Antibody purification method and composition thereof

Abstract

Described herein are methods for purifying humanized α4β7 antibodies, such as vedolizumab, produced in mammalian cell culture, and compositions obtained from the purification process.

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 to U.S. Provisional Application No. 62 / 859,580, filed June 10, 2019, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 5, 2020, is named T103022_1120WO_SL.txt and is 10,015 bytes in size. [Background technology]

[0004] Large-scale, economical purification of proteins is becoming an increasingly important challenge in the biotechnology industry. Typically, biopharmaceuticals are produced by cell culture using prokaryotic cell lines, e.g., bacterial cells, or eukaryotic cell lines, e.g., mammalian or fungal cells, engineered to produce large quantities of the therapeutic protein of interest. Because the cell lines used are living organisms, they must be fed a complex cell culture medium containing sugars, amino acids, and, in some cases, growth factors provided by preparations of animal serum. Separating the desired recombinant therapeutic protein from process-related impurities, e.g., cell culture medium components, host cell proteins (HCPs), host nucleic acids, and / or chromatography materials, as well as product-related impurities, e.g., aggregates, misfolded species, or fragments of the protein of interest, to a purity sufficient for use as a human therapeutic has been a challenging task.

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

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

[0007] The present invention specifically provides methods for purifying anti-α4β7 antibodies, such as vedolizumab, for example, from a liquid solution.

[0008] In one aspect, the invention features a method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution containing the anti-α4β7 antibody and one or more impurities, the method comprising: contacting a matrix comprising Protein A with the liquid solution containing the anti-α4β7 antibody and one or more impurities to bind the anti-α4β7 antibody to Protein A; washing the matrix comprising Protein A with a wash solution; and 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; wherein the anti-α4β7 antibody is a humanized IgG1 antibody and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[0009] In one embodiment, a method is used to obtain a composition comprising less than 1% high molecular weight (HMW) aggregates 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 Protein A, washing the matrix comprising Protein A with a wash solution, and 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 less than 1% HMW aggregates.

[0010] In one embodiment, Protein A is immobilized on a solid phase, hi one embodiment, the solid phase comprises one or more of beads, a gel, and a resin.

[0011] In one embodiment, the wash solution has a pH of about 7. In one embodiment, the elution solution comprises citric acid.

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

[0013] In another aspect, the invention features a method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution containing the anti-α4β7 antibody and one or more impurities, the composition comprising the anti-α4β7 antibody being obtained by contacting the solution containing the anti-α4β7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow the anti-α4β7 antibody to flow through the HIC resin, wherein the HIC resin is characterized as a highly hydrophobic HIC resin, and 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.

[0014] In one embodiment, the method is for obtaining a composition comprising an anti-α4β7 antibody and less than 0.6% HMW aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities, the method comprising contacting the solution containing the anti-α4β7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow the anti-α4β7 antibody to flow through the HIC resin, thereby obtaining a composition comprising the anti-α4β7 antibody and less than 0.6% HMW aggregates, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[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 comprises about 0.35 mM to about 0.15 mM potassium phosphate.

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

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

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

[0019] In one embodiment, the highly hydrophobic HIC resin has an average pore size of 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 100 μm.

[0021] In another aspect, the invention features 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 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 wash 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 formulation comprising a purified anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises 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.

[0022] In one embodiment of the above aspect, a method is for obtaining a formulation comprising less than 1% high molecular weight (HMW) aggregates from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising 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 wash 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 pH 3.9 or greater, thereby obtaining a formulation comprising less than 1% HMW aggregates.

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

[0025] In some embodiments, the elution solution comprises 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.

[0027] 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 bind / elute mode.

[0028] In another aspect, the invention features 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 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 wash 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 purified anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises 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.

[0029] In some embodiments of the above aspects, a method is for obtaining a preparation comprising an anti-α4β7 antibody in high yield 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 mixed-mode chromatography resin to bind the anti-α4β7 antibody to the resin; washing the mixed-mode chromatography resin with a wash 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, thereby obtaining a preparation comprising an anti-α4β7 antibody in high yield.

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

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

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

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

[0034] In some embodiments of the above aspects, the mixed-mode chromatography resin is Capto 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 bind / elute mode.

[0036] In another aspect, the invention features a method for producing a preparation comprising an anti-α4β7 antibody from a liquid solution comprising the anti-α4β7 antibody and one or more impurities, the method comprising contacting the liquid solution comprising the anti-α4β7 antibody and one or more impurities with a cation exchange (CEX) resin to bind the anti-α4β7 antibody to the 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 16 mS / cm or less to obtain a preparation comprising a purified anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises 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.

[0037] In one embodiment of the above aspect, a method is for obtaining a preparation comprising a reduced level of HMW aggregates from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising: contacting a liquid solution comprising the anti-α4β7 antibody and one or more impurities with a CEX resin to bind the anti-α4β7 antibody to the 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 16 mS / cm or less, thereby obtaining a preparation comprising a reduced level of HMW aggregates.

[0038] In some embodiments of the above aspects, 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 yet other embodiments, the elution solution has a conductivity of about 12-14 mS / cm.

[0039] In some embodiments of the above aspects, the elution solution comprises NaCl at a concentration of about 70 mM to about 110 mM.

[0040] In some embodiments of the above aspects, 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.

[0041] In some embodiments of the above aspects, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25-70 g of antibody per L 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 L 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 bind / elute mode.

[0044] In another aspect, the invention features a method for producing a preparation comprising an anti-α4β7 antibody from a liquid solution containing the major isoform and one or more basic isoform species of the anti-α4β7 antibody, the method comprising contacting the liquid solution containing the anti-α4β7 antibody and one or more basic isoform species with a cation exchange (CEX) resin to bind the anti-α4β7 antibody to the 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 greater to obtain a preparation comprising a purified anti-α4β7 antibody, wherein the anti-α4β7 antibody comprises 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.

[0045] In one embodiment of the above aspect, a method is for obtaining a preparation containing a reduced level of a basic isoform species of an anti-α4β7 antibody from a liquid solution containing a major isoform and one or more basic isoform species of an anti-α4β7 antibody, the method comprising: 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 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 preparation containing a reduced level of basic isoform species.

[0046] In one embodiment, the purified composition comprises about 4% to about 20% basic isoform.

[0047] In some embodiments of the above aspects, the elution solution has a conductivity of 12 mS / cm or greater. 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.

[0048] In some embodiments of the above aspects, 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.

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

[0050] In some embodiments, the elution solution comprises sodium chloride, for example, 70-110 mM sodium chloride.

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

[0052] 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 bind / elute mode.

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

[0054] In one embodiment, the host cell is a GS-CHO cell.

[0055] In one embodiment of any of the above aspects, 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:5.

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

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

[0058] 1. A method for obtaining a composition comprising less than 1% HMW aggregates from a liquid solution comprising 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, thereby binding the anti-α4β7 antibody to Protein A; washing the matrix containing the Protein A with a wash solution; eluting the anti-α4β7 antibody from the Protein A-containing matrix by contacting the matrix with an elution solution having a pH of 3.2 to 4 to obtain a composition containing less than 1% HMW aggregates; The method, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[0059] 2. The method according to item 1, wherein the protein A is immobilized on a solid phase.

[0060] 3. The method according to item 2, wherein the solid phase comprises one or more of beads, a gel, and a resin.

[0061] 4. The method according to any one of items 1 to 3, wherein the pH of the washing solution is about 7.

[0062] 5. The method according to any one of items 1 to 4, wherein the elution solution comprises citric acid.

[0063] 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.

[0064] 7. A method for obtaining a composition comprising an anti-α4β7 antibody and less than 0.6% HMW aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities, comprising: contacting a solution containing an 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, thereby obtaining a composition comprising the anti-α4β7 antibody and less than 0.6% HMW aggregates; the HIC resin is characterized as a highly hydrophobic resin; The method, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6.

[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 about 0.35 mM to about 0.15 mM potassium phosphate.

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

[0068] 11. The method of 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 of any one of items 7 to 11, wherein the composition contains less than about 0.3 ppm host cell protein (HCP).

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

[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 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: 5.

[0075] 18. The method of any one of items 1 to 16, wherein the anti-α4β7 antibody is vedolizumab. [Brief explanation of the drawings]

[0076] [Figure 1] Aggregation as a function of pH in Protein A eluates. [Figure 2-1] 1 is a plot showing the results of predictive profiler assays screened for performance results. [Figure 2-2] 1 is a plot showing the results of predictive profiler assays screened for performance results. [Figure 2-3] 1 is a plot showing the results of predictive profiler assays screened for performance results. [Figure 3] 1 is a graph showing a comparison of vedolizumab with three other IgG antibodies and the pH profile for eluting each antibody from a cation exchange column. [Figure 4] 1 is a surface plot of a linear regression model showing step recovery of Capto Adhere ImpRes versus pH and conductivity of the elution buffer. [Figure 5] 1 is a surface plot of a linear regression model showing the step recovery of Capto Adhere ImpRes versus elution buffer pH and load amount. [Figure 6] 1 is a surface plot of a linear regression model showing the percentage of HMW species of Capto Adhere ImpRes versus the pH and conductivity of the elution buffer. [Figure 7] 1 is a surface plot of a linear regression model showing the percentage of HMW species of Capto Adhere ImpRes versus elution buffer pH and load amount. [Figure 8] 1 is a surface plot of HMW showing the effect of elution buffer pH and elution buffer conductivity on the percentage of HMW species. [Figure 9] 1 is a surface plot of HMW clearance showing the effect of elution buffer pH and elution buffer conductivity on HMW clearance. [Figure 10] 1 is a surface plot of monomers showing the effect of elution buffer pH and elution buffer conductivity on the percentage of monomer species. [Figure 11] 1 is an acidic surface plot showing the effect of elution buffer pH and elution buffer conductivity on the percentage of acidic isoform species. [Figure 12] 1 is a primary surface plot showing the effect of elution buffer pH and elution buffer conductivity on the percentage of the major isoform species. [Figure 13] 1 is a basic surface plot showing the effect of elution buffer pH and elution buffer conductivity on the percentage of basic isoform species. DETAILED DESCRIPTION OF THE INVENTION

[0077] The present invention particularly relates to purification methods for controlling the amount of product-related materials (e.g., aggregates such as high molecular weight (HMW) aggregates, misfolded species, or protein fragments) and / or process-related impurities (e.g., host cell proteins (HCPs), host cell nucleic acids, viruses, chromatography materials, and / or media components) present in purified preparations of anti-α4β7 antibodies or antigen-binding fragments thereof, such as vedolizumab.

[0078] I. Definition In order that the present invention may be more readily understood, certain terms are first defined.

[0079] The cell surface molecule "α4β7 integrin," or "α4β7" (used interchangeably throughout this specification), is a heterodimer of the α4 chain (CD49D, ITGA4) and the 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. Typical of many integrins, α4β7 can exist in either a resting or activated state. Ligands for α4β7 include vascular cell adhesion molecule (VCAM), fibronectin, and mucosal addressin (MAdCAM, e.g., MAdCAM-1). Antibodies that bind to α4β7 integrin are referred to herein as "anti-α4β7 antibodies."

[0080] As used herein, an antibody or antibody-binding fragment that has "binding specificity for the α4β7 complex" binds to α4β7 but also binds to α4β1 and α E It also does not bind to β7. Vedolizumab is an example of an antibody with binding specificity for the α4β7 complex.

[0081] The term "antibody" as used herein refers to an immunoglobulin molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. 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) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody has a "fragment crystallizable" (Fc) region. In certain embodiments, the antibody is of the IgG1 isotype and has a kappa light chain.

[0082] "CDRs" or "complementarity determining regions" are regions of hypervariability interspersed within more conserved regions called framework regions (FR).

[0083] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to Fab, Fab', F(ab')2, and Fv fragments, single-chain antibodies, functional heavy-chain antibodies (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 the complementarity-determining regions having sufficient framework sequence to specifically bind to the epitope). Antigen-binding fragments can be produced by recombinant techniques or by enzymatic or chemical cleavage of antibodies.

[0084] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and functionality. In some cases, framework region (FR) residues of the humanized antibody are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or nearly all of the hypervariable CDR loops correspond to those of a non-human antibody and all or nearly all of the FRs are from human antibody sequences. Humanized antibodies optionally further comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Struct. Biol. 2:593-596 (1992).

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

[0086] The term "recombinant host cell" (used interchangeably herein with the term "host cell") includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Furthermore, it should be understood that unless otherwise specified, when the term "cell" is used, e.g., host cell or mammalian cell or mammalian host cell, it 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 linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0088] The term "upstream processing" as used herein in reference to a protein refers to activities that result in the production and recovery of a protein (e.g., an antibody) from a host cell (e.g., in cell culture to produce a protein of interest such as an antibody).

[0089] As used herein, the term "downstream processing" refers to one or more techniques used after upstream processing to purify a protein, such as, for example, an antibody of interest. For example, downstream processing techniques include purification of protein products using affinity chromatography, including, for example, 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 displacement chromatography.

[0090] As used herein, the terms "cultivation" and "cell culture" generally refer to a process in which cells are grown under controlled conditions, generally outside their native environment. "Culturing" cells refers to contacting the cells with a cell culture medium under conditions suitable for the survival and / or growth and / or proliferation of the cells. In certain embodiments, cell culture refers to methods and techniques 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 producing and recovering the protein of interest. For example, after an expression vector is introduced into a suitable host, e.g., host cells in culture, the host can be maintained under conditions suitable for the expression of the relevant nucleotide coding sequence and the recovery and purification of the desired recombinant protein. "Cell culture" can also refer to a solution containing cells.

[0091] 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 cellular debris and particulate impurities, from the material. After cell culture, the harvest is typically purified to remove cells and cellular debris using separation techniques such as centrifugation and filtration. The initial clarification, particle removal step results in 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.

[0092] As used herein, "chromatographic support" refers to a solid or porous matrix having a specific chemical composition or a specific three-dimensional structure, or onto which specific chemical groups or macromolecules can be immobilized for chromatography, including affinity chromatography, gel filtration (size exclusion chromatography), or ion exchange chromatography. Examples of chromatographic supports include, but are not limited to, resins (e.g., agarose) or membranes. As used herein, "chromatographic housing" refers to a structure that houses a chromatographic support. Examples of chromatographic housings include columns or cartridges or other containers.

[0093] As used herein, a "buffer" refers to an aqueous solution that resists changes in pH by the action of its acid-base conjugate components. A "buffer" is used to establish a specific set of conditions that regulate the process step or control of a chromatographic support, such as a chromatographic resin or membrane.

[0094] As used herein, the term "equilibration solution" refers to an aqueous liquid formulated to establish the initial operating conditions of a process step or chromatographic support, e.g., a chromatographic run. The equilibration solution is used, e.g., to prepare a solid phase, e.g., a chromatographic support, e.g., a resin or membrane, for loading with a protein of interest, e.g., an antibody.

[0095] As used herein, the term "wash fluid" or "wash solution" refers to an aqueous liquid formulated to displace unbound contaminants from a chromatographic support, such as a resin or membrane. In some embodiments, the wash fluid is passed through a solid support, e.g., a resin or membrane, after loading a protein of interest, e.g., an antibody, and before eluting the protein of interest, e.g., an antibody. In one embodiment, the wash fluid has similar biochemical properties as the equilibration solution.

[0096] As used herein, "flow-through operation" refers to a process in which proteins do not substantially bind to and / or elute from a matrix, e.g., a hydrophobic chromatography resin, during washing, while impurities remain bound to the chromatography support.

[0097] As used herein, the term "elution solution" or "eluate" refers to an aqueous liquid formulated to displace a protein of interest, e.g., an antibody, from a chromatographic support, e.g., a resin or membrane. In one embodiment, the elution solution has different biochemical properties than the equilibration solution and / or wash solution, such that the protein of interest, e.g., an antibody, is more likely to associate with the elution solution than with the chromatographic support, e.g., a resin or membrane.

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

[0099] As used herein, the term "process-related impurity" refers to an impurity (or impurities) that is present in a composition, e.g., a solution, containing a protein, but that is not derived from the protein itself. For example, process-related impurities include, but are not limited to, cell culture medium components, host cell components (e.g., proteins (HCPs), host cell nucleic acids, or lipid-containing intracellular structures or fragments thereof), viruses, trace metals or ions from buffers, and leachable substances from material handling vessels or chromatographic supports. Process-related impurities can be generated during the preparation (upstream and / or downstream processes) of a protein, e.g., an antibody.

[0100] As used herein, the term "host cell impurities" refers to any proteinaceous, nucleic acid, or lipid contaminants or by-products introduced by the host cell line, cell culture medium, or cell culture. Examples of impurities include, but are not limited to, Chinese hamster ovary protein (CHOP), E. coli proteins, enzyme proteins, monkey COS proteins, or myeloma cell proteins (e.g., NS0 protein (a murine plasmacytoma derived from BALB / c mice)).

[0101] As used herein, the term "product-related impurities" includes impurities derived from the protein of interest, e.g., an 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] As used herein, the term "aggregate" or "aggregates" refers to the association of multiple antibodies or antibody fragments. For example, aggregates can be dimers, trimers, tetramers, or multimers larger than tetramers of antibodies and / or antibody fragments. Antibody aggregates can be soluble or insoluble. The bonds between the aggregated molecules can be covalent or noncovalent, regardless of the mechanism by which the molecules bind. The bonds can be direct between the aggregated molecules or indirect through other molecules that link the molecules together. 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 binding with multiple components. Aggregates can form irreversibly during protein expression in cell culture, during downstream protein purification, or during storage of pharmaceutical products. The presence of aggregates in solution can be determined, for example, using size exclusion chromatography (SEC) (e.g., SEC with UV detection, SEC with light scattering detection (SEC-LSD)), field-flow fractionation, sedimentation velocity in analytical ultracentrifugation, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced).

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

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

[0105] The term "about" means that the value that follows is not the exact value, but is the center 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 value that follows is not the exact value, but is the center of a range that is ±5% of that value, and the upper limit of the range cannot exceed the 100% value.

[0106] II. Methods and Compositions Related to Antibody Purification Provided herein are methods for purifying anti-α4β7 antibodies, such as vedolizumab, from, for example, a liquid solution, e.g., from a clarified harvest of a mammalian cell culture. The invention is based, at least in part, on certain aspects of an antibody purification process that reduces the levels of impurities present in an antibody solution, including process-related impurities, such as cell culture media components, host cell proteins (HCPs), host cell nucleic acids, viruses, and chromatography materials, as well as product-related impurities, such as aggregates (including HMW aggregates), misfolded species, or fragments of the protein of interest. The methods of the invention are useful for purifying anti-α4β7 antibodies, particularly vedolizumab or antibodies having the binding region (i.e., CDRs or variable region) of vedolizumab, so that the antibodies can be formulated for use in human patients.

[0107] In particular, the methods disclosed herein are useful for obtaining low levels of antibody aggregation, e.g., HMW antibody aggregates. In certain embodiments, the methods disclosed herein provide for a low level of antibody aggregation, e.g., HMW antibody aggregates, of about 0% to 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%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8 In some embodiments, compositions are provided having aggregates, e.g., HMW aggregates, of 0.5%, 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%. In certain embodiments, the methods disclosed herein provide compositions having 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., HMW aggregates. The invention also includes compositions comprising an anti-α4β7 antibody and such low levels of HMW aggregates. Specifically, the methods disclosed herein can be used to produce the anti-α4β7 antibody vedolizumab, or antibodies having the antigen-binding region of vedolizumab. Vedolizumab is also known by the trade name ENTYVIO® (Takeda Pharmaceutical Company). Vedolizumab is a humanized antibody containing the framework and constant regions of human IgG1 and the antigen-binding CDRs derived from the murine antibody Act-1. The CDRs, variable region, and mutated Fc region (mutated to eliminate Fc effector function) of vedolizumab are described in U.S. Patent No. 7,147,851, the entire contents of which are incorporated herein by reference.

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

[0109] The 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 primarily expressed on intestinal endothelial cells and plays a key role in homing T lymphocytes to intestinal lymphoid tissues. The interaction between α4β7 integrin and MAdCAM-1 has been implicated as an important contributor to mucosal inflammation, such as the chronic inflammation characteristic of ulcerative colitis and Crohn's disease. Vedolizumab can be used to treat inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, pouchitis, including chronic pouchitis, graft-versus-host disease, and HIV.

[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 comprises a heavy chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4. Vedolizumab comprises a light chain variable region comprising CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 8. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. Vedolizumab and vedolizumab sequences are also described in U.S. Patent Application Publication Nos. 2014 / 0341885 and 2014-0377251, both of which are incorporated herein by reference in their entireties. The methods disclosed herein can be practiced using antibodies comprising binding regions (e.g., CDRs or variable regions) described above and in the accompanying sequence listing.

[0111] Methods for producing 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 for producing monoclonal antibodies 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.

[0112] When using cell culture methods, the anti-α4β7 antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In embodiments in which the anti-α4β7 antibody is produced intracellularly, particulate debris from 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 is first concentrated using a commercially available protein concentration filter.

[0113] The medium or lysate is subjected to one or more processing steps, such as precipitation, flocculation, centrifugation, and / or filtration, to remove particulate cell debris to form a clarified cell culture supernatant, or clarified harvest. The antibody, e.g., an anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab), is then purified as described in detail below to remove impurities (e.g., process-related impurities such as cell medium components, host cell proteins (HCPs), host cell nucleic acids, viruses, and chromatographic materials, and product-related impurities such as aggregates (including HMW aggregates), misfolded species, or fragments of the protein of interest).

[0114] The purification process can begin after the antibody has been produced using the upstream manufacturing methods described above and / or by other conventional manufacturing methods. After a clarified solution or mixture containing the antibody is obtained, the antibody of interest is separated from process-related impurities, such as other proteins produced by the cells, as well as product-related impurities. In certain non-limiting embodiments, such separation is performed using CEX, AEX, and / or MM chromatography. In certain embodiments, a combination of one or more different purification methods can be used, including affinity separation step(s), ion exchange separation step(s), mixed-mode separation step(s), and / or hydrophobic interaction separation step(s). Such further purification steps separate the antibody mixture based on the charge, hydrophobicity, and / or size of the antibodies. In one aspect of the invention, such further separation steps are performed using chromatography involving hydrophobic, anionic, or cationic interactions (or a combination thereof). Many chromatography resins are commercially available for each of these methods, allowing the purification scheme to be precisely tailored to the particular antibody of interest. Each of these separation methods involves flowing the antibodies through a column at different rates, achieving increasing physical separation as the antibodies pass further through the column, or allowing them to selectively adhere to the separation resin (or medium). The antibodies are then differentially eluted using different eluents. In some cases, the antibody of interest is separated from the impurities when the impurities specifically bind to the column resin and the antibody of interest does not (i.e., the antibody of interest is contained in the flow-through); in other cases, the antibody of interest adheres to the column resin, and the impurities and / or product-related materials are eluted from the column resin during a wash cycle, after which the antibody is released by changing the liquid surrounding the resin and the antibody of interest is eluted from the column.

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

[0116] In certain embodiments, the affinity chromatography step described herein involves subjecting the clarified collection containing anti-α4β7 antibodies to a Protein A matrix (e.g., a chromatography column containing Protein A resin). In certain embodiments, Protein A is useful for affinity purification and isolation of various antibody isotypes, particularly IgG1, IgG2, and IgG4. Protein A is a bacterial cell wall protein that binds mammalian IgG primarily via its Fc region. In its native state, Protein A has five IgG-binding domains and other domains of unknown function.

[0117] Purification of anti-α4β7 antibodies using Protein A resin In one aspect, the methods described herein involve purifying an anti-α4β7 antibody (e.g., vedolizumab) from a liquid solution, e.g., a clarified harvest, containing the antibody and one or more impurities using Protein A. 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 may be approximately 10g / L, 11g / L, 12g / L, 13g / L, 14g / L, 15g / L, 16g / L, 17g / L, 18g / L, 19g / L, 20g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L , 33g / L, 34g / L, 35g / L, 36g / L, 37g / L, 38g / L, 39g / L, 40g / L, 41g / L, 42g / L, 43g / L, 44g / L, 45g / L, 46g / L, 47g / L, 48g / L, 49g / L, or 50g / L can be loaded onto a Protein A affinity chromatography matrix.

[0118] Protein A resins are commercially available from several sources. One suitable resin is MabSelect™, sold by GE Healthcare. Suitable resins include, but are not limited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelect Xtra, rProtein A Sepharose, and MabSelect™ ProA resin, all sold by GE Healthcare; ProSep HC, ProSep Ultra, and ProSep Ultra Plus, sold by EMD Millipore; and MabCapture, sold by Life Technologies.

[0119] The Protein A column can be equilibrated with an appropriate equilibration solution prior to loading the sample. After loading the column, the column can be washed one or more times with an appropriate set of solutions to reduce one or more impurities, while the anti-α4β7 antibody remains bound to the 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 comprises phosphate. In one embodiment, the affinity column can be washed with a first wash solution comprising PBS, followed by a second wash solution comprising NaCl and PBS, and then a third wash solution comprising PBS. In one embodiment, the first and third wash solutions are the same. In one embodiment, the second wash solution comprises 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 wash solutions is about 7.0-7.4. In one embodiment, the pH of one or more of the wash solutions is about 7.2.

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

[0122] The Protein A column can then be eluted using an appropriate 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 a citric acid, e.g., sodium citrate, elution solution. In some embodiments, the elution solution can have a pH of about 3.0-4.0 (e.g., about 3.1-4.0, 3.2-4.0, 3.3-4.0, 3.4-4.0, 3.5-4.0, 3.6-4.0, 3.7-4.0, 3.8-4.0, or 3.9-4.0). In certain embodiments, the elution solution has a pH of about 3.0-3.4. 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 the eluate fraction of interest is collected, it can be prepared for further processing.

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

[0124] The buffers and methods described herein can reduce the level of host cell proteins (HCPs) in a composition, such as a composition comprising an anti-α4β7 antibody, eluted from Protein A compared to the level of HCPs when an elution buffer is used that does not have one or more of the parameters described herein. In some embodiments, the eluate of the Protein A resin comprises a composition comprising an anti-α4β7 antibody and less than about 250 ppm (e.g., less than about 240 ppm, 230 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, 40 ppm, 30 ppm, 20 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2 ppm, or 1 ppm) HCP. In some embodiments, the eluate from 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, 1-1 and a composition comprising: an HCP at a concentration of 1 to 20 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 ppm, 1 to 5 ppm, 1 to 4 ppm, 1 to 3 ppm, or 1 to 2 ppm.

[0125] In one embodiment, the anti-α4β7 antibody bound to Protein A is eluted with an elution buffer having a pH higher than 3.3 (e.g., pH 3.3-4.0, pH 3.4-4.0, pH 3.5-4.0, pH 3.6-4.0, pH 3.7-4.0, pH 3.8-4.0, or pH 3.9-4.0) 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 HCPs. In one embodiment, the elution solution has a pH of 3.3-3.9. In one embodiment, the elution buffer has a pH of 3.3-3.8. In one embodiment, the pH of the elution buffer is 3.4-3.6. In one embodiment, the pH of the elution buffer is 3.4-4.0. In one embodiment, the elution buffer contains citric acid, e.g., 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, the wash buffer having a pH of 5.6-6.2, 5.7-5.9, or 5.8, and the elution buffer having 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 deleted in the gene encoding glutamine synthetase. In some embodiments, the CHO cells can be DHFR-CHO cells deleted in the gene encoding dihydrofolate reductase.

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

[0128] Purification of anti-α4β7 antibodies using HIC Antibodies, e.g., anti-α4β7 antibodies (e.g., vedolizumab or antibodies having a binding region corresponding to vedolizumab), can also be purified using downstream processing techniques after purification with Protein A, as described in detail below and in Example 2. Purification steps performed later in the downstream process, often referred to as "polishing" steps, present unique challenges in that while impurity levels can be relatively low, even lower levels are desirable given the nature of antibodies intended for human use.

[0129] In one aspect, the methods described herein involve the purification of anti-α4β7 antibodies from a liquid solution, e.g., a clarified harvest, containing the antibody and one or more impurities using a hydrophobic interaction chromatography (HIC) resin.

[0130] In one embodiment, the present invention provides a method for reducing high molecular weight (HMW) aggregates from an anti-α4β7 antibody solution, comprising contacting the antibody solution with a hydrophobic interaction chromatography (HIC) resin. Hydrophobic interaction chromatography (HIC) separates proteins based on differences in their surface hydrophobicity by utilizing reversible interactions between the proteins and the hydrophobic surface of an HIC resin (e.g., a polymer matrix modified with hydrophobic ligands). Given the hydrophobic nature of anti-α4β7 antibodies, such as vedolizumab, using a highly hydrophobic HIC resin in the purification process can remove HMW aggregates, residual protein A, and / or host cell protein (HCP) contaminants, while the anti-α4β7 antibody flows through the HIC resin without binding. In some embodiments, a highly hydrophobic HIC resin suitable for use in the methods described herein comprises a polymethacrylate-based material coupled to C6 groups, such as Toyopearl Hexyl-650C (Tosoh Biosciences).

[0131] In some embodiments, HIC is used in "flow-through mode." Thus, as used herein, "flow-through fraction" refers to proteins in the mobile phase buffer that are collected in fractions that pass through a column containing a resin provided herein.

[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 includes flowing an anti-α4β7 antibody-containing solution through the column, i.e., the purification includes recovering the anti-α4β7 antibody in the column flow-through, while contaminants remain bound to the column, and the anti-α4β7 antibody and column are in a solution containing phosphate, e.g., potassium phosphate, at a concentration of 150-300 mM, 175-250 mM, or about 200 mM, at a pH of 5.2-6.5, 5.7-6.2, or about 5.9.

[0134] In some embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions comprising an anti-α4β7 antibody and about 0% to 2.0% (e.g., less than 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 less than 2%) HMW aggregates. In some embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions comprising an 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) HMW aggregates. In certain embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions 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 methods using highly hydrophobic HIC resins can be used to obtain compositions 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. Additionally, the composition can contain less than about 0.3 ppm host cell protein (HCP), where the host cells are Chinese hamster ovary (CHO) cells, such as GS-CHO cells. In one embodiment, the composition contains less than about 0.22 ppm residual protein A.

[0135] Purification of anti-α4β7 antibodies using mixed-mode resin In one aspect, provided herein are methods for purifying an anti-α4β7 antibody, e.g., vedolizumab, from a liquid solution containing the antibody and one or more impurities, e.g., a clarified cell culture harvest, using a mixed-mode chromatography resin in bind / elute mode. In some embodiments, the mixed-mode chromatography resin has properties suitable for high impurity rejection and high capacity. In one embodiment, the mixed-mode chromatography resin for purifying an anti-α4β7 antibody has strong anion exchange, hydrogen bonding, and hydrophobic binding capabilities. In another embodiment, the mixed-mode chromatography resin for purifying an anti-α4β7 antibody has strong anion exchange, hydrogen bonding, and hydrophobic binding capabilities on smaller beads, e.g., beads with a diameter of about 35-45 μm. In certain embodiments, the mixed-mode chromatography resin used in the methods and compositions described herein is CAPTO™ Adhere ImpRes (GE Healthcare 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 Healthcare Life Sciences, now Global Life Sciences Solutions, LLC). The clarified cell culture harvest can be obtained from host cells recombinantly expressing anti-α4β7 antibodies. 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 involve binding an anti-α4β7 antibody to a mixed-mode chromatography resin. Additional 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 mixed-mode chromatography can include Protein A eluate, AEX eluate, CEX eluate, or HIC eluate or collected HIC flow-through material. In some embodiments, the mixed-mode chromatography methods provided herein can further include washing the mixed-mode resin with a wash 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, 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) of antibody solution can be loaded onto the mixed-mode chromatography resin. For example, at least 55 g / L of antibody solution can be loaded onto the mixed-mode chromatography resin. In certain embodiments, an antibody solution of about 25 g / L to about 100 g / L, e.g., about 25 g / L to about 95 g / L, about 25 g / L to about 90 g / L, about 25 g / L to about 85 g / L, about 25 g / L to about 80 g / L (e.g., about 30 g / L to about 80 g / L, about 35 g / L to about 80 g / L, about 40 g / L to about 80 g / L, about 45 g / L to about 80 g / L, about 50 g / L to about 80 g / L, about 55 g / L to about 80 g / L, about 60 g / L to about 80 g / L, about 65 g / L to about 80 g / L, about 70 g / L to about 80 g / L, or about 75 g / L to about 80 g / L) can be loaded onto the mixed-mode chromatography resin. For example, about 55 g / L to about 80 g / L of antibody solution 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 bound antibody from 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 comprise 10 mM sodium phosphate, 25 mM sodium phosphate, 50 mM sodium phosphate, or 75 mM sodium phosphate, with a neutral pH or near neutral (e.g., pH 6-8). Other suitable wash buffers compatible with mixed-mode chromatography are widely available.

[0139] Described herein are buffers that increase the yield of anti-α4β7 antibody and / or reduce the level of aggregates (e.g., HMW species (%)) in a formulation of anti-α4β7 antibody after elution from a mixed-mode chromatography resin. The pH and / or conductivity of the mixed-mode elution buffer can be adjusted to increase the yield of anti-α4β7 antibody and / or reduce the level of aggregates (e.g., HMW species (%)). Suitable elution solutions compatible with mixed-mode chromatography are widely available. In some embodiments, the mixed-mode chromatography elution solution comprises a buffer such as citrate, acetate, or phosphate.

[0140] In some embodiments, an elution buffer for use with a mixed-mode chromatography resin in the methods described herein has a pH of 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, an elution buffer for use with a mixed-mode chromatography resin can have a pH of 3.9 or higher. In certain embodiments, an elution buffer for use with a mixed-mode chromatography resin in the methods described herein has a pH of about pH 3.9 to about pH 4.5 (e.g., a pH of 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). In some embodiments, an elution buffer for use in the mixed-mode chromatography methods provided herein can have a pH of about pH 3.9 to about pH 4.4.

[0141] In additional or alternative embodiments, elution buffers for use with mixed-mode chromatography resins in the methods described herein have a pH of 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, elution buffers for use with mixed-mode chromatography resins can have a pH of 4.2 or lower. In certain embodiments, an elution buffer for use with a mixed-mode chromatography resin in the methods described herein has a pH of about pH 4.2 to about pH 3.5 (e.g., about pH 4.2 to about pH 3.6, about pH 4.2 to about pH 3.7, about pH 4.2 to about pH 3.8, about pH 4.2 to about pH 3.9, about pH 4.2 to about pH 4.0, or about pH 4.2 to about pH 4.1). For example, an elution buffer for use with a mixed-mode chromatography resin can have a pH of about pH 4.2 to about pH 3.8.

[0142] In some embodiments, the elution buffer for use with the mixed-mode chromatography resin in the methods described herein has a solubility 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, The elution buffer for use with the mixed-mode chromatography resin may have a conductivity of about 30 mS / cm or less. In certain embodiments, the elution buffer for use with the mixed-mode chromatography resin in the methods described herein has a conductivity of about 10 mS / cm to about 40 mS / cm, e.g., about 15 mS / cm to about 35 mS / cm or about 20 mS / cm to about 30 mS / cm. For example, an elution buffer for use with a mixed-mode chromatography resin can have a conductivity of about 20 mS / cm to about 30 mS / cm.

[0143] In additional or alternative embodiments, elution buffers for use with mixed-mode chromatography resins in the methods described herein 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 10 mS / cm or less). For example, elution buffers for use with mixed-mode chromatography resins can have a conductivity of 28 mS / cm or less. In certain embodiments, the elution buffer for use with the mixed-mode chromatography resin in the methods described herein has a saturation of about 10 mS / cm to about 40 mS / cm (e.g., about 15 mS / cm to about 35 mS / cm, about 18 mS / cm to about 35 mS / cm, about 11 mS / cm to about 30 mS / cm, about 12 mS / cm to about 30 mS / cm, about 13 mS / cm to about 30 mS / cm, about 14 mS / cm to about 30 mS / cm, about 15 mS / cm to about 30 mS / cm, about 16 mS / cm to about The elution buffer may have a conductivity of about 30 mS / cm, about 17 mS / cm to about 30 mS / cm, about 18 mS / cm to about 30 mS / cm, about 19 mS / cm to about 30 mS / cm, about 20 mS / cm to about 30 mS / cm, about 21 mS / cm to about 30 mS / cm, about 22 mS / cm to about 30 mS / cm, about 23 mS / cm to about 30 mS / cm, about 24 mS / cm to about 30 mS / cm, about 25 mS / cm to about 30 mS / cm, about 26 mS / cm to about 30 mS / cm, or about 27 mS / cm to about 30 mS / cm. For example, in some embodiments, an elution buffer for use with a mixed-mode chromatography resin can have a conductivity of about 18 mS / cm to about 28 mS / cm.

[0144] In some embodiments, an elution buffer for use with a mixed-mode chromatography resin in the methods described herein can include an ionic salt, such as NaCl, at a concentration of about 100-300 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, an elution buffer for use with a mixed-mode chromatography resin can have a concentration of NaCl of about 160 mM to about 240 mM. In certain embodiments, an elution buffer for use with a mixed-mode chromatography resin in the methods described herein has a concentration of NaCl of about 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 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 mM, 290 mM, or 300 mM.

[0145] In some embodiments, a method for purifying an anti-α4β7 antibody, e.g., 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, e.g., a sodium citrate buffer, at a pH of 3.5-4.5, 3.9-4.4, or about 4.1. In some embodiments, the method further includes including an ionic salt, e.g., NaCl, such that the conductivity of the elution buffer is 15-35, 20-30, or about 24 mS / cm. In some embodiments, the method includes loading the antibody onto the column containing the mixed-mode resin at a concentration of 53-77 g of protein per liter of resin. In some embodiments, the method comprises eluting the antibody from the column with an elution buffer having a pH of about 3.9 to 4.4 and a conductivity of about 20 to 28 mS / cm.

[0146] In some embodiments, purification of anti-α4β7 antibodies can be achieved using the mixed-mode chromatography methods described herein in combination with cation exchange (CEX) chromatography.

[0147] In some embodiments, the methods described herein can improve the yield of anti-α4β7 antibody eluted from a mixed-mode chromatography column compared to the yield of a suitable control process using an elution buffer lacking one or more of the parameters described herein, e.g., 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 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% or more higher. In some embodiments, the buffers and methods described herein can result in a recovery of 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of anti-α4β7 antibody eluted from a mixed-mode chromatography column. In some embodiments, the buffers and methods described herein can result in a recovery of 50%-95% (e.g., 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, 90-95%, or more) of anti-α4β7 antibody eluted from a mixed-mode chromatography column.

[0148] In some embodiments, such compositions and methods using mixed-mode chromatography resins can be used to obtain compositions comprising an 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%, less than 1.9%, or 2%) HMW aggregates. In some embodiments, such methods using mixed-mode chromatography resins can be used to obtain compositions comprising an 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) HMW aggregates. In certain embodiments, such methods using mixed-mode chromatography resins can be used to obtain compositions 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%, 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 aggregates. In other embodiments, the level of HMW aggregates 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 relative to the level of HMW aggregates in the load material.In some embodiments, the mixed-mode chromatography methods described herein can be used to reduce the level of HMW aggregates in a composition comprising an anti-α4β7 antibody compared to the level of HMW aggregates obtained from an appropriate control process using an elution buffer that does not have one or more of the parameters described herein, e.g., 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 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 compared to a suitable control.

[0149] Purification of anti-α4β7 antibodies using cation exchange (CEX) resin In one aspect, provided herein are methods for purifying an anti-α4β7 antibody, e.g., vedolizumab, from a liquid solution containing the antibody and one or more impurities, e.g., a clarified cell culture harvest, using a cation exchange (CEX) resin in bind / elute mode. In some embodiments, the CEX resin for purifying the anti-α4β7 antibody is a strong cation exchange resin. In certain embodiments, the CEX resin adapted for use in the methods and compositions described herein is a strong cation exchange resin. - The CEX resin contains a 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 recombinantly expressing an 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.

[0150] The CEX methods provided herein involve binding an anti-α4β7 antibody to a cation exchange chromatography resin. Additional 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 methods described herein. Thus, in some embodiments, the load material used in CEX chromatography can include a Protein A eluate, an AEX eluate, a mixed-mode eluate, or an HIC eluate. In some embodiments, the CEX methods provided herein can further include washing the CEX resin with a wash solution and eluting the antibody from the resin. Suitable solutions for loading, washing, and eluting proteins compatible with CEX chromatography, such as anti-α4β7 antibodies, are widely available. In some embodiments, the CEX chromatography solution includes a buffer such as citrate, acetate, or phosphate.

[0151] In certain embodiments, 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) of antibody solution can be loaded onto the CEX resin. For example, at least 25 g / L of antibody solution can be loaded onto the CEX resin. In certain embodiments, 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) of antibody solution can be loaded onto the CEX resin. In certain embodiments, the CEX resin can be loaded with 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) of antibody solution. For example, about 30-60 g / L of antibody solution 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 bound antibody 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 a 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 5-7, such as 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 widely available.

[0153] The pH and / or conductivity of the elution buffer can be adjusted to control the level of HMW aggregates, the level of major (predominant) isoform species, the level of acidic isoform species, and / or the level of basic isoform species in the anti-α4β7 antibody preparation eluted from the CEX resin. In some embodiments, the elution buffer for use with the CEX resin in the methods described herein has a pH of 6.0 or less (e.g., 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 pH 6.0 or less). In certain embodiments, an elution buffer for use with a CEX resin in the methods described herein has a pH of about pH 4.5 to about pH 6.0 (e.g., about pH 4.5 to about pH 5.8, about pH 4.9 to about pH 5.9, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.9, about pH 5.0 to about pH 5.8, about pH 5.0 to about pH 5.7, about pH 5.0 to about pH 5.6, or about pH 5.0 to about pH 5.5). For example, an elution buffer for use with a CEX resin can have a pH of about pH 5.1 to about pH 5.8. In some embodiments, the pH of the CEX elution buffer is the same as the wash buffer.

[0154] In additional or alternative embodiments, elution buffers for use with CEX resins in the methods described herein have 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, elution buffers for use with CEX resins can have a conductivity of 16 mS / cm or less. In some embodiments, an elution buffer for use with a CEX resin in the methods described herein has a conductivity of about 10 mS / cm to about 20 mS / cm (e.g., about 10 mS / cm to about 19 mS / cm, about 10 mS / cm to about 18 mS / cm, about 10 mS / cm to about 17 mS / cm, about 10 mS / cm to about 16 mS / cm, about 10 mS / cm to about 15 mS / cm, about 10 mS / cm to about 14 mS / cm, about 10 mS / cm to about 13 mS / cm, or about 10 mS / cm to about 12 mS / cm). In some embodiments, an elution buffer for use with a CEX resin can have a conductivity of about 11 mS / cm to about 16 mS / cm. Additionally or alternatively, an elution buffer for use with a CEX resin can have a conductivity of 14 mS / cm or less. In certain embodiments, an elution buffer for use with a CEX resin in the methods described herein has a conductivity of about 11 mS / cm to about 14 mS / cm, e.g., about 12 mS / cm to about 14 mS / cm or about 13 mS / cm to about 14 mS / cm. For example, an elution buffer for use with a CEX resin can have a conductivity of about 12 mS / cm to about 14 mS / cm. Additionally or alternatively, an elution buffer for use with a CEX resin can have a conductivity of 11 mS / cm or greater (e.g., 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm, 17 mS / cm, 18 mS / cm, 19 mS / cm, or 20 mS / cm or greater). For example, an elution buffer for use with a CEX resin can have a conductivity of 12 mS / cm or greater in some embodiments.

[0155] In some embodiments, an elution buffer for use with a CEX resin in the methods described herein can have an NaCl 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, an elution buffer for use with a CEX resin can have an NaCl concentration of about 90 mM to about 120 mM. In certain embodiments, an elution buffer for use with a CEX resin in the methods described herein has an NaCl concentration of about 50 to 150 mM (e.g., 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, an elution buffer for use with a CEX resin in the methods described herein has an NaCl concentration of about 70-120 mM (e.g., about 70-110 mM, 70-100 mM, 70-90 mM, or 70-80 mM). For example, an elution buffer for use with a CEX resin can have an NaCl concentration of about 70 mM to about 1100 mM.

[0156] In some embodiments, a method for purifying an anti-α4β7 antibody, e.g., vedolizumab, from a liquid solution, e.g., a clarified cell culture harvest, using a CEX resin includes loading the anti-α4β7 antibody onto a column containing a CEX resin at a concentration of 40-90, 50-65, or about 57 g of protein per L of resin, washing the column, and eluting the column with a buffer, e.g., a sodium acetate buffer, at a pH of 5-6, 5.2-5.6, or about 5.4. In some embodiments, the method further includes including an ionic salt, e.g., NaCl, such that the conductivity of the elution buffer is 5-25, 10-17, or about 13 mS / cm. In other embodiments, a method for purifying an anti-α4β7 antibody, e.g., vedolizumab, from a liquid solution, e.g., a clarified cell culture harvest, using a CEX resin, e.g., a strong cation exchange resin, includes eluting the column with a buffer having a pH of 5-6, 5.2-5.6, or about 5.4 and a conductivity of 5-25, 10-15, or about 13 mS / cm, e.g., a sodium acetate buffer. In one embodiment, the method includes eluting the column 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 the antibody at about 57 g of protein per L of resin.

[0157] In some embodiments, purification of anti-α4β7 antibodies can be achieved using the CEX resins described herein in combination with mixed-mode chromatography.

[0158] In some embodiments, the CEX methods described herein can be used to obtain a composition comprising an 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. In some embodiments, such methods using a CEX resin can be used to obtain a composition comprising an 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) HMW aggregates. In certain embodiments, such methods using a CEX resin can be 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, 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 aggregates, e.g., HMW aggregates.In other embodiments, the level of HMW aggregates 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 relative to the level of HMW aggregates in the load material. In some embodiments, the CEX methods provided herein can be used to reduce the level of HMW aggregates in a composition comprising an anti-α4β7 antibody 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 that does not have one or more of the parameters described herein, e.g., compared to a process performed using an elution buffer having a pH of 6.3 or greater, 6.5 or greater, 6.7 or greater, or 6.9 or greater, and / or a conductivity of 18 mS / cm or greater, 19 mS / cm or greater, 20 mS / cm or greater, 22 mS / cm or greater, or 24 mS / cm or greater. In some embodiments, the level of HMW aggregates 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 compared to a suitable control.

[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 can be used to adjust the isoform distribution of the anti-α4β7 antibody present in the eluate. For example, the pH and / or conductivity of the elution buffer can be used to increase the percentage of the major antibody isoform, decrease the percentage of acidic isoform species, and / or decrease the percentage of basic isoform species.

[0160] In some embodiments, an elution buffer can be selected that has a pH of 6.0 or less, e.g., 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, e.g., pH 4.5-6.0, pH 4.5-5.5, or pH 5.0-6.0. In some embodiments, an elution buffer can be selected having a conductivity of at least 10 mS / cm, e.g., 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 or more, e.g., 10 to 17 mS / cm, 12 to 17 mS / cm, 13 to 17 mS / cm, 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 15 to 16 mS / cm. In some embodiments, the above elution buffer conditions can be used to obtain a composition comprising at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% or more of the major isoform of an anti-α4β7 antibody. In some embodiments, the above elution buffer conditions can be used to obtain a composition comprising 20% ​​or less (e.g., 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) basic isoform species. In certain embodiments, such methods using CEX resin can be used to obtain compositions comprising a major isoform of an 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 a basic isoform species.In other embodiments, the level of basic isoform species 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 relative to the level of basic isoform species in the load material.

[0161] III.Analysis method In certain embodiments, the levels of aggregates, monomers, and fragments in chromatography samples generated using the methods described herein are analyzed. In certain embodiments, aggregates, monomers, and fragments are measured for each molecule using size exclusion chromatography (SEC). For example, without limitation, a TSK-Gel G3000SWxL, 5 μm, 125 Å, 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 Å, 4.6 × 300 mm column (Tosoh Bioscience) can be used. In certain embodiments, the above columns are used with Agilent or Shimadzu HPLC systems. In certain embodiments, sample injection is performed under isocratic elution conditions, for example, using a mobile phase consisting of 100 mM sodium sulfate and 100 mM sodium phosphate at pH 6.8, and detection is performed by UV absorbance at 214 nm. In certain embodiments, the mobile phase consists of 1×PBS at pH 7.4 and the elution profile is detected by UV absorbance at 280 nm. In certain embodiments, quantitation is based on the relative areas of the detected peaks.

[0162] Any further technique, such as mass spectrometry, can be used to assay the size variants.

[0163] 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.

[0164] In various embodiments described herein, cation exchange chromatography (CEX) can be used to determine the relative amounts of the major isoform, basic isoform(s), and acidic isoform(s) present in a population of an antibody or antigen-binding portion thereof, e.g., vedolizumab. The CEX method fractionates antibody species based on overall surface charge. After dilution to low ionic strength with a mobile phase, the test sample can be injected onto a CEX column, such as a Dionex Pro-Pac™ WCX-10 column (Thermo Fisher Scientific, Waltham, MA, USA), equilibrated with an appropriate buffer, e.g., 10 mM sodium phosphate (pH 6.6). The antibody can be eluted using a sodium chloride gradient in the same buffer. Protein elution is monitored at 280 nm, and each peak can be assigned to the acidic, basic, or major isoform category. The acidic peak elutes from the column with a shorter retention time than the major isoform peak, and the basic peak elutes from the column with a longer retention time than the major isoform peak. The percentage of the major isoform, the total percentage of acidic species, and the total percentage of basic species are reported. The retention time of the major isoform in the sample is compared to that of a reference standard to determine suitability. In one embodiment, the CEX assay method involves diluting the test sample to low ionic strength, injecting it onto a CEX column equilibrated with 10 mM sodium phosphate (pH 6.6), eluting the column with a NaCl gradient in this buffer, monitoring the peaks at 280 nm, and assigning each peak as acidic, major, or basic, where the acidic peak elutes with the shortest retention time, the major peak elutes second, and the basic peak elutes with the longest retention time; the area of ​​each peak is quantified and calculated as a percentage of the total peak area.

[0165] In various embodiments described herein, size exclusion chromatography (SEC) can be used to determine the relative levels of monomer, high molecular weight (HMW) aggregates, and low molecular weight (LMW) degradation products present in a population of an antibody or antigen-binding portion thereof, e.g., vedolizumab. SEC allows for size-based separation of antibody monomer 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, SEC analysis can be performed using a G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA (USA)) or two G3000 SWxl columns connected in series with an isocratic phosphate-sodium chloride buffer system (pH 6.8). Elution of protein species is monitored at 280 nm. Purity is determined by assessing the main peak (monomer) and total peak areas. In one embodiment, SEC analysis involves injecting a sample onto two G3000 SWxl columns connected in series and running in an isocratic phosphoric acid-sodium chloride system (pH 6.8), monitoring the elution of protein species at 280 nm, and measuring the main peak (monomer) and total peak areas. Sample purity (calculated as % monomer), HMW aggregates (%), and / or LMW degradation products (%) are reported.

[0166] Residual CHO host cell protein (HCP) impurities present in antibody formulations can be measured, if desired, by enzyme-linked immunosorbent assay (ELISA) using standard techniques. Many ELISA kits designed for this purpose are commercially available, such as the CHO HCP ELISA Kit 3G from Cygnus Technologies (Southport, NC, USA). Host cell proteins in test samples can be captured using immobilized polyclonal anti-CHO HCP antibodies. The captured proteins can then be detected using an appropriate detection reagent, such as the same antibody conjugated to horseradish peroxidase. In this exemplary embodiment, the amount of captured peroxidase, which is directly proportional to the concentration of CHO HCP, can be measured colorimetrically at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the CHO HCP assay involves capturing HCP using a polyclonal anti-CHO HCP antibody, which is detected after binding to a horseradish peroxidase-conjugated antibody, which converts the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a substrate that is measured colorimetrically at 450 nm. The concentration of HCP can be determined by comparison to a CHO HCP standard curve, such as that included in the test kit, and is reported as a percentage of the total protein level in the antibody formulation.

[0167] IV. Downstream Processing and Formulation Anti-α4β7 antibodies (e.g., vedolizumab or antibodies having a binding region corresponding to vedolizumab) can be further purified from contaminating soluble proteins and polypeptides. Suitable purification methods include, for example, affinity chromatography using a resin that binds to the Fc region of the antibody, such as Protein A; fractionation using an ion exchange column or resin, such as cation exchange chromatography (CEX), for example, SP-Sepharose™ or CM-Sepharose™ hydroxyapatite; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed-mode chromatography; ethanol precipitation; chromatofocusing; ammonium sulfate precipitation; gel filtration, for example, using Sephadex G-75™; ultrafiltration and / or diafiltration, or a combination of the above. Exemplary purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the recombinant protein is highly pure and suitable for human therapeutic use, for example, in pharmaceutical antibody formulations as described below. Following purification, the highly pure recombinant protein can be subjected to ultrafiltration / diafiltration (UF / DF) to produce a pharmaceutical formulation suitable for administration to humans.

[0168] After ultrafiltration / diafiltration, the antibody formulation may remain liquid or may be lyophilized to form a dry antibody formulation. In one aspect, the dry, lyophilized antibody formulation is provided in a single-dose vial containing 180 mg, 240 mg, 300 mg, 360 mg, 450 mg, or 600 mg of anti-α4β7 antibody and can be reconstituted with a liquid, such as sterile water, for administration. In another aspect, the anti-α4β7 antibody, e.g., vedolizumab, is in a stable liquid pharmaceutical composition that is stored at about 2-8°C in a container, e.g., a vial, syringe, or cartridge, until administration to a subject in need thereof. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody contains about 0%-5.0%, 0%-2%, 2% or less, 1% or less, 0.6% or less, or 0.5% or less aggregates.

[0169] Thus, in some embodiments, provided herein are reconstituted lyophilized formulations or stable liquid pharmaceutical compositions of anti-α4β7 antibodies comprising a humanized anti-α4β7 antibody, or an antigen-binding portion thereof. An example of a lyophilized formulation comprising an anti-α4β7 antibody, such as vedolizumab, is described in U.S. Patent No. 9,764,033, the contents of which are incorporated herein by reference. An example of a liquid formulation comprising an anti-α4β7 antibody, such as vedolizumab, is described in U.S. Patent No. 10,040,855, the contents of which are incorporated herein by reference. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of an anti-α4β7 antibody contains about 11% to 16%, 12% to 15%, 14% or less, 13% or less, 12% or less, or 11% or less of basic isoform species. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody comprises 65%-75%, 66%-74%, 67%-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] A purified antibody, e.g., an anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab), can be concentrated to provide a concentrated protein composition, e.g., a protein composition having an antibody concentration of at least 100 mg / mL, 125 mg / mL, or 150 mg / mL, or a concentration of about 100 mg / mL, 125 mg / mL, or 150 mg / mL. It will be understood that the concentrated antibody product can be concentrated to any level possible under the concentration conditions, e.g., to a concentration where the polypeptide is no longer soluble in solution.

[0171] In some embodiments, compositions provided herein comprise purified anti-α4β7 antibodies, such as vedolizumab, which are then formulated for human use. In one embodiment, the purified antibodies are formulated as a dry, lyophilized formulation, which can be administered by reconstitution with a liquid, such as sterile water. Administration of the reconstituted formulation can be by parenteral injection via one of the routes described above. Intravenous injection can be administered by infusion, with further dilution in sterile isotonic saline, a buffer, such as phosphate-buffered saline or Ringer's (lactate or dextrose) solution. In some embodiments, the purified antibodies are formulated as liquid formulations, and the anti-α4β7 antibodies are administered by subcutaneous injection, e.g., at a dose of about 54 mg, 108 mg, 165 mg, or about 216 mg.

[0172] Containers that can be used to store and freeze the purified compositions described herein include polycarbonate bottles (for IV formulations) or PETG bottles (for subcutaneous formulations). After the formulations are dispensed into the bottles, they can be frozen (e.g., at -60°C or below).

[0173] The following examples illustrate improved methods and compositions for purifying antibodies. Examples 1-5 below can be used to obtain purified compositions of anti-α4β7 antibodies, particularly vedolizumab. The present specification includes the methods described in the examples below, including the various parameters described in the examples below.

[0174] Example The following example describes the purification process for vedolizumab produced in cell culture using CHO cells as an expression system.

[0175] Example 1: Effect of elution buffer on the purification of vedolizumab using Protein A resin This example demonstrates an antibody purification method using Protein A resin that can be used to produce therapeutic anti-α4β7 antibodies, such as vedolizumab. As described herein, adjusting the elution pH of the Protein A resin reduced the level of aggregates in purified vedolizumab compositions.

[0176] Vedolizumab 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 vedolizumab 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 harvest obtained from the upstream primary recovery process. This step also removed process-related impurities such as host cell proteins (HCPs).

[0178] The Protein A resin was first equilibrated with PBS equilibration solution (pH 7.2). The clarified harvest was then loaded onto the resin. 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 1 M NaCl in PBS wash solution (pH 7.2). Wash 3 was performed with both Wash 1 (PBS) and the same solution used for equilibration. Because the antibody remained bound to the resin, each wash served to wash away impurities from the antibody. The antibody was then eluted from the resin using elution buffers of different pHs. As shown in Figure 1, elution buffers ranging from pH 3 to 3.5 were tested. The results, shown in Figure 1, demonstrate that the percentage of aggregates decreases with increasing pH. As shown in Figure 1, elution of vedolizumab from Protein A at pH 3 resulted in an eluate with high levels of aggregates (i.e., approximately 1-1.2%), whereas elution of the antibody using an elution buffer with a higher pH (e.g., pH approximately 3.5) resulted in an eluate with approximately 0.6-0.85% aggregates.

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

[0180] As shown in Table 1, linear regression models indicated that elution pH had a significant effect on all assay results (p<0.05). Data variability was slightly greater for LMW percentage (%) and HCP, but load volume affected HCP clearance. The combination of load volume and load flow rate slightly affected LMW (%).

[0181] ·monomer(%): The elution pH had 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 increased, the monomer percentage (%) increased.

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

[0183] LMW ratio (%): The elution pH and the combination of load volume and load flow rate affected the LMW percentage (p<0.05), but the influence of the input parameters on the LMW percentage (%) appears to be minimal.

[0184] HCP: Dissolution pH and load had a significant effect on HCPs in both ppm and log reduction factors (p<0.05).

[0185] [Table 1] Vedolizumab has properties, such as high hydrophobicity, that make it superior to other IgG antibodies. Figure 3 compares vedolizumab with three other IgG antibodies, showing the amount of aggregates (HMW fraction (%)) found in the eluate when each antibody (vedolizumab (MLN0002), IgG A, IgG B, or IgG C) was eluted from a cation exchange column (Nuvia S, BioRad) using elution buffers of increasing pH (increasing pH from left to right). The aggregate clearance performance of vedolizumab was more variable than that of the other three tested IgGs under their respective optimal conditions. Therefore, as can be seen in Figure 3, pH can affect aggregate levels during vedolizumab purification.

[0186] Example 2: Purification of vedolizumab using hydrophobic HIC resin Given the hydrophobic nature of vedolizumab, reducing HMW aggregates can be challenging in downstream processes. Furthermore, minimizing host cell protein (HCP) levels is essential when vedolizumab is produced in mammalian cells, such as CHO cells. HIC, mixed-mode, and anion-exchange resins and membranes were screened for performance using a high-throughput method. Eight HIC resins were then tested under different equilibration, loading, and elution conditions for their respective abilities to reduce aggregates and minimize HCPs in vedolizumab purification. The only resin that demonstrated acceptable aggregate clearance and minimized HCPs was Toyopearl Hexyl-650C (Tosoh Biosciences) hydrophobic HIC resin. More specifically, Hexyl-650C reduced aggregate levels to approximately 1.5% to 0.35% 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, Superbutyl-55C, Phenyl-650M, Phenyl-600M, PPG-600M, and Ether-650M, but were unable to achieve such low levels of aggregates.

[0187] Hexyl-650C was the most hydrophobic resin compared to the other resins tested, including ether, PPG, phenyl, and butyl. Hexyl-650C had 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 both bind / elute and flow-through modes. In bind / elute experiments, Hexyl-650C was able to reduce aggregates to approximately 0.30% HMW, but the binding capacity was low (approximately 20 mg / ml of resin). In contrast, flow-through mode using Hexyl-650C and vedolizumab demonstrated both aggregate reduction and increased load capacity. Flow-through experiments were performed using an initial unadjusted load of 108 mg / ml in 0.2 M sodium chloride in 10 mM sodium phosphate (pH 6.7). These flow-through conditions resulted in a reduction of HMW from 1.39% to 0.71%. Increasing the salt, including replacing sodium chloride with calcium phosphate, improved aggregate reduction even further. A low load of 67.5 mg / ml using 250 mM potassium phosphate, 50 mM potassium chloride (for equilibration and load adjustment) resulted in a reduction of aggregates from approximately 0.72% HMW to approximately 0.3% HMW (95.5% recovery).

[0189] A column-based design of experiments (DOE) study was conducted to further evaluate the ability of Hexyl-650C in flow-through mode to reduce aggregate levels in vedolizumab purification. As shown in Table 2, low HMW fraction (%) and low HCP levels (ppm) were obtained using low pH and high phosphate load equilibration conditions. The experiments in Table 2 were performed using a 60 mg / ml resin load. At all low pH conditions, HMW reduction ranged from 1.0% to less than 0.34% HMW. The average antibody recovery was approximately 91.5%. The combination of high pH and high potassium phosphate likely increased the affinity of the major vedolizumab species to the resin, resulting in lower recovery. In contrast, the combination of higher phosphate levels and low pH resulted in higher HMW clearance. At low phosphate and low pH, low HMW, low HCP, and low residual protein A leaching were observed (see, e.g., line 12 below). Therefore, the highly hydrophobic HIC resin was able to effectively remove aggregated (HMW) vedolizumab to a level below 0.5%.

[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 high antibody titers (≥5.0 g / L) was generated, which requires the development of a purification process designed to handle large quantities of this highly hydrophobic antibody.

[0192] Capto Adhere ImpRes is a mixed-mode (MXM) chromatography resin with strong anion exchange, hydrogen bonding, and hydrophobic interaction functionalities in a smaller bead size, allowing for improved impurity removal and increased capacity.

[0193] Capto Adhere ImpRes mixed-mode resin used in flow-through mode was capable of purifying vedolizumab, but with lower-than-ideal yields and impurity removal levels. Preliminary characterization experiments using Capto Adhere ImpRes in bind-elute mode demonstrated significant losses in step yield and / or impurity removal performance related to three process input parameters: resin load capacity, elution buffer pH, and elution buffer conductivity. This example describes studies designed to further explore the impact of variations in these parameters on the performance of Capto Adhere ImpRes in purifying vedolizumab, as well as its impact on various product quality attributes.

[0194] Materials and Methods 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 pH 7.8 sodium phosphate buffer, and the antibody was eluted from the column under various conditions described below.

[0195] 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 frozen retentates (-80°C). The methods used for the analyses are shown in Table 3 below and described in detail below. Load material sampled after queued runs was analyzed to confirm no significant changes in the quality characteristics of the load material.

[0196] [Table 3]

[0197] Experimental design A full factorial design was used with three levels for resin load, 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. An additional center-point condition was 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 sodium chloride concentration of the elution buffer was used to design the experiment, and the measured conductivity of the elution buffer was used as the input parameter value for statistical analysis. The parameter ranges tested and a summary of the design are listed in Tables 4 and 5, respectively.

[0198] [Table 4] [Table 5] DOE pattern (3): upper level, (2): middle level, (1): lower level, (0): midpoint of each input parameter range. Experiments were designed using NaCl concentration as an input parameter, and actual conductivity measurements were used in the statistical analysis.

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

[0200]

number

[0201] statistical analysis Assay results and product quality representing KPIs for all experiments in the design were analyzed using JMP 11 statistical software (SAS Institute, Cary, NC). Each response was analyzed by fitting to a linear model 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 with a relatively large number of potential inputs often results in overfitting. One characteristic of overfitting is a high R 2The model had a significant value but multiple scientifically meaningless (and statistically insignificant) terms. To determine the best statistically significant model while avoiding overfitting, each model was developed using a forward regression of the input parameters and the response of interest. The stopping rule for the regression was a p-value threshold, and input parameters were included in the model if their p-value was <0.05. The algorithm for this analysis determined (i) the highest possible R 2 A model was generated that (ii) achieved the values, (iii) included as few input parameters as possible, and (iv) described the physically possible behavior of antibodies undergoing multimodal chromatography (even when such findings appear inconsistent with initial technical expectations).

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

[0205] [Table 6]

[0206] [Table 7]

[0207] Effect of pH and conductivity of elution buffer on antibody yield The effect of elution buffer pH and elution buffer conductivity on the process recovery or yield of vedolizumab is shown in Figures 4 and 5. As shown in Figures 4 and 5, the observed process recovery data was significantly higher than R 2 There is a good fit to the linear regression model as indicated by the value of 0.930.

[0208] Figures 4 and 5 show plots of Capto Adhere ImpRes process recovery versus elution buffer pH and conductivity or load amount. Recovery was significantly affected by elution buffer pH (p<0.0001) and resin load amount (p=0.0021), and to a lesser extent by elution buffer conductivity (p=0.0189). At any level of elution buffer conductivity and load amount, step recovery was below 83.91% at an elution buffer pH of approximately 4.40 (Runs 1, 4, 11-14, 16, 19, and 27). Low resin load amount negatively impacted recovery. Breakthrough was observed during the post-sample loading wash step in all runs with a load amount of 77 g per L of resin. The effect of load amount depended on elution buffer pH (p=0.0170). At low elution buffer pH (i.e., pH 3.8), the load amount had no substantial effect on recovery, whereas as the elution buffer pH increased, recoveries decreased at lower resin loads (81.94–83.91% at a load of 77 g / L of resin, compared with 73.36–78.84% at a load of 53 g / L of resin, at an elution buffer pH of approximately 4.4). The conductivity of the elution buffer had only a small effect on recovery, according to experimental results and model predictions. The lowest recovery of 73.36% was observed with an elution buffer pH of 4.40, an elution buffer conductivity of 28.89 mS / cm, and a load of 53 g / L of resin (Run 13, predicted by the model as 76.22%).

[0209] Thus, 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.

[0210] Effect of pH and conductivity of elution buffer on HMW aggregates The effect of elution buffer pH and conductivity on the level of aggregates is shown in Figures 6 and 7, which show the effect of input parameters on HMW content.

[0211] According to the linear regression model, the amount of HMW species in the eluate was affected by 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) (R 2 = 0.962). Increasing elution pH and conductivity decreased the amount of HMW species in the eluate, while increasing the load resulted in higher levels. The model also predicted a statistically significant (p = 0.0462) interaction between elution buffer pH and conductivity.

[0212] As shown in Figures 6 and 7 below, HMW species contents of approximately 1% or greater were observed at an elution buffer pH of approximately 3.80. The highest HMW content of 1.23% was obtained (predicted by the model to be 1.19%) at an elution pH of 3.80, an elution conductivity of 19.67 mS / cm (160 mM NaCl), and a load of 65 g per L of resin (Run 2). 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, elution buffer pH (p<0.0001), elution buffer conductivity (p=0.0003), and load amount (p=0.0016) each had a statistically significant effect on HMW clearance performance. A consistent level of clearance (12.50-72.79%) was achieved across all conditions evaluated in this study. Among these inputs, elution pH had the greatest effect. Increasing elution buffer pH improved HMW clearance, which was opposite to its effect on recovery. According to the model, increasing elution buffer conductivity and decreasing load amount resulted in higher HMW clearance.

[0214] Thus, as shown in Figures 6 and 7, the level of aggregates (percentage of HMW species) in purified vedolizumab compositions 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 aggregate levels in vedolizumab formulations. Studies focused on the suitability of CEX chromatography using Nuvia HR-S resin (Bio-Rad, Hercules, CA, USA) for the purification of vedolizumab are described herein, with particular emphasis on reducing aggregate levels from this hydrophobic antibody. Elution conditions for the CEX process, conducted in bind / elute mode, were evaluated. A design of experiments (DoE) approach was used to evaluate the effect of several parameters, including elution buffer pH and elution buffer conductivity, on process outcomes.

[0216] Materials and Methods The load materials and analytical methods used in this study were similar to those described in Example 3 above.

[0217] Experimental design Initial screening tests and preliminary risk assessments identified elution buffer pH and elution buffer conductivity as having known or potential impacts on the process performance outcome (PPO) of Nuvia HR-S when the resin is used in bind / elute mode. This study was conducted to characterize the impact of these process parameters. The parameter ranges and experimental design for the study are shown in Tables 8 and 9, respectively. The elution buffer conductivity was varied by adjusting the sodium chloride (NaCl) concentration, and the NaCl ranges evaluated are shown in Table 8.

[0218] [Table 8]

[0219] [Table 9-1] [Table 9-2]

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

[0221] Effect of pH and conductivity of elution buffer on HMW aggregates The effect of elution buffer pH and conductivity on the level of aggregates is shown in Figures 8 to 10, which show the effect of input parameters on HMW content.

[0222] As shown in Figures 8-10, the variations in HMW, monomer, and LMW of the eluate were 0.01-0.88%, 98.33-99.27%, and 0.62-1.35%, respectively. The model for HMW includes a strong linear dependence on the pH and conductivity of the elution buffer, along with interaction terms involving both parameters. The model surface (shown in Figure 8) shows that HMW is lowest at values ​​of elution buffer pH and conductivity where HMW is very low. The highest values ​​are shown for very high elution buffer pH and conductivity.

[0223] HMW clearance was measured for each run to account for variations in the HMW content of the load material throughout the study. Similar to the HMW content of the eluate, HMW clearance varied widely throughout the study (-30.65% to 98.61%), and the HMW clearance model included linear and interaction terms for the pH and conductivity of the elution buffer. Figure 9 illustrates the behavior of the model. While the highest HMW clearance values ​​were obtained at conditions with low elution buffer pH and conductivity, many of the conditions tested showed HMW clearances greater than 70%. However, the negative HMW clearance values ​​reported for Runs 9, 20, and 24 (-1.18%, -20.55%, and -30.65%, respectively) indicate that significant variations in aggregate removal were observed across the wide range of conditions evaluated, and that certain conditions may generate aggregate species rather than remove them.

[0224] In runs 40–42, Capto Adhere ImpRes eluate containing higher aggregate levels than those typically used for load materials processed by CEX at centerpoint conditions was used as the load material. Modeling of HMW and HMW clearance indicated that increasing the elution buffer pH and conductivity resulted in eluates with higher HMW content. The conditions selected for runs 40–42 were intended to explore potential elution conditions using the "worst-case" aggregate levels of the CEX load material. At elution buffer pH values ​​of 5.50–5.54 and elution buffer conductivity of 13.40 mS / cm, the resulting eluate HMW was 0.31–0.34%. However, the eluate HMW increased to 0.59% when using an eluate at pH 5.60 (whereas conductivity remained unchanged). At that aggregate level, further processing would likely not meet vedolizumab's acceptance criteria for HMW.

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

[0226] Therefore, as shown in Figures 8 to 10, the pH and conductivity of the elution buffer can be used to adjust the aggregate level (percentage (%) of HMW species) in a composition comprising vedolizumab purified using a CEX resin. Furthermore, as shown in Figures 8 to 10, the aggregate level in a purified vedolizumab composition can be reduced when a CEX resin is used with an elution buffer having a low pH and / or low conductivity.

[0227] Effect of pH and conductivity of elution buffer on basic isoform species The effect of elution buffer pH and conductivity on the levels of basic isoform species is shown in Figures 11-13, which show the effect of input parameters on the content of acidic, major, and basic isoform species.

[0228] As shown in Figures 11-13, the acidic, major, and basic content results ranged from 12.49 to 30.27%, 64.32 to 73.82%, and 5.42 to 18.04%, respectively. The model for acidic content included elution buffer pH, elution buffer conductivity, and load amount, along with interaction terms for all three parameters. Elution buffer pH and elution buffer conductivity had the strongest effect on acidic content. As can be seen from the model surface in Figure 11, the highest acidic content was obtained using a combination of very low elution buffer pH and conductivity.

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

[0230] The linear terms of elution buffer pH, elution buffer conductivity, and load amount most strongly influence the basic isoform content of the eluate, with the interaction terms contributing only slightly. As can be seen from the model surface in Figure 13, the basic isoform content increased with increasing elution buffer pH and elution buffer conductivity.

[0231] Thus, as shown in Figures 11-13, the pH and conductivity of the elution buffer can be 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 a purified vedolizumab composition can be reduced when a CEX resin is used with an elution buffer having a high pH and / or high conductivity. Furthermore, as shown in Figure 13, the level of basic isoform species in a purified vedolizumab composition can be reduced when a CEX resin is used with an elution 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 attributes of vedolizumab.

[0233] Cation exchange chromatography (CEX) fractionates vedolizumab antibody species (major isoform, basic species, and acidic species) based on overall surface charge. After dilution to low ionic strength with a mobile phase, test samples were injected onto a Dionex Pro-Pac™ WCX-10 column (Thermo Fisher Scientific, Waltham, MA, USA) equilibrated in 10 mM sodium phosphate (pH 6.6) and eluted with a sodium chloride gradient in the same buffer. Protein elution was monitored at 280 nm, and each peak was assigned to the acidic, basic, or major isoform category. The percentage of major isoform, the total percentage of acidic species, and the total percentage of basic species are reported. The retention time of the major isoform in the sample was compared to that of a reference standard to determine suitability.

[0234] Vedolizumab purity is determined using size exclusion chromatography (SEC). Reference standards and test samples (75 μg) are analyzed 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). The method provides separation of antibody monomer from high molecular weight (HMW) species and low molecular weight (LMW) degradation products. Elution of protein species is monitored at 280 nm. Purity is determined by assessing the main peak (monomer) and total peak area. Sample purity (%) (calculated as % monomer) and aggregates (%) are reported.

[0235] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. The present invention includes the following embodiments. [1] 1. A method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution containing the 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, thereby binding the anti-α4β7 antibody to Protein A; washing the matrix containing the Protein A with a wash solution; and 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 Protein A, thereby obtaining a composition containing the anti-α4β7 antibody. The method, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6. [2] The method according to [1] above, wherein the composition comprising the anti-α4β7 antibody contains less than 1% high molecular weight (HMW) aggregates. [3] The method according to [1] or [2] above, wherein the protein A is immobilized on a solid phase. [4] The method according to [3] above, wherein the solid phase comprises one or more of beads, gel, and resin. [5] The method according to any one of the above [1] to [4], wherein the pH of the washing solution is about 7. [6] The method according to any one of the above [1] to [5], wherein the elution solution contains citric acid. [7] The method according to any one of the above [1] to [6], wherein the pH of the elution solution is 3.2 to 3.7 or 3.3 to 3.8. [8] 1. A method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution containing the antibody and one or more impurities, comprising: contacting a solution containing an 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, thereby obtaining a composition comprising the anti-α4β7 antibody; the HIC resin is characterized as a highly hydrophobic resin; The method, wherein the anti-α4β7 antibody is a humanized antibody, an IgG1 antibody, and comprises a heavy chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 4, the CDR2 domain set forth in SEQ ID NO: 3, and the CDR1 domain set forth in SEQ ID NO: 2, and a light chain variable region comprising the CDR3 domain set forth in SEQ ID NO: 8, the CDR2 domain set forth in SEQ ID NO: 7, and the CDR1 domain set forth in SEQ ID NO: 6. [9] The method according to [8] above, wherein the composition comprises the anti-α4β7 antibody and contains less than 0.6% HMW aggregates.

[10] The method according to [8] or [9] above, wherein the HIC resin is equilibrated with a phosphate buffer having a pH of less than about 7.2.

[11] The method according to

[10] above, wherein the phosphate buffer contains about 0.35 M to about 0.15 M potassium phosphate.

[12] The method according to any one of the above [8] to

[11] , wherein the resin load is about 55 to 75 mg / ml.

[13] The method according to any one of the above [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 [8] to

[13] above, wherein the composition contains less than about 0.3 ppm of host cell protein (HCP).

[15] The method according to any one of the above [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 the above [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] 1. A method for producing a composition comprising an anti-α4β7 antibody from a liquid solution containing the anti-α4β7 antibody and one or more impurities, comprising: contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, thereby binding the anti-α4β7 antibody to the resin; washing the mixed-mode chromatography resin with a wash solution; 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, thereby obtaining a composition comprising the anti-α4β7 antibody; The method, wherein the anti-α4β7 antibody comprises 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.

[18] The method according to

[17] above, wherein the composition comprising the anti-α4β7 antibody contains less than 1% HMW aggregates.

[19] The method according to

[17] or

[18] above, wherein the elution solution has a pH of 4.1 or higher.

[20] The method according to any one of the above

[17] to

[19] , wherein the elution solution has a pH of about pH 3.9 to about pH 4.4.

[21] The method according to any one of the above

[17] to

[20] , wherein the elution solution has a conductivity of 30 mS / cm or less.

[22] The method according to

[21] above, wherein the elution solution has a conductivity of about 20 mS / cm to about 30 mS / cm.

[23] The method according to any one of the above

[17] to

[22] , wherein the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM.

[24] The method according to any one of the above

[17] to

[23] , wherein the mixed-mode chromatography resin is Capto Adhere ImpRes.

[25] The method according to any one of

[17] to

[24] above, further comprising purifying the anti-α4β7 antibody using a cation exchange (CEX) resin.

[26] The method according to

[26] above, wherein the CEX resin is operated in bind / elute mode.

[27] 1. A method for producing a composition comprising an anti-α4β7 antibody from a liquid solution containing the anti-α4β7 antibody and one or more impurities, comprising: contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatography resin, thereby binding the anti-α4β7 antibody to the resin; washing the mixed-mode chromatography resin with a wash 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, thereby obtaining a composition comprising the anti-α4β7 antibody; The method, wherein the anti-α4β7 antibody comprises 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.

[28] The method described in

[27] above, wherein the composition has a higher yield of anti-α4β7 antibody compared to a control composition containing anti-α4β7 antibody similarly obtained using a control elution solution having a pH higher than pH 4.2 and / or a control conductivity higher than 28 mS / cm.

[29] The method according to

[27] or

[28] above, wherein the elution solution has a pH of 4.0 or less.

[30] The method according to

[27] or

[28] above, wherein the elution solution has a pH of about pH 4.2 to about pH 3.8.

[31] The method according to any one of the above

[27] to

[30] , wherein the elution solution has a conductivity of about 18 mS / cm to about 28 mS / cm.

[32] The method according to any one of the above

[27] to

[31] , wherein the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM.

[33] The method according to any one of

[27] to

[32] above, wherein the mixed-mode chromatography resin is contacted with at least 55 g of the anti-α4β7 antibody per 1 L of resin.

[34] The method according to

[33] above, wherein the mixed-mode chromatography resin is contacted with about 55 g to about 80 g of the anti-α4β7 antibody per 1 L of resin.

[35] The method according to any one of the above

[27] to

[34] , wherein the mixed-mode chromatography resin has a smaller bead size and strong anion exchange, hydrogen bonding, and hydrophobic interactions, and optionally the mixed-mode chromatography resin is Capto Adhere ImpRes.

[36] The method according to any one of

[27] to

[35] above, further comprising purifying the anti-α4β7 antibody using a cation exchange (CEX) resin.

[37] The method according to

[36] above, wherein the CEX resin is operated in bind / elute mode.

[38] 1. A method for producing a composition comprising an anti-α4β7 antibody from a liquid solution containing the anti-α4β7 antibody and one or more impurities, comprising: contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a cation exchange (CEX) resin, thereby binding the anti-α4β7 antibody to the resin; washing the CEX resin with a washing solution; and 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, thereby obtaining a composition comprising the anti-α4β7 antibody; The method, wherein the anti-α4β7 antibody comprises 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.

[39] The method according to

[38] above, wherein the composition comprising the anti-α4β7 antibody contains about 1% or less HMW aggregates.

[40] The method according to

[38] or

[39] above, wherein the elution solution has a conductivity of 14 mS / cm or less.

[41] The method according to

[38] or

[39] above, wherein the elution solution has a conductivity of about 11 to 16 mS / cm.

[42] The method according to

[38] or

[39] above, wherein the elution solution has a conductivity of about 12 to 14 mS / cm.

[43] The method according to any one of the above

[38] to

[42] , wherein the elution solution contains NaCl at a concentration of about 70 mM to about 110 mM.

[44] The method according to any one of the above

[38] to

[43] , wherein the elution solution has a pH of about pH 5 to about pH 6.

[45] The method according to

[44] above, wherein the elution solution has a pH of about pH 5.1 to about pH 5.8.

[46] The method according to any one of

[38] to

[45] above, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 to 70 g of antibody per 1 L of resin.

[47] The method according to any one of

[38] to

[46] above, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 to 60 g of antibody per 1 L of resin.

[48] The method according to any one of the above

[38] to

[47] , wherein the CEX resin is a strong CEX resin, and optionally the CEX resin is Nuvia HR-S.

[49] The method according to any one of

[38] to

[48] above, further comprising purifying the anti-α4β7 antibody using a mixed-mode chromatography resin.

[50] 49. The method according to claim 49, wherein the mixed mode chromatography resin is operated in bind / elute mode.

[51] 1. A method for producing a composition comprising an anti-α4β7 antibody from a liquid solution containing a major isoform and one or more basic isoform species of the anti-α4β7 antibody, comprising: contacting the liquid solution containing the anti-α4β7 antibody and one or more basic isoform species with a cation exchange (CEX) resin, thereby binding 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 greater to elute the anti-α4β7 antibody from the CEX resin, thereby obtaining a composition comprising the anti-α4β7 antibody; The method, wherein the anti-α4β7 antibody comprises 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.

[52] The method according to

[51] above, wherein the composition comprising the anti-α4β7 antibody comprises about 4% to about 20% of the basic isoform.

[53] The method according to

[51] or

[52] above, wherein the elution solution has a conductivity of 12 mS / cm or more.

[54] The method according to

[51] or

[52] above, wherein the elution solution has a conductivity of about 11 to 16 mS / cm.

[55] The method according to

[51] or

[52] above, wherein the elution solution has a conductivity of about 12 to 14 mS / cm.

[56] The method according to any one of the above

[51] to

[55] , wherein the elution solution has a pH of about pH 5 to about pH 6.

[57] The method according to

[56] above, 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

[51] to

[57] above, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 25 to 70 g of antibody per 1 L of resin.

[59] The method according to

[58] above, wherein the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of about 30 to 60 g of antibody per 1 L of resin.

[60] The method according to any one of the above

[51] to

[59] , wherein the CEX resin is Nuvia HR-S.

[61] The method according to any one of

[51] to

[60] above, further comprising purifying the anti-α4β7 antibody using a mixed-mode chromatography resin.

[62] 61. The method according to claim 61, wherein the mixed mode chromatography resin is operated in bind / elute mode.

[63] The method according to any one of [1] to

[62] above, wherein the antibody is produced in Chinese hamster ovary (CHO) cells.

[64] The method according to

[63] above, wherein the host cell is a GS-CHO cell.

[65] The method described in any of [1] to

[64] above, wherein the obtained composition contains a purified anti-α4β7 antibody, and the method further comprises a subsequent step of formulating the anti-α4β7 antibody into a preparation suitable for human use.

[66] The method according to any one of [1] to

[65] above, which comprises formulating the purified anti-α4β7 antibody as a lyophilized formulation.

[67] The method according to

[66] above, further comprising reconstituting the dried lyophilized formulation with a liquid to make it suitable for administration.

[68] The method according to any one of [1] to

[65] above, comprising formulating the purified anti-α4β7 antibody as a liquid preparation so that the anti-α4β7 antibody is suitable for administration by subcutaneous injection.

[69] The method according to any one of [1] to

[68] above, wherein the anti-α4β7 antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 1 and a light chain variable region sequence shown in SEQ ID NO: 5.

[70] The method according to any one of the above-mentioned [1] to

[68] , wherein the anti-α4β7 antibody is vedolizumab.

[71] A composition comprising an anti-α4β7 antibody, obtained by the method according to any one of [1] to

[70] above.

[0236] Sequence Listing [Table 10-1]

Table 10-2

Claims

1. 1. A method for obtaining a composition comprising an anti-α4β7 antibody from a liquid solution containing the 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, thereby binding the anti-α4β7 antibody to Protein A, wherein the Protein A is immobilized on a solid phase; washing the matrix containing the Protein A with a wash solution; and eluting the anti-α4β7 antibody from the matrix containing Protein A by contacting the matrix with an elution solution having a pH of 3.2 to 4 to obtain a composition containing the anti-α4β7 antibody; the composition comprising the anti-α4β7 antibody contains less than 1% high molecular weight (HMW) aggregates; The method, wherein the anti-α4β7 antibody is vedolizumab and is produced in Chinese hamster ovary (CHO) cells.

2. The method of claim 1 , wherein the pH of the wash solution is about 7.

3. The method of claim 1 or 2, wherein the elution solution comprises citric acid.

4. The method according to any one of claims 1 to 3, wherein the pH of the elution solution is from 3.2 to 3.

7.

5. The method according to any one of claims 1 to 4, wherein the pH of the elution solution is from 3.3 to 3.

8.

6. The method according to any one of claims 1 to 4, wherein the pH of the elution solution is from 3.7 to 4.

7. The method of any one of claims 1 to 6, wherein the solid phase comprises one or more of beads, a gel, and a resin.

8. 4. The method of any one of claims 1 to 3, wherein the resulting composition comprises a purified anti-α4β7 antibody, and the method further comprises a subsequent step of formulating the anti-α4β7 antibody into a formulation suitable for human use.

9. The method according to any one of claims 1 to 8, wherein the CHO cells are GS-CHO cells.

10. 10. The method of any one of claims 1 to 9, wherein the composition comprising vedolizumab contains no more than 0.9% high molecular weight (HMW) aggregates.

11. The method of any one of claims 1 to 10, wherein the composition comprising vedolizumab comprises greater than 97% monomer.

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