody isoform can be increased, the percentage of acidic isoform species can be decreased, and / or the percentage of basic isoform species can be decreased.

[0160] In some embodiments, an elution buffer having a pH of 6.0 or less, such as 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, or 5.2 or less, for example, pH 4.5-6.0, pH 4.5-5.5, or pH 5.0-6.0 can be selected. In some embodiments, an elution buffer having a conductivity of at least 10 mS / cm, such as at least 11 mS / cm, at least 12 mS / cm, at least 13 mS / cm, at least 14 mS / cm, at least 15 mS / cm, at least 16 mS / cm, cm or more, for example, 10 to 17 mS / cm, 12 to 17 mS / cm, 13 to 17 mS / c m, 14 to 17 mS / cm, 15 to 17 mS / cm, 16 to 17 mS / cm, 10 to 16 mS / cm, 12 to 16 mS / cm, 13 to 16 mS / cm, 14 to 16 mS / cm, or a elution buffer having a conductivity of 15 to 16 mS / cm can be selected. In some embodiments, using the above elution buffer conditions, at least 60%, 61% , 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71% , 72%, 73%, 74%, 75%, or more of the major iso form of the anti-α4β7 antibody can be obtained. In some embodiments, using the above elution buff er conditions, a composition containing 20% or less (for example, about 19% or less, 18% or less, 17% or less, 16 % or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less) of the basic isoform species can be obtained. In certain embodiments, using such a method using a CEX resin, the major iso form of the anti-α4β7 antibody and about 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less , 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 salt basic isoform species can be obtained. In other embodiments, the level of the basic iso form species is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 1 with respect to the level of the basic isoform species in the loading material, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 1 2%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 3 0%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 8 0%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or reduced by more than that will be.

[0161] III. Analytical Methods In certain embodiments, the chromatography generated using the methods described herein is used to analyze the levels of aggregates, monomers, and fragments in a sample. In certain embodiments, aggregates , monomers, and fragments are measured for each molecule using size exclusion chromatography (SEC) methods . For example, but not intending to be limiting, a TSK-Gel G3000 SWxL, 5 μm, 125 angstroms, 7.8 × 300 mm column (Tosoh Bioscience) can be used in combination with certain embodiments, and in another embodiment , a TSK-Gel Super SW3000, 4 μm, 250 angstroms, 4.6 × 300 mm column (Tosoh Bioscience) can be used . In certain embodiments, the above columns are used with an Agilent or Shimazhu HP LC system. In certain embodiments, sample injection is performed, for example, using a mobile phase consisting of 100 mM sodium sulfate and 100 mM sodium phosphate at pH 6. 8 under isocratic elution conditions and detected at a UV absorbance of 214 nm. In certain embodiments, the mobile phase consists of 1X PBS at pH 7.4 and the elution profile is detected at a UV absorbance of 28 0 nm. In certain embodiments, quantification is based on the relative area of the detected peaks. ​

[0162] Any additional techniques, such as mass spectrometry, can be used to assay size variants. This can be done.

[0163] The various parameters of the antibodies or antigen-binding portions thereof reported herein can be measured using standard analytical methods and techniques such as those described below. This can be done.

[0164] In different embodiments described herein, cation exchange chromatography (CEX ) can be used to determine the relative amounts of the major isoforms, basic isoform(s), and acidic isoform(s) present in a population of an antibody or antigen-binding portion thereof, such as bevacizumab. The CEX method fractionates antibody species based on their overall surface charge. After dilution to low ionic strength with the mobile phase, the test sample is equilibrated with a suitable buffer, e.g., 10 mM sodium phosphate (pH 6.6), and then injected onto a CEX column such as a Dionex P ro-Pac (trademark) WCX-10 column (Thermo Fisher Scient ific, Waltham, MA (USA)). The antibody can be eluted using a sodium chloride gradient in the same buffer. Elution of the protein is monitored at 280 nm, and each peak can be assigned to the category of acidic, basic, or major isoform. Acidic peaks elute from the column with a shorter retention time than the peak of the major isoform, and basic peaks elute from the column with a longer retention time than the peak of the major isoform. Report the percentage of the major isoform, the sum of the percentages of acidic species, and the sum of the percentages of basic species. The major a of the sample ro-Pac (trademark) WCX-10 column (Thermo Fisher Scient ific, Waltham, MA (USA)) can be done. The antibody can be eluted using a sodium chloride gradient in the same buffer. Elution of the protein is monitored at 280 nm, and each peak can be assigned to the category of acidic, basic, or major isoform. Acidic peaks elute from the column with a shorter retention time than the peak of the major isoform, and basic peaks elute from the column with a longer retention time than the peak of the major isoform. Report the percentage of the major isoform, the sum of the percentages of acidic species, and the sum of the percentages of basic species. Acidic peaks elute from the column with a shorter retention time than the peak of the major isoform, and basic peaks elute from the column with a longer retention time than the peak of the major isoform. Acidic peaks elute from the column with a shorter retention time than the peak of the major isoform, and basic peaks elute from the column with a longer retention time than the peak of the major isoform. Report the percentage of the major isoform, the sum of the percentages of acidic species, and the sum of the percentages of basic species. Report the percentage of the major isoform, the sum of the percentages of acidic species, and the sum of the percentages of basic species. The major a Determine the conformity by comparing the retention time of the isoform with the retention time of the reference standard. In one embodiment the CEX assay method involves diluting the test sample to a low ionic strength and injecting it into a CEX column equilibrated with 10 mM sodium phosphate (pH 6.6), eluting the column with an NaCl gradient in this buffer monitoring the peaks at 280 nm, and assigning each peak as acidic, major, or basic, with acidic peaks eluting with the shortest retention time, major peaks eluting second, and basic peaks eluting with the longest retention time, quantifying the area of each peak, and calculating the amount as a percentage of the total peak area .

[0165] In different embodiments described herein, size exclusion chromatography (CEX) is 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 an antibody or its antigen-binding portion, e.g., vedolizumab. The SEC method enables separation based on the size of antibody monomers from HMW species and LMW degradation products. Each test sample and reference standard can be analyzed using a commercially available SEC column with an appropriate buffer . For example, in some embodiments the SEC analysis can be performed using a G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA (USA)), or two G3 000 SWxl columns connected in series, with an isocratic phosphate-sodium chloride buffer system (pH 6.8). The elution of protein species is monitored at 280 nm . The main peak (monomer) and total peak area are evaluated to determine purity. In one embodiment . ​​In the SEC analysis, samples were injected onto two G3000 SWxl columns connected in series. and in an isocratic phosphoric acid-sodium chloride system (pH 6.8). The elution of protein species was monitored at 280 nm, with the main peak (monomer ) and total peak area are measured. Purity (%) of the sample (calculated as monomer (%)), H MW aggregates (%) and / or LMW degradation products (%) are reported.

[0166] Residual CHO host cell protein (HCP) impurities present in antibody formulations are removed as necessary. and measured by enzyme-linked immunosorbent assay (ELISA) using standard techniques. Cygnus Technologies (Southport, NC(U There are several kits designed for this purpose, such as the CHO HCP ELISA Kit 3G available from SA. Many ELISA kits that have been developed for this purpose are commercially available. Capture can then be performed using a fixed polyclonal anti-CHO HCP antibody. The captured proteins were then analyzed using the same antibody, but with a horseradish peroxidase label. It can be detected using an appropriate detection reagent. In this exemplary embodiment, CHO H The amount of captured peroxidase, which is directly proportional to the concentration of CP, was determined by measuring the amount of the peroxidase substrate. Colorimetry at 450 nm using 3,3',5,5'-tetramethylbenzidine (TMB) Therefore, the CHO HCP assay can be used to measure the The method includes capturing the HCPs using a peroxygenase anti-CHO HCP antibody, The enzyme substrate, 3,3',5,5'-tetramethylbenzidine (TMB), was measured at 450 nm. is converted to a substrate measured by colorimetric analysis, and detected after binding a polyclonal anti-CHO HCP antibody labeled with horseradish peroxidase. The concentration of HCP is determined by comparison with a CHO HCP calibration curve such as those included in the test kit, and is reported as a percentage of the total protein level in the antibody preparation. After binding an antibody labeled with horseradish peroxidase, it is detected. The concentration of HCP is determined by comparison with a CHO HCP calibration curve such as those included in the test kit, and is reported as a percentage of the total protein level in the antibody preparation. can be determined by comparison with a CHO HCP calibration curve such as those included in the test kit, and is reported as a percentage of the total protein level in the antibody preparation. can be determined by comparison with a CHO HCP calibration curve such as those included in the test kit, and is reported as a percentage of the total protein level in the antibody preparation.

[0167] IV. Downstream Processes and Formulation The anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab) can be further purified from contaminating soluble proteins and polypeptides, and examples of suitable purification methods include the following methods, which can be used alone or in combination as needed, and in combination with one or more of the methods provided herein: for example, affinity chromatography using a resin that binds to the Fc region of an antibody such as Protein A; fractionation using an ion exchange chromatography (CEX) such as cation exchange chromatography using SP-Sepharose (trademark) or CM-Sepharose (trademark) hydroxyapatite or resin; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. The anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab) can be further purified from contaminating soluble proteins and polypeptides, and examples of suitable purification methods include the following methods, which can be used alone or in combination as needed, and in combination with one or more of the methods provided herein: for example, affinity chromatography using a resin that binds to the Fc region of an antibody such as Protein A; fractionation using an ion exchange chromatography (CEX) such as cation exchange chromatography using SP-Sepharose (trademark) or CM-Sepharose (trademark) hydroxyapatite or resin; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. The anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab) can be further purified from contaminating soluble proteins and polypeptides, and examples of suitable purification methods include the following methods, which can be used alone or in combination as needed, and in combination with one or more of the methods provided herein: for example, affinity chromatography using a resin that binds to the Fc region of an antibody such as Protein A; fractionation using an ion exchange chromatography (CEX) such as cation exchange chromatography using SP-Sepharose (trademark) or CM-Sepharose (trademark) hydroxyapatite or resin; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. The anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab) can be further purified from contaminating soluble proteins and polypeptides, and examples of suitable purification methods include the following methods, which can be used alone or in combination as needed, and in combination with one or more of the methods provided herein: for example, affinity chromatography using a resin that binds to the Fc region of an antibody such as Protein A; fractionation using an ion exchange chromatography (CEX) such as cation exchange chromatography using SP-Sepharose (trademark) or CM-Sepharose (trademark) hydroxyapatite or resin; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. affinity chromatography using a resin that binds to the Fc region of an antibody such as Protein A; fractionation using an ion exchange chromatography (CEX) such as cation exchange chromatography using SP-Sepharose (trademark) or CM-Sepharose (trademark) hydroxyapatite or resin; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75 (trademark); ultrafiltration and / or diafiltration, or a combination of the above. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. At the end of the purification process, the purity of the recombinant protein is high and is suitable for human therapeutic use, for example, in the pharmaceutical antibody formulations described below. After production, ultrafiltration / diafiltration (UF / DF ) can be performed to obtain a pharmaceutical preparation suitable for administration to humans.

[0168] After ultrafiltration / diafiltration, the antibody preparation may remain in liquid form, or it may be lyophilized to obtain a dried antibody preparation. In one embodiment, the dried lyophilized antibody preparation is provided in a single-dose vial containing 180 mg, 240 mg, 300 mg, 360 mg, 450 mg or 600 mg of anti-α 4β7 antibody and can be reconstituted with a liquid such as sterile water for administration. In another embodiment, the anti-α4β7 antibody, such as vedolizumab, is, for example, in a container such as a vial, syringe, or cartridge, about 2- 8 °C until administered to a subject in need of administration, in a stable liquid pharmaceutical composition. In some embodiments, the reconstituted lyophilized preparation or stable liquid pharmaceutical composition of the anti-α4β 7 antibody contains about 0% to 5.0%, 0 % to 2%, 2% or less, 1% or less, 0.6% or less, or 0.5% or less aggregates. % aggregates.

[0169] Thus, in some embodiments, as used herein, a reconstituted lyophilized preparation or stable liquid pharmaceutical composition of a humanized anti-α4β7 antibody, or an anti-α4β7 antibody comprising an antigen-binding portion thereof, is provided. Examples of lyophilized preparations containing an anti-α4β7 antibody, such as vedolizumab, are described in U.S. Patent No. 9,764,033, the contents of which are incorporated herein by reference. Examples of liquid preparations containing an anti-α4β7 antibody, such as vedolizumab, are described in U.S. Patent No. 10 ,040,855, the contents of which are incorporated herein by reference. In some embodiments, a reconstituted lyophilized preparation or stable liquid pharmaceutical composition of an anti-α4β7 antibody is provided. The composition contains about 11% to 16%, 12% to 15%, 14% or less, 13% or less, 12% or less, or 11% or less of the basic isoform species. In some embodiments, the returned lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β 7 antibody contains 65% to 75%, 66 % to 74%, 67% to 73%, at least 65%, at least 66%, at least 67% at least 68%, at least 69%, or at least 70% of the major isoform.

[0170] Purified antibodies, such as anti-α4β7 antibodies (e.g., antibodies having a binding region corresponding to vedolizumab or vedolizumab are concentrated to provide a concentrated protein composition, such as an antibody concentration of at least 100 mg / mL or 125 mg / mL or 150 mg / mL, or a protein composition having a concentration of about 100 mg / mL or 125 mg / mL or 150 mg / mL. The concentrated antibody product can be concentrated to levels possible under concentration conditions, e.g., to a concentration at which the polypeptide becomes insoluble in solution. It will be understood that this is possible.

[0171] In some embodiments, the compositions obtained herein contain a purified anti-α4β7 antibody, such as vedolizumab, and are then formulated for use in humans. In one embodiment the purified antibody is formulated as a dry lyophilized formulation, which can be reconstituted with a liquid such as sterile water and administered. Administration of the reconstituted formulation can be by parenteral injection by one of the routes described above. Intravenous injection can be with sterile isotonic saline, a buffer, e.g., phosphate buffered saline or Ringer's (lactate or dextrose) solution. By further diluting with a liquid or the like, it can be administered by intravenous drip. Some embodiments In, the purified antibody is formulated as a liquid preparation, and the anti-α4β7 antibody is, for example, about 54 m g, 108 mg, 165 mg or about 216 mg and administered by subcutaneous injection .

[0172] The purified compositions described herein can be used for storing and freezing Containers include polycarbonate bottles (for IV preparations) or PETG bottles (for subcutaneous Preparations). After dispensing the preparation into the bottle, freezing can be performed (e.g., - 60 °C or lower).

[0173] The following examples illustrate improved methods and compositions for purifying antibodies Using the following Examples 1-5, purified compositions of anti-α4β7 antibodies, particularly vedolizumab Can be obtained. This specification includes the methods described in the following examples, including various parameters Described in the following examples.

[0174] Example In the following examples, the purification process of vedolizumab produced in a cell culture using CHO cells as an expression system is described .

[0175] Example 1: Effect of elution buffer on the purification of vedolizumab using Protein A resin Effect In this example, an antibody purification method using Protein A resin that can be used for the production of therapeutic anti-α4β7 antibodies, such as vedolizumab, is shown. As described herein , by adjusting the elution pH of the Protein A resin, the level of aggregates in the purified composition of vedolizumab was reduced . .

[0176] Berdilizumab was produced by cell culture of recombinant Chinese hamster -ovary (CHO) cells (GS-CHO) genetically engineered to express the antibody (for general cell culture methods, see Li et al. (2010) mAbs 2:5,466-477). )

[0177] After cell culture in CHO cells, selective capture of Berdilizumab was performed after primary recovery using a Protein A affinity column. Affinity chromatography using recombinant Protein A resin was performed to selectively remove the antibody from the clarified recovered material obtained from the upstream primary recovery process. In this step, process-related impurities

[0178] such as host cell proteins (HCP) were also removed. The Protein A resin was first equilibrated with a PBS equilibration solution (pH 7.2). Subsequently, the clarified recovered material was loaded. Three washes were performed. Wash 1 was performed with the same PBS wash solution (pH 7.2) as the equilibration solution, Wash 2 was performed with a PBS wash solution of 1 M NaCl (pH 7.2), and Wash 3 was performed with the same solution as Wash 1 (PBS) and the solution used for equilibration. Since the antibody remained bound to the resin, each wash served to wash away impurities from the antibody. Next, the antibody was eluted from the resin using elution buffers of different pHs. As shown in Figure 1, elution buffers of pH 3-3.5 The eluate obtained by eluting the antibody with an elution buffer having 5) was about 0.6 to 0. It had 85% aggregates.

[0179] Further tests were conducted to identify process parameters related to Protein A purification that significantly affect the product quality of vedolizumab. The clarified harvest from GS-CHO cells expressing vedolizumab recombinantly was loaded onto MabSelect SuReLX resin (GE Healthcare, Pittsburgh, PA). The bound antibody was washed with sodium citrate buffer and PBS prior to elution. The elution pH was evaluated in the range of pH 3.3 to pH 3.9. Sodium citrate buffer was used for elution. The effects of the elution buffer on the level of aggregates (percentage of HMW species (%)) and the level of HCP in the purified composition of vedolizumab are shown in Table 1 and Figure 2. HO cells was loaded onto MabSelect SuReLX resin (GE Healthcare, Pittsburgh, PA). The bound antibody was washed with sodium citrate buffer and PBS prior to elution. The elution pH was evaluated in the range of pH 3.3 to pH 3.9. Sodium citrate buffer was used for elution. The effects of the elution buffer on the level of aggregates (percentage of HMW species (%)) and the level of HCP in the purified composition of vedolizumab are shown in Table 1 and Figure 2.

[0180] As shown in Table 1, the linear regression model showed that the elution pH had a significant effect on the results of all assays (p < 0.05). The data variability was slightly greater for the LMW percentage (%) and HCP, but the load amount affected the clearance of HCP. The combination of the load amount and the load flow rate slightly affected the LMW (%). · Monomer (%):

[0181] The elution pH showed a significant effect on the monomer percentage (%) (p < 0.05). None of the other input parameters showed a correlation with the monomer percentage (%). As the elution pH was increased, the monomer percentage (%) increased.

[0182] · HMW percentage (%): ​​​​​​​The dissolution pH showed a significant effect on the HMW ratio (%) (p < 0.05). None of the other input parameters showed a correlation with the HMW ratio (%). As the dissolution pH increased, the HMW ratio (%) decreased.

[0183] · LMW ratio (%): The dissolution pH, and the combination of the loading amount and the loading flow rate, affected the LMW ratio (%) ( p < 0.05), but the influence of the input parameters on the LMW ratio (%) was considered minimal.

[0184] · HCP: The dissolution pH and the loading amount had a significant effect on both the ppm and the logarithmic reduction coefficient for HCP (p < 0.05).

[0185]

Table 1

[0186] Example 2: Purification of Bevacizumab Using Hydrophobic HIC Resin Considering the hydrophobic nature of bevacizumab, reducing HMW aggregates can be difficult in downstream processes. Furthermore, when bevacizumab is produced intracellularly in mammalian cells such as CHO cells, it is also essential to minimize the level of host cell protein (HCP). HIC, mixed mode, and anion exchange resins, and membranes were screened for performance by a high-throughput method. Subsequently, eight types of HIC resins were tested for their respective abilities to reduce aggregates and minimize HPC in the purification of bevacizumab under different equilibration, loading, and elution conditions. The only resin that showed acceptable aggregate clearance and was able to minimize HCP was the Toyopearl Hexyl-650C (Tosoh Biosciences) hydrophobic HIC resin. More specifically, Hexyl-650C reduced the aggregate level to about 1.5% - 0.35% of HMW aggregates under appropriate binding conditions (e.g., 0.5 M (NH4)2SO4 at pH 6.7). Other resins tested included Butyl-650M, Butyl-600M, Super Butyl-55C, Phenyl-650M, Phenyl-600M, PPG-600M, and Ether-650M, but such low levels of aggregates could not be achieved. Hexyl-650C was the most hydrophobic resin among the other resins tested, including ether, PPG, phenyl, and butyl. Hexyl-650C has an average pore size of about 1000 Å and an average particle size of about 100 μm. Considering the hydrophobic nature of bevacizumab, reducing HMW aggregates can be difficult in downstream processes. Furthermore, when bevacizumab is produced intracellularly in mammalian cells such as CHO cells, it is also essential to minimize the level of host cell protein (HCP). HIC, mixed mode, and anion exchange resins, and membranes were screened for performance by a high-throughput method. Subsequently, eight types of HIC resins were tested for their respective abilities to reduce aggregates and minimize HPC in the purification of bevacizumab under different equilibration, loading, and elution conditions. The only resin that showed acceptable aggregate clearance and was able to minimize HCP was the Toyopearl Hexyl-650C (Tosoh Biosciences) hydrophobic HIC resin. More specifically, Hexyl-650C reduced the aggregate level to about 1.5% - 0.35% of HMW aggregates under appropriate binding conditions (e.g., 0.5 M (NH4)2SO4 at pH 6.7). Other resins tested included Butyl-650M, Butyl-600M, Super Butyl-55C, Phenyl-650M, Phenyl-600M, PPG-600M, and Ether-650M, but such low levels of aggregates could not be achieved. Hexyl-650C was the most hydrophobic resin among the other resins tested, including ether, PPG, phenyl, and butyl. Hexyl-650C has an average pore size of about 1000 Å and an average particle size of about 100 μm. (NH 4 ) 2 SO 4 ) Other resins tested included Butyl-650M, Butyl-600M, Super Butyl-55C, Phenyl-650M, Phenyl-600M, PPG-600M, and Ether-650M, but such low levels of aggregates could not be achieved. Hexyl-650C was the most hydrophobic resin among the other resins tested, including ether, PPG, phenyl, and butyl. Hexyl-650C has an average pore size of about 1000 Å and an average particle size of about 100 μm.

[0187] Hexyl-650C was the most hydrophobic resin among the other resins tested, including ether, PPG, phenyl, and butyl. Hexyl-650C has an average pore size of about 1000 Å and an average particle size of about 100 μm. Hexyl-650C has an average pore size of about 1000 Å and an average particle size of about 100 μm.

[0188] Further experiments were performed using Hexyl-650C in bind / elute and flow-through modes. In the bind / elute experiments, Hexyl-650C reduced the aggregates to HMW approximately 0.3. Although it was possible to reduce the binding capacity to 0%, the binding capacity was low (approximately 20 mg / ml of resin). In contrast, the flow-through mode using Hexyl-650C and vedolizumab showed both a reduction in aggregates and an increase in loading capacity. Initial unadjusted load of 108 mg / ml in 0.2 M sodium chloride in 100 mM NaCl (pH 6.7) Flow-through experiments were performed using these conditions. The substitution of sodium chloride with calcium phosphate resulted in a decrease in HMW to 0.71%. Increasing the salt, including phosphate phosphate, improved aggregate reduction even further. 67.5 mg / mL with potassium chloride, 50 mM potassium chloride (for equilibration and load adjustment) Loading less ml reduced aggregates from HMW 0.72% to HMW 0.3% (Recovery rate 95.5%).

[0189] A column-based design of experiments (DOE) study was performed to evaluate the efficacy and safety of coagulation in the purification of vedolizumab. Further demonstrating the ability of Hexyl-650C in flow-through mode to reduce aggregate levels As shown in Table 2, low pH and high phosphate load equilibration conditions were used. When the 60m The experiments in Table 2 were performed using resin loads of 1000 µg / ml. The reduction rate was 1.0% to 0.34% HMW or less. The average recovery rate of antibodies was about 91%. It was 0.5%. The combination of high pH and high potassium phosphate seems to have increased the affinity of vedolizumab for the resin, and the recovery rate decreased. In contrast, with the combination of higher levels of phosphate and low pH, the HMW clearance increased. At low phosphate and low pH, low HMW, low HCP, and low residual protein A elution were observed (see, for example, line 12 below). Therefore, the highly hydrophobic HIC resin was able to effectively remove aggregated (HMW) vedolizumab to levels lower than 0.5%. and the recovery rate decreased. In contrast, with the combination of higher levels of phosphate and low pH, the HMW clearance increased. At low phosphate and low pH, the HMW clearance increased. At low phosphate and low pH, low HMW, low HCP, and low residual protein A elution were observed (see, for example, line 12 below). Therefore, the highly hydrophobic HIC resin was able to effectively remove aggregated (HMW) vedolizumab to levels lower than 0.5%. and the recovery rate decreased. In contrast, with the combination of higher levels of phosphate and low pH, the HMW clearance increased. At low phosphate and low pH, low HMW, low HCP, and low residual protein A elution were

[0190]

Table 2

[0191] Example 3: Effect of column load, elution buffer pH, and conductivity on the purification of vedolizumab using mixed-mode chromatography resin A production CHO cell line expressing vedolizumab at a high antibody titer (≧5.0 g / L) was generated A production CHO cell line expressing vedolizumab at a high antibody titer (≧5.0 g / L) was generated . For this production CHO cell line, the development of a purification process designed to handle large amounts of this highly hydrophobic antibody was required. . For this production CHO cell line, the development of a purification process designed to handle large amounts of this highly hydrophobic antibody was required.

[0192] Capto Adhere ImpRes is a mixed-mode (MXM) chromatography resin with functional properties of strong anion exchange, hydrogen bonding, and hydrophobic interaction with a smaller bead diameter, enabling improved impurity removal and increased capacity. Capto Adhere ImpRes is a mixed-mode (MXM) chromatography resin with functional properties of strong anion exchange, hydrogen bonding, and hydrophobic interaction with a smaller bead diameter, enabling improved impurity removal and increased capacity. Capto Adhere ImpRes is a mixed-mode (MXM) chromatography resin with functional properties of strong anion exchange, hydrogen bonding, and hydrophobic interaction with a smaller bead diameter, enabling improved impurity removal and increased capacity.

[0193] The Capto Adhere ImpRes mixed-mode resin used in flow-through mode was able to purify vedolizumab, but the levels of yield and impurity removal were The Capto Adhere ImpRes mixed-mode resin used in flow-through mode was able to purify vedolizumab, but the levels of yield and impurity removal were It was lower than the ideal value. Capto Adhere ImpRes was used in the binding-elution mode In the preliminary characterization experiments conducted using it, the resin loading capacity, the pH of the elution buffer, and the conductivity of the elution buffer were related to three process input parameters of step yield and / or a significant loss in impurity removal performance was shown. In this example, the changes in these parameters in the performance of Capto Adhere ImpRes in the purification of vedolizumab, as well as its impact on various product quality characteristics, will be further investigated as described in the planned tests.

[0194] Materials and Methods The clarified cell culture harvest was loaded onto a Capto Adhere ImpRes (GE Healthcare, Chicago, IL, USA) chromatography column after purification with Protein A. The mixed-mode resin was washed with sodium phosphate buffer at pH 7.8 and the antibody was eluted from the column under various conditions described below.

[0195] The samples were immediately submitted for analysis by SEC, stored at 2 - 8 °C, and processed within one week . The remaining assays (CEX, CHO HCP ELISA) were performed using the frozen aliquots (-80 °C). The methods used for analysis are shown in Table 3 below and described in detail below. After the implementation put into the waiting matrix, the loaded materials sampled were analyzed to confirm that there were no significant changes in the quality characteristics of the loaded materials.

[0196]

Table 3

[0197] Experimental Design A full factorial design was used with three levels for the resin load amount, elution buffer pH, and elution buffer conductivity. A sodium citrate elution buffer was used in these experiments. The resulting design included 30 runs with three center point conditions. Additional center point conditions were made part of the experimental design shown as DOE pattern 222 in Table 5. All other process input parameters were maintained at the center point conditions. The experiments were planned using the sodium chloride concentration of the elution buffer, and the measured conductivity values of the elution buffer were used as input parameter values for the statistical analysis. The parameter ranges tested and an overview of the design are described in Tables 4 and 5, respectively.

[0198]

Table 4

Table 5

[0199] Calculation HMW clearance (%) = (1 - (eluate HMW / load HMW)) * 100 The logarithmic reduction factor (LRF) was determined as follows.

[0200]

Equation

[0201] Statistical Analysis The generated article quality showing assay results and KPIs in all experiments within the plan was analyzed using JMP 1 1 statistical software (SAS Institute, Cary, NC). Each response was analyzed by fitting it to the linear model shown in Equation 1. Shown in Equation 1.

[0202] Equation 1 General linear model of regression

Number

[0203] In statistical analysis and modeling, fitting a relatively small data set to a model containing a relatively large number of potential inputs often results in overfitting. One characteristic of overfitting is a model that has a high R value but contains multiple scientifically meaningless (and statistically insignificant) terms. To determine the best statistically significant model while avoiding overfitting, forward regression of the input parameters and the response of interest were used to develop each model. The stopping rule for regression was a p-value threshold, and input 2 parameters were incorporated into the model when the p-value < 0.05. By this algorithm for this analysis, (i) the highest possible R value was achieved, (ii) the model contained the fewest possible number of input parameters, and (iii was used to develop each model. The stopping rule for regression was a p-value threshold, and input parameters were incorporated into the model when the p-value < 0.05. By this algorithm for this analysis, (i) the highest possible R value was achieved, (ii) the model contained the fewest possible number of input parameters, and (iii possible R 2 value was achieved, (ii) the model contained the fewest possible number of input parameters, and (iii Describe the physically possible behavior of the antibody for which multimode chromatography processing is performed (Even if such findings do not appear to match the initial technical expectations) the model was generated.

[0204] Results and Discussion For the model of each response obtained by statistical analysis, the parameter estimated value, the corresponding p-value , and R 2 values are shown in Tables 6 and 7. The experimental results include the experimental results.

[0205]

Table 6

[0206]

Table 7

[0207] Effect of pH and conductivity of elution buffer on antibody yield Effect of pH of elution buffer and conductivity of elution buffer on process recovery rate or yield of vedolizumab is shown in Figures 4 and 5. As shown in Figures 4 and 5, the observed process recovery rate data fits well with the linear regression model, as indicated by an R value of 0.930. 2 value of 0.930. rule.

[0208] Figures 4 and 5 show plots of the process recovery rate by Capto Adhere ImpRes against the pH and conductivity or loading amount of the elution buffer. The recovery rate is significantly affected by the pH of the elution buffer (p < 0.0001) and the loading amount of the resin (p = 0.0021), and although not as much, also by the conductivity of the elution buffer (p = 0.0189). At any level of elution buffer conductivity and loading amount, about 4.40 0.0189). At any level of elution buffer conductivity and loading amount, about 4.40 At the elution buffer pH, the process recovery rate was 83.91% or less (runs 1, 4, 11~ 14, 16, 19, and 27). The low resin loading amount had a negative impact on the recovery rate. In the washing process after sample loading, breakthrough was observed in all runs with a loading amount of 77 g per liter of resin. The effect of the loading amount was dependent on the elution buffer pH ( p = 0.0170). At a low elution buffer pH (i.e., pH 3.8), the loading amount did not substantially affect the recovery rate, while as the elution buffer pH increased, the recovery rate decreased with a low resin loading (at an elution buffer pH of about 4.4, it was 81.94 - 83.91% for a loading amount of 77 g per liter of resin, compared to 73.36 - 78.84% for a loading amount of 53 g per liter of resin). The conductivity of the elution buffer had only a slight effect on the recovery rate according to the experimental results and model predictions. The lowest recovery rate of 73.36% was observed at an elution buffer pH of 4.40, an elution buffer conductivity of 28.89 mS / cm, and a loading amount of 53 g per liter of resin (run 13, predicted by the model to be 76 22%). Therefore, as shown in Figures 4 and 5, the yield of vedolizumab is higher when the mixed - mode chromatography resin is used with an elution buffer having optimized pH and conductivity. Effect of pH and conductivity of elution buffer on HMW aggregates The effect of the pH and conductivity of the elution buffer on the level of aggregates is shown in Figures 6 and 7, which show the effect of the input parameters on the HMW amount.

[0209]

[0210]

[0211] According to the linear regression model, the amount of HMW species in the eluate is related to the pH of the elution buffer (p < 0 .0001), the conductivity of the elution buffer (p < 0.0001), and the load amount (p = 0. 0015), with an R 2 value of 0.962. As the elution pH and conductivity increase, the amount of HMW species in the eluate decreases, and increasing the load amount raises the level. This model also predicted a statistically significant (p = 0.0462 ) interaction between the pH and conductivity of the elution buffer.

[0212] As shown in Figures 6 and 7 below, an HMW species content of about 1% or more was observed at an elution buffer pH of about 3.80. At an elution pH of 3.80, an elution conductivity of 19.67 mS / cm (16 0 mM of NaCl), and a load amount of 65 g per liter of resin (Run 2), the highest HMW content of 1.23% was obtained (predicted by the model to be 1.19%). The predicted worst-case HMW content was 1.24% at 77 g / L under the same elution buffer conditions.

[0213] According to the linear regression model, the elution buffer pH (p < 0.0001), the elution buffer conductivity (p = 0.0003), and the load amount (p = 0.0016) each have a statistically significant effect on the HMW clearance performance. A constant level of clearance (12.50 - 72.79%) was achieved under all conditions evaluated in this study. Among these inputs, the elution pH had the greatest effect. Increasing the elution buffer pH improved the HMW clearance, which is the opposite of the effect on the recovery rate. According to the model, increasing the elution buffer conductivity and decreasing the load amount increased the HMW clearance. ​

[0214] Therefore, as shown in FIGS. 6 and 7, the level (percentage of HMW species) of aggregates in the purified composition of vedolizumab can be adjusted by the selection of the elution buffer pH and elution buffer conductivity used to elute the antibody from the mixed-mode chromatography resin. Furthermore, the level of aggregates can be reduced when the mixed-mode chromatography resin is used with an elution buffer having a high pH and / or high conductivity.

[0215] Example 4: Effect of elution buffer on the purification of vedolizumab using cation exchange (CEX) Cation exchange (CEX) chromatography was also investigated as a means to further reduce the level of aggregates in the vedolizumab formulation. In this specification, a test focusing on adapting CEX chromatography using Nuvia HR-S resin (Bio-Rad, Hercules, CA, USA) for the purification of vedolizumab is described with particular emphasis on reducing the level of aggregates from this hydrophobic antibody. The elution conditions of the CEX process performed in binding / elution mode were evaluated. Using a design of experiments (DoE) approach, the effects of several parameters including the pH of the elution buffer and the conductivity of the elution buffer on the process results were evaluated.

[0216] Materials and Methods The load materials and analytical methods used in this test were the same as those described in Example 3 above.

[0217] Experimental Design Based on the initial screening tests and preliminary risk assessments, the resin was used in binding / elution mode​​​​​​​​​​ When used, the pH of the elution buffer and the conductivity of the elution buffer are such that they have a known or potential impact on the process performance results (PPO) of Nuvia HR -S and are specified. This test was conducted to characterize the impact of these process parameters The test parameters range and experimental design are shown in Tables 8 and 9 respectively. The conductivity of the elution buffer was varied by adjusting the concentration of sodium chloride (NaCl), and the range of NaCl evaluated is shown in Table 8

[0218]

Table 8

[0219]

Table 9-1

Table 9-2

[0220] Results and Discussion The impact of the pH of the elution buffer and the conductivity of the elution buffer on the process performance results (PPO) of Nuvia HR-S is shown in Figures 8 to 13

[0221] Impact of the pH and conductivity of the elution buffer on HMW aggregates The impact of the pH and conductivity of the elution buffer on the level of aggregates is shown in Figures 8 to 10, which show the impact of the input parameters on the amount of HMW

[0222] As shown in Figures 8 to 10, the variations in HMW, monomer, and LMW in the eluate are 0.01 - 0.88%, 98.33 - 99.27%, and 0.62 - 1.35% respectively ​​​​There was. The HMW model includes an interaction term containing both parameters and shows a strong linear dependence on the pH and conductivity of the elution buffer. The model surface (shown in Figure 8) indicates that HMW is lowest at extremely low pH and conductivity values of the elution buffer and highest at extremely high pH and conductivity values of the elution buffer. For each run, the HMW clearance was measured to account for the variation in the HMW content of the load material throughout the study. Similar to the HMW in the eluate, the HMW clearance varied widely throughout the study (-30.65 to 98.61%), and the HMW clearance model includes linear and interaction terms for the pH and conductivity of the elution buffer. Figure 9 shows the behavior of the model. The highest HMW clearance values were obtained at low pH and conductivity conditions of the elution buffer, while HMW clearances exceeding 70% were shown under many of the tested conditions. However, the negative HMW clearance values reported for runs 9, 20, and 24 (-1.18%, -20.55%, and -30.65%, respectively) indicate that significant variability in the removal rate of aggregates was observed over the wide range of conditions evaluated, and that under certain conditions, aggregate species may not be removed but rather generated. HMW was lowest at extremely low pH and conductivity values of the elution buffer and highest at extremely high pH and conductivity values of the elution buffer. This indicates that HMW is lowest at extremely low pH and conductivity values of the elution buffer and highest at extremely high pH and conductivity values of the elution buffer.

[0223] Throughout the test, the HM W clearance was measured for each run to explain the variation in the HMW content of the load material. Similar to the HMW in the eluate, the HMW clearance varied widely throughout the test (-30.65 to 98.61%), and the HMW clearance model includes linear and interaction terms for the pH and conductivity of the elution buffer. throughout the test (-30.65 to 98.61%), and the HMW clearance model includes linear and interaction terms for the pH and conductivity of the elution buffer. Figure 9 shows the behavior of the model. The highest HMW clearance values were obtained at low pH and conductivity conditions of the elution buffer, while HMW clearances exceeding 70% were shown under many of the tested conditions. However, the negative HMW clearance values reported for runs 9, 20, and 24 (-1.18%, -20. 55%, and -30.65%) indicate that significant variability in the removal rate of aggregates was observed over the wide range of conditions evaluated, and that under certain conditions, aggregate species may not be removed but rather generated. The highest HMW clearance values were obtained at low pH and conductivity conditions of the elution buffer, while HMW clearances exceeding 70% were shown under many of the tested conditions. However, the negative HMW clearance values reported for runs 9, 20, and 24 (-1.18%, -20. 55%, and -30.65%) indicate that significant variability in the removal rate of aggregates was observed over the wide range of conditions evaluated, and that under certain conditions, aggregate species may not be removed but rather generated. This indicates that significant variability in the removal rate of aggregates was observed over the wide range of conditions evaluated, and that under certain conditions, aggregate species may not be removed but rather generated. This indicates that significant variability in the removal rate of aggregates was observed over the wide range of conditions evaluated, and that under certain conditions, aggregate species may not be removed but rather generated.

[0224] In runs 40 - 42, Capto Adhere ImpR es eluate containing higher aggregate levels than typically used in the process by CEX at the central point conditions was used as the load material. The models for HMW and HMW clearance predict that increasing the pH and conductivity of the elution buffer results in eluates with high HMW content. For each run, the HMW clearance was measured to account for the variation in the HMW content of the load material throughout the study. Similar to the HMW in the eluate, the HMW clearance varied widely throughout the study (-30.65 to 98.61%), and the HMW clearance model includes linear and interaction terms for the pH and conductivity of the elution buffer. Figure 9 shows the behavior of the model. The highest HMW clearance values were obtained at low pH and conductivity conditions of the elution buffer, while HMW clearances exceeding 70% were shown under many of the tested conditions. However, the negative HMW clearance values reported for runs 9, 20, and 24 (-1.18%, -20. It was shown. The conditions selected for runs 40 - 42 were aimed at exploring potential elution conditions using the aggregate level at the "worst case" of the CEX load material. The eluate HMW obtained at a pH value of the elution buffer of 5.50 - 5.54 and a conductivity of the elution buffer of 13.40 mS / cm was 0.31 - 0.34%. However, the eluate HMW increased to 0.59% when using an elution buffer at pH 5.60 (whereas the conductivity did not change). At that aggregate level, further processing might not meet the acceptance criteria for vedolizumab with respect to HMW.

[0225] LMW was shown to decrease linearly with an increase in the pH of the elution buffer or the conductivity of the elution buffer. This model also included interaction terms for elution buffer pH / elution buffer conductivity, elution buffer pH / load amount, and elution buffer conductivity / load amount. Similar to the model for HMW, the monomer model included a strong dependence on the pH and conductivity of the elution buffer in the form of linear and interaction terms. The model surface (shown as a saddle function in Figure 10) indicates that the lowest monomers are obtained when combining very high pH and conductivity conditions of the elution buffer.

[0226] Therefore, as shown in Figures 8 - 10, the level of aggregates (percentage of HMW species) in the composition containing vedolizumab purified using a CEX resin can be adjusted using the pH and conductivity of the elution buffer. Furthermore, as shown in Figures 8 - 10, the level of aggregates in the purified composition of vedolizumab is lower when using a CEX resin at a low pH and / or ​​​​​​​​​​​​​​It can be reduced when used with an elution buffer having a low conductivity.

[0227] Effect of pH and conductivity of elution buffer on basic isoform species The effects of the pH and conductivity of the elution buffer on the level of the basic isoform species, the effects of the input parameters on the contents of acidic, major, and basic isoform species are shown in Figure 1. are shown in Figures 1 to 13.

[0228] As shown in Figures 11 to 13, the results of the acidic, major, and basic contents are 12.49 to 30.27%, 64.32 to 73.82%, and 5.42 to 18.04%, respectively, in the ranges of. The model for the acidic content includes the pH of the elution buffer, the conductivity of the elution buffer, and the load amount, along with the interaction terms of all three parameters. The pH of the elution buffer and the conductivity of the elution buffer most strongly affected the acidic content. As seen on the model surface in Figure 11, the highest acidic content was obtained when used in combination with extremely low values of the pH and conductivity of the elution buffer. As seen on the model surface in Figure 11, the highest acidic content was obtained when used in combination with extremely low values of the pH and conductivity of the elution buffer. As seen on the model surface in Figure 11, the highest acidic content was obtained when used in combination with extremely low values of the pH and conductivity of the elution buffer.

[0229] The model developed for the major isoform content includes the interaction terms between the pH of the elution buffer, the conductivity of the elution buffer, and the load amount, and the interaction of the pH of the elution buffer / conductivity of the elution buffer shows the greatest influence on the model behavior. As shown in Figure 12, the highest major isoform content was obtained with the highest elution buffer conductivity and the lowest elution buffer pH, and the lowest major isoform content is predicted with a combination of extremely low elution buffer pH and conductivity. is predicted with a combination of extremely low elution buffer pH and conductivity.

[0230] The linear terms of the pH of the dissolution buffer, the conductivity of the dissolution buffer, and the loading amount most strongly affect the basic isoform content in the eluate, and the contribution of the interaction terms is minor. As seen on the model surface in Figure 13, the basic isoform content increased in response to an increase in the pH of the dissolution buffer and the conductivity of the dissolution buffer.

[0231] Therefore, as shown in Figures 11 to 13, the pH and conductivity of the dissolution buffer are used to adjust the charged isoform distribution in a composition containing vedolizumab purified using a CEX resin. As shown in Figure 11, the level of acidic isoform species in the purified composition of vedolizumab can be reduced when using a CEX resin with a dissolution buffer having a high pH and / or high conductivity. Furthermore, as shown in Figure 13, the level of basic isoform species in the purified composition of vedolizumab can be reduced when using a CEX resin with a dissolution buffer having a low pH and / or low conductivity.

[0232] Example 5: Determination of Product Quality Characteristics The following analytical assays and methods were used in the above examples to determine the product quality characteristics of vedolizumab.

[0233] Cation exchange chromatography (CEX) fractionates vedolizumab antibody species (major isoform, basic species, and acidic species) based on the overall surface charge. After dilution to low ionic strength with the mobile phase, the test sample is equilibrated in 10 mM sodium phosphate (pH 6.6) on a Dionex Pro-Pac™ WCX-10 column (Thermo Fi sciences). ​​​​Inject into sher Scientific, Waltham, MA (USA)), and elute in the same buffer using a sodium chloride gradient. Monitor the elution of the protein at 280 nm, and assign each peak to a category of acidic, basic, or major isoform. Report the sum of the percentage of the major isoform (%), the sum of the percentage of acidic species (%), and the percentage of basic species (%). Determine the compliance by comparing the retention time of the major isoform of the sample with the retention time of the reference standard. Use size exclusion chromatography (SEC) to determine the purity of vedolizumab. Analyze the reference standard and test sample (75 μg) using two G3000 SWxl columns (Tosoh Bioscience, King of Prussia, PA (USA)) connected in series and an isocratic phosphate-sodium chloride buffer system (pH 6.8). This method provides separation of the antibody monomer from high molecular weight (HMW) species and low molecular weight (LMW) degradation products. Monitor the elution of protein species at 280 nm. Evaluate the main peak (monomer) and the total peak area to determine the purity. Report the purity (%) of the sample (calculated as monomer (%)) and aggregates (%). Equivalents One of ordinary skill in the art will recognize or be able to ascertain many equivalents of the specific embodiments of the invention described herein without undue experimentation. Such equivalents are intended to be encompassed by the following claims. The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference into this specification.

[0234]

[0235]

[0236] Sequence Listing

Table 10-1

Table 10-2

Claims

1. A method for obtaining a composition comprising the anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, comprising: contacting the liquid solution comprising the anti-α4β7 antibody and the one or more impurities with a matrix comprising Protein A to bind the anti-α4β7 antibody to the Protein A; washing the matrix comprising Protein A with a washing solution; eluting the anti-α4β7 antibody from the matrix comprising Protein A by contacting the matrix with an elution solution having a pH of 3.2 to 4 to obtain a composition comprising the anti-α4β7 antibody, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 4, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO:

6.

2. The method according to claim 1, wherein the composition comprising the anti-α4β7 antibody comprises less than 1% high molecular weight (HMW) aggregates.

3. The method according to claim 1 or 2, wherein the Protein A is immobilized on a solid phase.

4. The method according to claim 3, wherein the solid phase comprises one or more of beads, gels, and resins.

5. The method according to any one of claims 1 to 4, wherein the pH of the washing solution is about 7.

6. The method according to any one of claims 1 to 5, wherein the elution solution comprises citric acid.

7. The method according to any one of claims 1 to 6, wherein the pH of the elution solution is 3.2 to 3.7 or 3.3 to 3.

8.

8. A method for obtaining a composition comprising the anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, comprising contacting a solution comprising the anti-α4β7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow flow-through of the anti-α4β7 antibody through the HIC resin to obtain a composition comprising the anti-α4β7 antibody, 。 wherein the HIC resin is characterized as a high hydrophobicity HIC resin, and the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and is set forth in SEQ ID NO:

4. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ A heavy chain variable region comprising a CDR3 domain, a CDR2 domain set forth in SEQ ID NO: 3, and a CDR1 domain set forth in SEQ ID NO: 2, and a CDR3 domain set forth in SEQ ID NO: 8, a CDR2 domain set forth in SEQ ID NO: 7, and a CDR1 domain set forth in SEQ ID NO: 6 A light chain variable region, and the method as described above.

9. The method according to claim 8, wherein the composition comprises the anti-α4β7 antibody and contains less than 0.6% HMW aggregates.

10. The method according to claim 8 or 9, wherein the HIC resin is equilibrated with a phosphate buffer having a pH of less than about 7.

2.

11. The method according to claim 10, wherein the phosphate buffer contains about 0.35 M to about 0.15 M potassium phosphate.

12. The method according to any one of claims 8 to 11, wherein the resin load is about 55 to 75 mg / ml.

13. The method according to any one of claims 8 to 12, wherein the composition contains less than about 0.22 ppm of residual Protein A.

14. The method according to any one of claims 8 to 13, wherein the composition contains less than about 0.3 ppm of host cell protein (HCP).

15. The method according to any one of claims 8 to 14, wherein the highly hydrophobic HIC resin has an average pore size of about 50 to 150 μm.

16. The method according to any one of claims 8 to 14, wherein the highly hydrophobic HIC resin has an average pore size of about 100 nm and / or a pore size of about 100 μm.

17. A method for producing a composition comprising the anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with a mixed mode chromatography resin to bind the anti-α4β7 antibody to the resin; washing the mixed mode chromatography resin with a washing solution; contacting the resin with an elution solution having a pH of 3.9 or higher to elute the anti-α4β7 antibody from the mixed mode chromatography resin, thereby obtaining a composition comprising the anti-α4β7 antibody, and wherein the anti-α4β7 antibody comprises a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO:

2.

18. ​ ​ ​ ​ ​ ​ ​ ​ 、 ​ ​ ​ ​ ​ ​ ​ The method according to claim 17, wherein the composition comprising the anti-α4β7 antibody comprises less than 1% HMW aggregates. **Claim 19** The method according to claim 17 or 1 8, wherein the elution solution has a pH of 4.1 or higher. **Claim 20** The method according to any one of claims 17 to 19, wherein the elution solution has a pH of about 3.9 to about 4.

4. **Claim 21** The method according to any one of claims 17 to 20, wherein the elution solution has a conductivity of 30 mS / cm or less. **Claim 22** The method according to claim 21, wherein the elution solution has a conductivity of about 20 mS / cm to about 30 mS / cm. **Claim 23** The method according to any one of claims 17 to 22, wherein the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM. **Claim 24** The method according to any one of claims 17 to 23, wherein the mixed-mode chromatography resin is Capto Adhere ImpRes. **Claim 25** The method according to any one of claims 17 to 24, further comprising purifying the anti-α4β7 antibody using a cation exchange (CEX) resin. **Claim 26** The method according to claim 26, wherein the CEX resin is operated in a bind / elute mode. **Claim 27** A method for generating a composition comprising an anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, comprising: binding the anti-α4β7 antibody to a mixed-mode chromatography resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the mixed-mode chromatography resin; washing the mixed-mode chromatography resin with a washing solution; eluting the anti-α4β7 antibody from the mixed-mode chromatography resin by contacting the resin with an elution solution having a pH of 4.2 or lower and a conductivity of 28 mS / cm or lower to obtain a composition comprising the anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO:

2. **Claim 28** The method according to claim 27, wherein the composition has a high yield of anti-α4β7 antibody as compared to a control composition comprising an anti-α4β7 antibody obtained in the same manner using a control elution solution having a pH higher than 4.2 and / or a conductivity higher than 28 mS / cm. ​ ​ ​ 、 ​ ​ ​ ​ ​ ​ ​ ​ Claim 29 The method according to claim 27 or 28, wherein the elution solution has a pH of 4.0 or less. Claim 30 The method according to claim 27 or 28, wherein the elution solution has a pH of from about pH 4.2 to about pH 3.

8. Claim 31 The method according to any one of claims 27 to 30, wherein the elution solution has a conductivity of from about 18 mS / cm to about 28 mS / cm. Claim 32 The method according to any one of claims 27 to 31, wherein the elution solution contains NaCl at a concentration of from about 160 mM to about 240 mM. Claim 33 The method according to any one of claims 27 to 32, wherein the mixed-mode chromatography resin is contacted with at least 55 g of the anti-α4β7 antibody per liter of resin. Claim 34 The method according to claim 33, wherein the mixed-mode chromatography resin is contacted with from about 55 g to about 80 g of the anti-α4β7 antibody per liter of resin. Claim 35 The mixed-mode chromatography resin has a smaller bead diameter and has strong anion exchange, hydrogen bonding, and hydrophobic interactions, and optionally, the mixed-mode chromatography resin is Capto Adhere ImpRes. The method according to any one of claims 27 to 34. Claim 36 The method according to any one of claims 27 to 35, further comprising purifying the anti-α4β7 antibody using a cation exchange (CEX) resin. Claim 37 The method according to claim 36, wherein the CEX resin is operated in a bind / elute mode. Claim 38 A method for producing a composition comprising an anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, comprising: binding the anti-α4β7 antibody to a cation exchange (CEX) resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with the CEX resin; washing the CEX resin with a wash solution; eluting the anti-α4β7 antibody from the CEX resin by contacting the resin with an elution solution having a conductivity of 16 mS / cm or less to obtain a composition comprising the anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO:

2. The method according to claim 38, wherein the composition comprising the anti-α4β7 antibody comprises HMW aggregates of about 1% or less. Claim 39 The method according to claim 38. Claim 40 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The method according to claim 38 or 39, wherein the elution solution has a conductivity of 14 mS / cm or less. **Claim 41** The method according to claim 38 or 39, wherein the elution solution has a conductivity of about 11 to 16 mS / cm. **Claim 42** The method according to claim 38 or 39, wherein the elution solution has a conductivity of about 12 to 14 mS / cm. **Claim 43** The method according to any one of claims 38 to 42, wherein the elution solution contains NaCl at a concentration of about 70 mM to about 110 mM. **Claim 44** The method according to any one of claims 38 to 43, wherein the elution solution has a pH of about pH 5 to about pH 6. **Claim 45** The method according to claim 44, wherein the elution solution has a pH of about pH 5.1 to about pH 5.

8. **Claim 46** The method according to any one of claims 38 to 45, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 to 70 g of antibody per liter of resin. **Claim 47** The method according to any one of claims 38 to 46, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 to 60 g of antibody per liter of resin. **Claim 48** The method according to any one of claims 38 to 47, wherein the CEX resin is a strong CEX resin, and optionally, the CEX resin is Nuviar HR-S. **Claim 49** The method according to any one of claims 38 to 48, further comprising purifying the anti-α4β7 antibody using a mixed-mode chromatography resin. **Claim 50** The method according to claim 49, wherein the mixed-mode chromatography resin is operated in a binding / elution mode. **Claim 51** A method for generating a composition comprising an anti-α4β7 antibody from a liquid solution comprising the major isoform and one or more basic isoform species of the anti-α4β7 antibody, comprising: binding the anti-α4β7 antibody to a cation exchange (CEX) resin by contacting the liquid solution comprising the anti-α4β7 antibody and one or more basic isoform species with the CEX resin; washing the CEX resin with a washing solution; eluting the anti-α4β7 antibody from the CEX resin by contacting the resin with an elution solution having a conductivity of 11 mS / cm or more to obtain a composition comprising the anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO:

2. ​ ​

52. The method according to claim 51, wherein the composition containing the anti-α4β7 antibody contains from about 4% to about 20% of a basic isoform.

53. The method according to claim 51 or 52, wherein the elution solution has a conductivity of 12 mS / cm or more.

54. The method according to claim 51 or 52, wherein the elution solution has a conductivity of about 11 to 16 mS / cm.

55. The method according to claim 51 or 52, wherein the elution solution has a conductivity of about 12 to 14 mS / cm.

56. The method according to any one of claims 51 to 55, wherein the elution solution has a pH of about pH 5 to about pH 6.

57. The method according to claim 56, wherein the elution solution has a pH of about pH 5.1 to about pH 5.

8.

58. The method according to any one of claims 51 to 57, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 to 70 g of antibody per liter of resin.

59. The method according to claim 58, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 to 60 g of antibody per liter of resin.

60. The method according to any one of claims 51 to 59, wherein the CEX resin is Nuvi a HR-S.

61. The method according to any one of claims 51 to 60, further comprising purifying the anti-α4β7 antibody using a mixed-mode chromatography resin.

62. The method according to claim 61, wherein the mixed-mode chromatography resin is operated in a binding / elution mode.

63. The method according to any one of claims 1 to 62, wherein the antibody is produced in Chinese hamster ovary (CHO) cells.

64. The method according to claim 63, wherein the host cell is a GS-CHO cell.

65. The method according to any one of claims 1 to 64, wherein the obtained composition contains a purified anti-α4β7 antibody, and the method further comprises a step of formulating the anti-α4β7 antibody into a formulation suitable for use in humans.

66. The method according to any one of claims 1 to 65, comprising formulating the purified anti-α4β7 antibody into a dry lyophilized formulation.

67. The method according to claim 66, further comprising reconstituting the dry lyophilized formulation with a liquid so as to be suitable for administration.

68. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ formulating the purified anti-α4β7 antibody as a liquid formulation such that the anti-α4β7 antibody is suitable for administration by subcutaneous injection The method according to any one of claims 1 to 65, comprising: method. **Claim 69** The method according to any one of claims 1 to 68, wherein the anti-α4β7 antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO:

5. method. **Claim 70** The method according to any one of claims 1 to 68, wherein the anti-α4β7 antibody is vedolizumab. method. **Claim 71** A composition comprising an anti-α4β7 antibody, obtained by the method according to any one of claims 1 to 70. composition.

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