Antibody purification methods and compositions thereof

Abstract

Described herein are methods of purifying humanized α4β7 antibodies, such as vedolizumab, produced in mammalian cell culture, as well as compositions resulting from the purification methods.

Description

TECHNICAL FIELD

[0001] The present invention relates to methods for purifying anti-a4b7 antibodies or fragments thereof.

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. provisional application 62 / 859,580, filed June 10, 2019. The entire contents of the foregoing application are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 5, 2020, is named T103022_1120WO_SL.txt and is 10,015 bytes in size. BACKGROUND

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

[0007] Product-related impurities and process-related impurities, including aggregates, can interfere with the purification process, affect the protein during storage, and / or can cause adverse reactions upon administration of the antibody as a drug to a subject (Shukla et al., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 848(1), 28-39).

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

[0009] The present invention provides, inter alia, methods for purifying anti-a4b7 antibodies, such as vedolizumab, e.g., from a liquid solution.

[0010] In one aspect, the application features a method for obtaining a composition comprising an anti-a4b7 antibody from a liquid solution comprising the anti-a4b7 antibody and one or more impurities, the method comprising contacting a matrix comprising Protein A with the liquid solution comprising the anti-a4b7 antibody and one or more impurities, thereby causing the anti-a4b7 antibody to bind to the Protein A; washing the matrix comprising Protein A with a wash solution; and eluting the anti-a4b7 antibody from the matrix by contacting the matrix comprising Protein A with an elution solution having a pH of 3.2 to 4, thereby obtaining a composition comprising the anti-a4b7 antibody, wherein the anti-a4b7 antibody is a humanized antibody, is an IgGl antibody, comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO:4, a CDR2 domain as set forth in SEQ ID NO:3, and a CDR1 domain as set forth in SEQ ID NO:2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO:8, a CDR2 domain as set forth in SEQ ID NO:7, and a CDR1 domain as set forth in SEQ ID NO:6.

[0011] In one embodiment, the method is for obtaining a composition comprising less than 1% high molecular weight (HMW) aggregate from a liquid solution comprising the anti-a4b7 antibody and one or more impurities, the method comprising contacting a matrix comprising Protein A with the liquid solution comprising the anti-a4b7 antibody and one or more impurities, thereby causing the anti-a4b7 antibody to bind to the Protein A; washing the matrix comprising Protein A with a wash solution; and eluting the anti-a4b7 antibody from the matrix by contacting the matrix comprising Protein A with an elution solution having a pH of 3.2 to 4, thereby obtaining a composition comprising less than 1% HMW aggregate.

[0012] In one embodiment, the Protein A is immobilized on a solid phase. In one embodiment, the solid phase comprises one or more of a bead, a gel, and a resin.

[0013] In one embodiment, the wash solution has a pH of about 7. In one embodiment, the elution solution comprises citric acid.

[0014] In one embodiment, the elution solution has a pH of 3.2 to 3.7.

[0015] In another aspect, the application features a method for obtaining a composition comprising an anti-a4b7 antibody from a liquid solution comprising the anti-a4b7 antibody and one or more impurities, the method comprising contacting a solution comprising an anti-a4b7 antibody and at least one impurity with a hydrophobic interaction chromatography (HIC) resin under conditions that allow the anti-a4b7 antibody to flow through the HIC resin, thereby obtaining a composition comprising the anti-a4b7 antibody, wherein the HIC resin is characterized as a high hydrophobic HIC resin, wherein the anti-a4b7 antibody is a humanized antibody, is an IgGl antibody, comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO:4, a CDR2 domain as set forth in SEQ ID NO:3, and a CDRl domain as set forth in SEQ ID NO:2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO:8, a CDR2 domain as set forth in SEQ ID NO:7, and a CDRl domain as set forth in SEQ ID NO:6.

[0016] In one embodiment, the method is for obtaining a composition comprising the anti-a4b7 antibody and less than 0.6% HMW aggregate from a liquid solution comprising the anti-a4b7 antibody and one or more impurities, the method comprising the contacting a solution comprising an anti-a4b7 antibody and at least one impurity with a HIC resin under conditions that allow the anti-a4b7 antibody to flow through the HIC resin, thereby obtaining a composition comprising the anti-a4b7 antibody and less than 0.6% HMW aggregate, wherein the anti-a4b7 antibody is a humanized antibody, is an IgGl antibody, comprises a heavy chain variable region comprising a CDR3 domain as set forth in SEQ ID NO:4, a CDR2 domain as set forth in SEQ ID NO:3, and a CDRl domain as set forth in SEQ ID NO:2; and comprises a light chain variable region comprising a CDR3 domain as set forth in SEQ ID NO:8, a CDR2 domain as set forth in SEQ ID NO:7, and a CDRl domain as set forth in SEQ ID NO:6.

[0017] a CDR2 domain as set forth in SEQ ID NO:7, and a CDRl domain as set forth in SEQ ID NO:6.

[0018] 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.

[0019] In one embodiment, the resin loading is about 55 to 75 mg / ml.

[0020] In one embodiment, the composition comprises less than about 0.22 ppm residual Protein A.

[0021] In one embodiment, the composition contains less than about 0.3 ppm of host cell protein (HCP).

[0022] In one embodiment, the average pore size of the highly hydrophobic HIC resin is about 50 to 150 μm.

[0023] 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.

[0024] In another aspect, the invention is characterized by a method for producing a formulation containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, the method comprising contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatographic resin, thereby binding the anti-α4β7 antibody to the resin; washing the mixed-mode chromatographic resin with a washing solution; and eluting the anti-α4β7 antibody from the resin by contacting the mixed-mode chromatographic resin with an elution solution having a pH equal to or higher than pH 3.9, thereby obtaining a formulation containing purified anti-α4β7 antibody, wherein the anti-α4β7 antibody contains the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:2.

[0025] In one embodiment of the foregoing aspect, the method is used to obtain a formulation containing less than 1% HMW aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities, the method comprising contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatographic resin, thereby binding the anti-α4β7 antibody to the resin; washing the mixed-mode chromatographic resin with a washing solution; and eluting the anti-α4β7 antibody from the resin by contacting the mixed-mode chromatographic resin with an elution solution with a pH equal to or higher than pH 3.9, thereby obtaining a formulation containing less than 1% HMW aggregates.

[0026] In one embodiment, the pH of the elution solution is equal to or higher than pH 4.1. In another embodiment, the pH of the elution solution is from about pH 3.9 to about pH 4.4.

[0027] In some embodiments, the conductivity of the elution solution is 30 mS / cm or lower. In some embodiments, the conductivity of the elution solution is about 20 mS / cm to about 30 mS / cm.

[0028] In some embodiments, the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM.

[0029] In some embodiments, the mixed-mode chromatographic resin is Capto Adhere ImpRes.

[0030] In some embodiments of the foregoing aspects, the method further includes purifying the anti-α4β7 antibody using a cation exchange (CEX) resin. In some such embodiments, the CEX resin is operated in a binding / elution mode.

[0031] In another aspect, the invention is characterized by a method for producing a formulation containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, the method comprising contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatographic resin, thereby binding the anti-α4β7 antibody to the resin; washing the mixed-mode chromatographic resin with a washing solution; and eluting the anti-α4β7 antibody from the resin by contacting the mixed-mode chromatographic resin with an elution solution having a pH equal to or lower than pH 4.2 and a conductivity equal to or lower than 28 mS / cm, thereby obtaining a formulation containing purified anti-α4β7 antibody, wherein the anti-α4β7 antibody contains the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:2.

[0032] In some embodiments of the foregoing aspects, the method is used to obtain a formulation containing an anti-α4β7 antibody in increased yield from a liquid solution containing an anti-α4β7 antibody and one or more impurities. The method includes contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a mixed-mode chromatographic resin, thereby binding the anti-α4β7 antibody to the resin; washing the mixed-mode chromatographic resin with a washing solution; and eluting the anti-α4β7 antibody from the resin by contacting the mixed-mode chromatographic resin with an elution solution having a pH equal to or lower than pH 4.2 and a conductivity equal to or lower than 28 mS / cm, thereby obtaining a formulation containing an anti-α4β7 antibody in increased yield.

[0033] In some embodiments, the pH of the elution solution is equal to or lower than 4.0. In other embodiments, the pH of the elution solution is from about pH 4.2 to about pH 3.8.

[0034] In some embodiments, the conductivity of the elution solution is from about 18 mS / cm to about 28 mS / cm.

[0035] In some embodiments, the elution solution contains NaCl at a concentration of about 160 mM to about 240 mM.

[0036] In some embodiments of the foregoing aspects, the mixed-mode chromatographic resin is contacted with at least 55 g of anti-α4β7 antibody per liter of resin. In some embodiments, the mixed-mode chromatographic resin is contacted with about 55 g to about 80 g of anti-α4β7 antibody per liter of resin.

[0037] In some embodiments of the foregoing aspects, the mixed-mode chromatographic resin is Capto Adhere ImpRes.

[0038] In some embodiments of the foregoing aspects, the method further includes purifying the anti-α4β7 antibody using a cation exchange (CEX) resin. In some such embodiments, the CEX resin is operated in a binding / elution mode.

[0039] In another aspect, the invention is characterized by a method for producing a formulation containing an anti-α4β7 antibody from a liquid solution containing an anti-α4β7 antibody and one or more impurities, the method 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 resin by contacting the CEX resin with an elution solution having a conductivity equal to or less than 16 mS / cm, thereby obtaining a formulation containing purified anti-α4β7 antibody, wherein the anti-α4β7 antibody contains the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:2.

[0040] In some embodiments of the foregoing aspects, the method is used to obtain a formulation containing reduced levels of HMW aggregates from a liquid solution containing an anti-α4β7 antibody and one or more impurities, the method comprising: contacting the liquid solution containing the anti-α4β7 antibody and one or more impurities with a 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 resin by contacting the CEX resin with an elution solution having a conductivity equal to or less than 16 mS / cm, thereby obtaining a formulation containing reduced levels of HMW aggregates.

[0041] In some embodiments described above, the conductivity of the elution solution is equal to or less than 14 mS / cm. In other embodiments, the conductivity of the elution solution is about 11-16 mS / cm. In still other embodiments, the conductivity of the elution solution is about 12-14 mS / cm.

[0042] In some embodiments of the above aspects, the elution solution contains NaCl at a concentration of about 70 mM to about 110 mM.

[0043] In some embodiments of the above aspects, the pH of the elution solution is from about pH 5 to about pH 6. In some embodiments, the pH of the elution solution is from about pH 5.1 to about pH 5.8.

[0044] In some embodiments of the above aspects, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of approximately 25-70 g of antibody per liter of resin. In some embodiments, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of approximately 30-60 g of antibody per liter of resin.

[0045] In some embodiments of the above aspects, the CEX resin is Nuvia HR-S.

[0046] In some embodiments of the above aspects, the method further includes purifying the anti-α4β7 antibody using a mixed-mode chromatographic resin. In some such embodiments, the mixed-mode chromatographic resin operates in a binding / elution mode.

[0047] In another aspect, the invention is characterized by a method for producing a formulation containing an anti-α4β7 antibody from a liquid solution containing a major isotype of an anti-α4β7 antibody and one or more basic isotypes, the method comprising contacting the liquid solution containing the anti-α4β7 antibody and one or more basic isotypes 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 resin by contacting the CEX resin with an elution solution having a conductivity equal to or greater than 11 mS / cm, thereby obtaining a formulation containing purified anti-α4β7 antibody, wherein the anti-α4β7 antibody contains the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:2.

[0048] In some embodiments of the foregoing aspects, the method is used to obtain a formulation containing a reduced level of a basic isotype of α4β7 antibody from a liquid solution containing a major isotype of anti-α4β7 antibody and one or more basic isotypes. The method includes contacting the liquid solution containing the anti-α4β7 antibody and one or more basic isotypes with a 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 resin by contacting the CEX resin with an elution solution having a conductivity equal to or greater than 11 mS / cm, thereby obtaining a formulation containing a reduced level of a basic isotype.

[0049] In one embodiment, the purified composition contains about 4% to about 20% of a basic isoform.

[0050] In some embodiments described above, the conductivity of the elution solution is equal to or greater than 12 mS / cm. In other embodiments, the conductivity of the elution solution is about 11-16 mS / cm. In still other embodiments, the conductivity of the elution solution is about 12-14 mS / cm.

[0051] In some embodiments of the above aspects, the pH of the elution solution is from about pH 5 to about pH 6. In some embodiments, the pH of the elution solution is from about pH 5.1 to about pH 5.8.

[0052] In some embodiments of the above aspects, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of approximately 25-70 g of antibody per liter of resin. In some embodiments, the anti-α4β7 antibody is loaded onto the CEX resin at a concentration of approximately 30-60 g of antibody per liter of resin.

[0053] In some embodiments, the elution solution contains sodium chloride, for example, 70 to 110 mM sodium chloride.

[0054] In some embodiments of the above aspects, the CEX resin is Nuvia HR-S.

[0055] In some embodiments of the above aspects, the method further includes purifying the anti-α4β7 antibody using a mixed-mode chromatographic resin. In some such embodiments, the mixed-mode chromatographic resin operates in a binding / elution mode.

[0056] In any of the above embodiments, the antibody is produced in Chinese hamster ovary (CHO) host cells.

[0057] In one embodiment, the host cell is a GS-CHO cell.

[0058] In any of the above embodiments, the anti-α4β7 antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:1 and a light chain variable region sequence as shown in SEQ ID NO:5.

[0059] In any of the above embodiments, the anti-α4β7 antibody is vedolizumab.

[0060] In addition, the present invention also includes the following embodiments:

[0061] 1. A method for obtaining a composition containing less than 1% HMW aggregates from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising...

[0062] The matrix containing protein A is contacted with the liquid solution containing the anti-α4β7 antibody and one or more impurities, thereby causing the anti-α4β7 antibody to bind to protein A;

[0063] The matrix containing protein A is washed with a washing solution; and

[0064] The anti-α4β7 antibody is eluted from the matrix by contacting the matrix containing protein A with an elution solution at a pH of 3.2 to 4, thereby obtaining a composition containing less than 1% HMW aggregates.

[0065] The anti-α4β7 antibody is a humanized antibody, specifically an IgG1 antibody, comprising a heavy chain variable region containing a CDR3 domain as shown in SEQ ID NO:4, a CDR2 domain as shown in SEQ ID NO:3, and a CDR1 domain as shown in SEQ ID NO:2; and a light chain variable region containing a CDR3 domain as shown in SEQ ID NO:8, a CDR2 domain as shown in SEQ ID NO:7, and a CDR1 domain as shown in SEQ ID NO:6.

[0066] 2. The method of Project 1, wherein the protein A is immobilized on a solid phase.

[0067] 3. The method of Project 2, wherein the solid phase comprises one or more of beads, gels and resins.

[0068] 4. The method of any one of items 1 to 3, wherein the pH of the washing solution is about 7.

[0069] 5. The method of any one of items 1 to 4, wherein the elution solution comprises citric acid.

[0070] 6. The method of any one of items 1 to 5, wherein the pH of the elution solution is 3.2 to 3.7.

[0071] 7. A method for obtaining a composition comprising anti-α4β7 antibody and less than 0.6% HMW aggregates from a liquid solution comprising anti-α4β7 antibody and one or more impurities, the method comprising

[0072] A solution containing an anti-α4β7 antibody and at least one impurity is contacted 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 containing the anti-α4β7 antibody and less than 0.6% HMW aggregates.

[0073] The HIC resin described therein is characterized as a highly hydrophobic HIC resin.

[0074] The anti-α4β7 antibody is a humanized antibody, specifically an IgG1 antibody, comprising a heavy chain variable region containing a CDR3 domain as shown in SEQ ID NO:4, a CDR2 domain as shown in SEQ ID NO:3, and a CDR1 domain as shown in SEQ ID NO:2; and a light chain variable region containing a CDR3 domain as shown in SEQ ID NO:8, a CDR2 domain as shown in SEQ ID NO:7, and a CDR1 domain as shown in SEQ ID NO:6.

[0075] 8. The method of Project 7, wherein the HIC resin is equilibrated with a phosphate buffer solution with a pH less than about 7.2.

[0076] 9. The method of Item 8, wherein the phosphate buffer contains about 0.35 mM to about 0.15 mM of potassium phosphate.

[0077] 10. The method of any one of items 7 to 9, wherein the resin loading is about 55 to 75 mg / ml.

[0078] 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.

[0079] 12. The method of any one of items 7 to 11, wherein the composition contains less than about 0.3 ppm of host cell protein (HCP).

[0080] 13. The method of any one of items 7 to 12, wherein the average pore size of the highly hydrophobic HIC resin is about 50 to 150 μm.

[0081] 14. The method of any one of items 7 to 12, wherein the average pore size of the highly hydrophobic HIC resin is about 100 nm and / or about 100 μm.

[0082] 15. The method of any one of items 1 to 14, wherein the antibody is produced in Chinese hamster ovary (CHO) cells.

[0083] 16. The method of Item 15, wherein the host cell is a GS-CHO cell.

[0084] 17. The method of any one of items 1 to 16, wherein the anti-α4β7 antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:1 and a light chain variable region sequence as shown in SEQ ID NO:5.

[0085] 18. The method of any one of items 1 to 16, wherein the anti-α4β7 antibody is vedolizumab. Attached Figure Description

[0086] Figure 1 Describe the changes in aggregate size with the pH of the protein A elution buffer.

[0087] Figure 2 A graph that filters performance output to depict the results of the predictive profile measurements.

[0088] Figure 3 The comparison of vedolizumab with three other IgG antibodies and the pH characteristics used to elute each antibody from the cation exchange column are depicted graphically.

[0089] Figure 4 A surface plot of a linear regression model to depict the recovery rate of the Capto Adhere ImpRes step compared to the pH and conductivity of the elution buffer.

[0090] Figure 5 A surface plot of a linear regression model was created to depict the recovery rate of the Capto Adhere ImpRes step compared to the pH value and loading of the elution buffer.

[0091] Figure 6 A surface plot of a linear regression model to depict the HMW content of Capto Adhere ImpRes relative to the pH and conductivity of the elution buffer.

[0092] Figure 7 A surface plot of a linear regression model to depict the HMW content of Capto Adhere ImpRes relative to the pH of the elution buffer and the loading.

[0093] Figure 8HMW surface plot to depict the effect of elution buffer pH and elution buffer conductivity on HMW material %.

[0094] Figure 9 HMW clearance surface plot to depict the effect of elution buffer pH and elution buffer conductivity on HMW clearance.

[0095] Figure 10 A monomer surface plot to depict the effect of elution buffer pH and elution buffer conductivity on monomer content (%).

[0096] Figure 11 A surface plot of acidic isotypes to depict the effect of elution buffer pH and elution buffer conductivity on the percentage of acidic isotypes.

[0097] Figure 12 Main surface plot to depict the effect of elution buffer pH and elution buffer conductivity on the percentage of major isotypes.

[0098] Figure 13 To depict the effect of elution buffer pH and elution buffer conductivity on the percentage of basic isotypes. Detailed Implementation

[0099] The present invention particularly relates to purification methods for controlling the amount of product-related substances (e.g., aggregates, such as high molecular weight (HMW) aggregates, misfolded substances, or protein fragments) and / or process-related impurities (e.g., host cell proteins (HCPs), host cell nucleic acids, viruses, chromatographic materials, and / or culture medium components) present in purified formulations of anti-α4β7 antibodies or their antigen-binding fragments, such as vedozimab.

[0100] I. Definition

[0101] To make the invention easier to understand, some terms are first defined.

[0102] The cell surface molecule “α4β7 integrin” or “α4β7” (used interchangeably throughout this document) 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 lymphocytes and T lymphocytes, particularly memory CD4+ lymphocytes. α4β7, a typical integrin, can exist in either a quiescent or activated state. Ligands for α4β7 include vascular cell adhesion molecules (VCAMs), fibronectin, and mucosal addressins (MAdCAMs, such as MAdCAM-1). Antibodies binding to α4β7 integrin are referred to herein as “anti-α4β7 antibodies.”

[0103] As used herein, antibodies or their antigen-binding fragments that are "specifically bound to the α4β7 complex" bind to α4β7 but not to α4β1 or α E B7. Vedolizumab is an example of an antibody that has binding specificity to the α4β7 complex.

[0104] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule comprising four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, namely CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody has a crystallizable fragment (Fc) region. In some embodiments, the antibody is an IgG1 isotype and has a κ light chain.

[0105] “CDR” or “complementary determination zone” refers to a highly variable region scattered within a more conservative region called the “frame zone” (FR).

[0106] As used herein, the term “antigen-binding fragment” or “antigen-binding moiety” for 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 competes with an intact antibody for specific binding to at least one desired epitope (e.g., a separated portion of the complementarity-determining region having sufficient frame sequence to specifically bind to the epitope). Antigen-binding fragments can be generated by recombinant techniques or by enzymatic or chemical cleavage of the antibody.

[0107] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human antibody. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody), wherein residues from the receptor hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capability. In some cases, the frame region (FR) residues of the human antibody are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable CDR loops correspond to the hypervariable CDR loops of the non-human antibody, and all or substantially all of the FRs are FRs of the human antibody sequence. The humanized antibody may also optionally contain at least a portion of the antibody constant region (Fc), typically a portion of the human antibody. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0108] As used herein, the term "recombinant antibody" refers to an antibody produced by the transcription and translation of a gene carried on a recombinant expression vector introduced into a host cell, such as a mammalian host cell. In some embodiments, the recombinant protein is an isotype antibody selected from the group consisting of: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In some embodiments, the recombinant antibody is IgG1.

[0109] The term “recombinant host cell” (which is used interchangeably with the term “host cell” herein) includes cells in which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to refer to a specific subject cell but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may actually differ from the parent cells but are still included within the scope of the term “host cell” as used herein. Furthermore, it should be understood that, unless otherwise stated, the use of the term “cell,” such as host cell or mammalian cell or mammalian host cell, is intended to include a population of cells.

[0110] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. The term includes vectors that exhibit a self-replicating nucleic acid structure, as well as vectors incorporated into the genome of a host cell, wherein the vector has been introduced into the host cell. Some vectors are capable of directing the expression of a nucleic acid operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0111] As used herein, in the case of protein (e.g., antibody) formulations, the term "upstream process" refers to activities involving the production and collection of proteins (e.g., antibodies) from host cells (e.g., the production of target proteins, such as antibodies, after cell culture).

[0112] As used herein, the term "downstream process" refers to one or more techniques used after upstream processes to purify a target protein, such as an antibody. For example, downstream process techniques include purifying protein products using, for example, affinity chromatography (including protein A affinity chromatography), ion exchange chromatography (such as anion or cation exchange chromatography), size exclusion chromatography, mixed-mode chromatography, hydrophobic interaction chromatography (HIC), or displacement chromatography.

[0113] As used herein, the terms “culture” and “cell culture” generally refer to the process of growing cells under controlled conditions, typically outside their natural environment. “Culturing” cells means contacting cells with a cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation. In some embodiments, cell culture refers to methods for producing and maintaining a population of host cells capable of producing a target recombinant protein, such as an anti-α4β7 antibody, and methods and techniques for producing and collecting the target protein. For example, after incorporating an expression vector into a suitable host, such as cultured host cells, the host can be maintained under conditions suitable for expressing the relevant nucleotide coding sequence and for collecting and purifying the desired recombinant protein. “Cell culture” can also refer to a solution containing cells.

[0114] As used herein, the term "clarified harvest" refers to a liquid material containing a target protein, such as an anti-α4β7 antibody, extracted from a cell culture, such as a fermentation bioreactor, after undergoing one or more processing steps to remove solid particles, such as cell debris and particulate impurities, from said material. Following cell culture, the harvest is typically purified using separation techniques, such as centrifugation and filtration, to remove cells and cell debris. The initial clarification, particulate removal step produces a "clarified harvest," which can be used, for example, in subsequent chromatographic steps (downstream processing). Clarified harvests are generally used as starting material for downstream processing, such as the downstream processing steps described herein.

[0115] As used herein, a “chromatographic support” refers to a solid or porous matrix having a specific chemical composition or a specific three-dimensional structure, or to which specific chemical groups or macromolecules can be immobilized for performing chromatographic methods, 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, a “chromatographic housing” refers to a structure that houses a chromatographic support. Examples of chromatographic housings include columns or filter cartridges or other containers.

[0116] As used herein, the term "buffer solution" refers to an aqueous solution that resists pH changes through the action of an acid-base conjugate component. Buffer solutions are used to establish a specific set of conditions to mediate control over processing steps or chromatographic supports, such as chromatographic resins or membranes.

[0117] As used herein, the term "equilibration solution" refers to an aqueous liquid prepared to establish initial operating conditions for a processing step or chromatographic carrier, such as a chromatographic operation. Equilibration solutions are used to prepare, for example, solid phases, chromatographic carriers, such as resins or membranes, for loading target proteins (e.g., antibodies).

[0118] As used herein, the terms "washing fluid" or "washing solution" refer to an aqueous liquid formulated to displace unbound contaminants from a chromatographic support, such as a resin or membrane. In some embodiments, the washing fluid is passed through a solid support, such as a resin or membrane, after loading a target protein (e.g., an antibody) and before eluting the target protein (e.g., the antibody). In one embodiment, the washing fluid has biochemical properties similar to those of an equilibrium solution.

[0119] As used herein, “flow-through operation” refers to a process in which proteins are substantially not bound to a matrix, such as a hydrophobic chromatographic resin, and / or eluted during washing, while impurities remain associated with the chromatographic carrier.

[0120] As used herein, the terms "elution solution" or "elution buffer" refer to an aqueous liquid prepared for displacing a target protein (e.g., an antibody) from a chromatographic carrier, such as a resin or membrane. In one embodiment, the elution solution has different biochemical properties than the equilibration solution and / or wash solution, thereby making the target protein (e.g., an antibody) more likely to associate with the elution solution than with the chromatographic carrier, such as a resin or membrane.

[0121] As used herein with respect to impurities contained in a solution containing the antibody to be purified, the term "impurity" includes both process-related impurities and product-related impurities.

[0122] As used herein, the term "process-related impurity" refers to one or more impurities present in a protein-containing composition, such as a solution, but not originating from the protein itself. Examples of process-related impurities include, but are not limited to, cell culture medium components, host cell components (such as proteins (HCPs), host cell nucleic acids, or lipid-containing subcellular structures or fragments thereof), viruses, trace metals or ions from buffer solutions, and leachable materials from material handling containers or chromatographic carriers. Process-related impurities may form during the preparation (upstream and / or downstream processing) of proteins, such as antibodies.

[0123] As used herein, the term "host cell impurity" refers to any protein, nucleic acid contaminant, lipid contaminant, or byproduct introduced by a host cell line, cell culture medium, or cell culture. Examples of impurities include, but are not limited to, Chinese hamster ovary protein (CHOP), E. coli protein, yeast protein, simian COS protein, or myeloma cell protein (e.g., NSO protein (derived from mouse plasmacytoma cells of BALB / c mice)).

[0124] As used herein, the term "product-related impurities" includes impurities originating from the target protein (e.g., antibody) itself. For example, product-related impurities include, but are not limited to, aggregates of the target antibody (e.g., HMW), misfolded substances, oxidized or deamidized substances, or low molecular weight fragments.

[0125] As used herein, the term "aggregate" refers to the association of two or more antibodies or antibody fragments. For example, aggregates can be dimers, trimers, tetramers, or larger polymers of antibodies and / or antibody fragments. Antibody aggregates can be soluble or insoluble. Association between aggregate molecules can be covalent or non-covalent, regardless of the mechanism of association. Association can be direct association between aggregate molecules or indirect association by linking said aggregate molecules together via other molecules. Examples of the latter include, but are not limited to, disulfide bonding with other proteins, hydrophobic association with lipids, charge association with DNA, affinity association with leached protein A, or association with a mixed pattern of multiple components. Aggregates can form irreversibly during protein expression in cell culture, during protein purification in downstream processing, or during storage of pharmaceutical products. The presence of aggregates in solution can be determined using, for example, size exclusion chromatography (SEC) (e.g., SEC with UV detection, SEC with light scattering detection (SEC-LSD)), field flow fractionation, analytical ultracentrifugation sedimentation rate, or capillary electrophoresis-sodium dodecyl sulfate (CE-SDS, reduced and non-reduced).

[0126] The term "high molecular weight" or "HMW" is used to indicate antibody complexes with a molecular weight greater than that of the monomeric antibody. In one embodiment, the molecular weight of the HMW aggregate is greater than about 147 kDa. The presence of high molecular weight aggregates can be determined by standard methods known in the art, such as size exclusion chromatography (SEC).

[0127] Regarding the desired protein, "substantially purified" means that the purified sample containing the protein contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% of the desired recombinant protein and less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5% of impurities.

[0128] The term "about" indicates that subsequent values ​​are not exact values, but rather the midpoint of a range of + / - 5% of the value. If the value is a relative value given as a percentage, the term "about" also indicates that subsequent values ​​are not exact values, but rather the midpoint of a range of + / - 5% of the value, whereby the upper limit of the range cannot exceed 100% of the value.

[0129] II. Methods and compositions related to antibody purification

[0130] This document provides a method for purifying anti-α4β7 antibodies, such as vedolizumab, from liquid solutions, such as clarified harvests from mammalian cell cultures. The invention is based, at least in part, on aspects of antibody purification processes that reduce the levels of impurities present in the antibody solution, including, for example, process-related impurities such as cell culture 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 substances, and fragments of the target antibody. The method of the invention can be used to purify anti-α4β7 antibodies, particularly vedolizumab, or antibodies having a binding region of vedolizumab, i.e., a CDR or variable region, thereby enabling the formulation of said antibodies for human patients.

[0131] In particular, the methods disclosed herein can be used to achieve lower levels of antibody aggregation, such as HMW antibody aggregates. In some embodiments, the methods disclosed herein provide antibody aggregates having a content of approximately 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%). Compositions of aggregates, such as HMW aggregates, in the form of 0.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5%. In certain embodiments, the methods disclosed herein provide compositions having aggregates, such as HMW aggregates, at about 0% to 2%, ≤2%, ≤1.9%, ≤1.8%, ≤1.7%, ≤1.6%, ≤1.5%, ≤1.4%, ≤1.3%, ≤1.2%, ≤1.1%, ≤1%, ≤0.9%, ≤0.8%, ≤0.7%, ≤0.6%, or ≤0.5%. The invention also includes compositions comprising an anti-α4β7 antibody and said low levels of HMW aggregates.

[0132] Specifically, the methods disclosed herein can be used to generate anti-α4β7 antibody vedolizumab or antibodies containing the antigen-binding region of vedolizumab. Vedolizumab is also marketed under the trade name... Takeda Pharmaceuticals, Inc. is known to have vedolizumab, a humanized antibody comprising a human IgG1 framework region and a constant region, as well as an antigen-binding CDR derived from the murine antibody Act-1. The CDR, variable region, and mutated Fc region of vedolizumab (mutated to eliminate Fc effector function) are described in U.S. Patent No. 7,147,851, which is incorporated herein by reference.

[0133] Vedolizumab is a humanized monoclonal antibody that specifically binds to α4β7 integrin, such as the α4β7 complex, and blocks the interaction between α4β7 integrin and mucosal addressing cell adhesion molecule-1 (MAdCAM-1), and inhibits the migration of memory T lymphocytes across the endothelium into inflamed gastrointestinal parenchyma. Vedolizumab does not bind to or inhibit the function of α4β1 and αEβ7 integrin, and does not antagonize the interaction between α4 integrin and vascular cell adhesion molecule-1 (VCAM-1).

[0134] α4β7 integrin is expressed on the surface of a small group of distinct memory T lymphocytes that preferentially migrate to the gastrointestinal tract. MAdCAM-1 is primarily expressed on intestinal endothelial cells and plays a crucial role in T lymphocyte homing to intestinal lymphoid tissue. The interaction between α4β7 integrin and MAdCAM-1 has been considered a significant contributing factor to mucosal inflammation, such as chronic inflammation that is a hallmark of ulcerative colitis and Crohn's disease. Vedolizumab is used to treat inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, pouchitis (including chronic pouchitis), graft-versus-host disease, and HIV.

[0135] The heavy chain variable region of vedolizumab is provided herein as SEQ ID NO:1, and the light chain variable region of vedolizumab is provided herein as SEQ ID NO:5. Vedolizumab comprises a heavy chain variable region containing CDR1 of SEQ ID NO:2, CDR2 of SEQ ID NO:3, and CDR3 of SEQ ID NO:4. Vedolizumab comprises a light chain variable region containing CDR1 of SEQ ID NO:6, CDR2 of SEQ ID NO:7, and CDR3 of SEQ ID NO:8. In one embodiment, the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:9 and a light chain containing the amino acid sequence of SEQ ID NO:10. Vedolizumab and its sequences are also described in U.S. Patent Publication No. 2014 / 0341885 and U.S. Patent Publication No. 2014-0377251, the entire contents of which are expressly incorporated herein by reference. The methods disclosed herein can be performed using antibodies containing binding regions, such as CDRs or variable regions, as described above and in the appended sequence listings.

[0136] Methods for generating antibodies are known in the art. Mammalian host cells are engineered to stably express anti-α4β7 antibodies (e.g., vedolizumab). Complete cell culture methods and considerations for generating monoclonal antibodies, such as vedolizumab, are described in Li et al. (2010) mAbs 2:5,466-477, which are incorporated herein by reference; and Birch and Racher (2006) Adv. Drug Delivery Rev. 58:671-685.

[0137] When using cell culture techniques, anti-α4β7 antibodies can be generated intracellularly, in the periplasmic space, or secreted directly into the culture medium. In embodiments where anti-α4β7 antibodies are generated intracellularly, particulate debris from host cells or lysed cells (e.g., generated by homogenization) can be removed in various ways, including but not limited to centrifugation or filtration. In cases where anti-α4β7 antibodies are secreted into the culture medium, the supernatant from such expression systems can first be concentrated using a commercially available protein concentrator filter.

[0138] The culture medium or lysate may undergo one or more processing steps, such as sedimentation, flocculation, centrifugation, and / or filtration, to remove particulate cell debris, thereby forming a clear cell culture supernatant, or a clear harvest. Subsequently, as described in detail below, the antibody, such as an anti-α4β7 antibody (e.g., vedolizumab or an antibody having a binding region corresponding to vedolizumab), is purified to remove impurities, such as process-related impurities, such as cell culture medium components, host cell proteins (HCP), host cell nucleic acids, viruses, and chromatographic materials, as well as product-related impurities, such as aggregates (including HMW aggregates), misfolded substances, and fragments of the target antibody.

[0139] The purification process can begin after the antibody has been produced using the upstream production methods described above and / or alternative production methods conventional in the art. After obtaining a clear solution or mixture containing the antibody, the target antibody can be separated from process-related impurities, such as other proteins produced by cells, and product-related substances. In some non-limiting embodiments, such separation is performed using CEX, AEX, and / or MM chromatography. In some embodiments, a combination of one or more different purification techniques may also be employed, including affinity separation steps, ion exchange separation steps, mixed-mode steps, and / or hydrophobic interaction separation steps. Such additional purification steps separate antibody mixtures based on the antibody's charge, degree of hydrophobicity, and / or size. In one aspect of the invention, such additional separation steps are performed using chromatography, including hydrophobic interaction, anionic interaction, or cation interaction (or combinations thereof). For each of these techniques, many chromatographic resins are commercially available, thereby allowing for precise customization of the purification protocol for the specific antibody involved. Each separation method allows the antibody to cross the column at different rates, thereby achieving physical separation that increases as the antibody further crosses the column, or selectively adheres to the separation resin (or medium). Next, the antibody was eluted differentially using different elution buffers. In some cases, the target antibody separated from the impurities when impurities specifically adhered to the resin of the column and the target antibody did not adhere to the resin, i.e., the target antibody was contained in the flow-through. In other cases, the target antibody adhered to the resin of the column, while impurities and / or product-related substances were extruded from the resin of the column during the wash cycle. Subsequently, the antibody was released due to changes in the liquid around the resin, and the target antibody eluted from the column.

[0140] In some embodiments, during the “capture step,” the antibody-containing solution undergoes affinity chromatography to purify the antibody and remove impurities. In some embodiments, the chromatographic material is capable of selectively or specifically binding to the target antibody (“capture”). Non-limiting examples of such chromatographic materials include: protein A, protein G, chromatographic materials containing, for example, an antigen bound to by the target antibody, and chromatographic materials containing Fc-binding proteins.

[0141] In specific embodiments, the affinity chromatography steps described herein involve passing a clarified harvest containing anti-α4β7 antibodies through a protein A matrix, such as a column containing a protein A resin. In some embodiments, the protein A resin can be used for the affinity purification and separation of various antibody isotypes, particularly IgG1, IgG2, and IgG4. Protein A is a bacterial cell wall protein that binds primarily to mammalian IgG via its Fc region. In its native state, protein A possesses five IgG-binding domains as well as other domains with unknown functions.

[0142] Anti-α4β7 antibody was purified using Protein A resin.

[0143] In one aspect, the method described herein includes purifying an anti-α4β7 antibody (e.g., vedolizumab) from a liquid solution containing an antibody and one or more impurities, such as a clarified harvest, using protein A. The method includes binding the anti-α4β7 antibody to an affinity chromatography matrix, such as protein A. In some embodiments, antibody solutions exceeding 10 g / L, such as 10 to 50 g / L, 20 to 45 g / L, or 30 to 40 g / L, can be loaded onto the affinity chromatography matrix. For example, concentrations of about 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, etc., can be used. Antibody solutions of g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, or 50 g / L were loaded onto the protein A affinity chromatography matrix.

[0144] Protein A resins are available from several commercial sources. One suitable resin is MabSelect™ from GE Healthcare. Suitable resins include, but are not limited to, MabSelect SuRe from GE Healthcare. TM , MabSelect SuRe LX, MabSelect, MabSelect Xtra, rProtein A Sepharose; MabSelect from EMDMillipore TMProA resin, ProSep HC, ProSep Ultra, and ProSep Ultra Plus; from MabCapture, Life Technologies.

[0145] Before loading the sample, the protein A column can be equilibrated with a suitable equilibration solution. After column loading, the column can be washed once or multiple times with a suitable set of solutions to reduce one or more impurities, while the anti-α4β7 antibody remains bound to protein A.

[0146] In some embodiments, the protein A matrix is ​​washed more than once. In some embodiments, the protein A matrix is ​​washed three times. In one embodiment, one or more washing solutions contain phosphate. In one embodiment, the affinity column may be washed with an initial washing solution containing PBS, followed by a second washing solution containing NaCl and PBS, and then a third washing solution containing PBS. In one embodiment, the first and third washing solutions are the same. In one embodiment, the second washing solution contains NaCl (e.g., 1M NaCl) and PBS, and has a pH of 7.2. In another embodiment, one or more washing solutions have a pH of about 7.0-7.4. In one embodiment, one or more washing solutions have a pH of about 7.2.

[0147] In other embodiments, the affinity column is washed with an initial wash solution containing PBS, followed by second and third washes containing buffers such as citrate, acetate, or phosphate. In one embodiment, the second and third solutions contain sodium citrate buffer. In one embodiment, the sodium citrate buffer in the second and third wash solutions is the same. In another embodiment, the sodium citrate buffer in the second wash solution is different from that in the third wash solution. In one embodiment, the molar concentration of sodium citrate buffer in the second wash solution is higher than that in the third wash solution. In one embodiment, the molar concentration of sodium citrate buffer in the second wash solution is 75 mM to 125 mM or 100 mM and the molar concentration of sodium citrate buffer in the third wash solution is 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.

[0148] Next, the protein column A can be eluted using a suitable elution solution. For example, glycine-HCl, acetic acid, or citric acid can be used as the elution solution. In one embodiment, the elution solution is citric acid, such as sodium citrate. In some embodiments, the pH of the elution solution can be about 3.0 to 4.0 (e.g., about 3.1 to 4.0, 3.2 to 4.0, 3.3 to 4.0, 3.4 to 4.0, 3.5 to 4.0, 3.6 to 4.0, 3.7 to 4.0, 3.8 to 4.0, or 3.9 to 4.0). In some embodiments, the pH of the elution solution is about 3.0 to 3.4. In some embodiments, the pH of the elution solution can be 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 pH of the eluent is equal to or higher than 3.3. In some embodiments, the pH of the eluent is equal to or higher than 3.4. In some embodiments, the pH of the eluent is equal to or higher than 3.5. In some embodiments, the pH of the eluent is equal to or higher than 3.6. In some embodiments, the pH of the eluent is equal to or higher than 3.7. In some embodiments, the pH of the eluent is equal to or higher than 3.8. In some embodiments, the pH of the eluent is equal to or higher than 3.9. The eluent can be monitored using techniques well known to those skilled in the art. Target eluent fractions can be collected and then prepared for further processing.

[0149] As shown in the examples, compared to embodiments with a lower pH value, such as 2.9 to 3.3, the protein A affinity column elution buffer has a higher pH value, such as 3.3 to 4.0, and the elution buffer containing the anti-α4β7 antibody has fewer impurities, such as HMW aggregates. In some embodiments, the eluent contains anti-α4β7 antibody and contains about 0% to 5.0% (e.g., 0%-0.1%, 0%-0.2%, 0%-0.3%, 0%-0.4%, 0%-0.5%, 0%-0.6%, 0%-0.7%, 0%-0.8%, 0%-0.9%, 0%-1%, 0%-1.1%, 0%-1.2%, 0%-1.3%, 0%-1.4%, 0%-1.5%, 0%-1.6%, 0%-1.7%, 0%-1.8%, 0%-1.9%, 0%-2%, 0%-2.5%, 0%-3%, 0%-3.5%, 0%-4%, 0%-4.5%, or 0%-5%) of HMW aggregates. In some embodiments, the eluent contains anti-α4β7 antibody and contains about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In a particular embodiment, the protein A resin eluent contains anti-α4β7 antibody and about 0% to 2%, ≤2%, ≤1.9%, ≤1.8%, ≤1.7%, ≤1.6%, ≤1.5%, ≤1.4%, ≤1.3%, ≤1.2%, ≤1.1%, ≤1%, ≤0.9%, ≤0.8%, ≤0.7%, ≤0.6%, ≤0.5%, ≤0.4%, ≤0.3%, ≤0.2%, or ≤0.1% of aggregates, such as HMW aggregates. In one embodiment, the eluent contains anti-α4β7 antibody and about 1.2% or less of HMW aggregates. In another embodiment, the eluent contains anti-α4β7 antibody and about 1.1% or less of HMW aggregates. In one embodiment, the eluent contains an anti-α4β7 antibody and contains about 1% or less (e.g., about 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In another embodiment, the eluent contains an anti-α4β7 antibody and contains about 0.9% or less of HMW aggregates.

[0150] The buffers and methods described herein reduce the level of host cell protein (HCP) in compositions, such as those containing anti-α4β7 antibody eluted from a protein A resin, relative to the level of HCP when using an elution buffer that does not have one or more of the parameters described herein. In some embodiments, the protein A resin eluent comprises a composition containing 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). In some embodiments, the protein A resin eluent comprises a composition containing an anti-α4β7 antibody and about 1-250 ppm (e.g., about 1-240 ppm, 1-230 ppm, 1-220 ppm, 1-210 ppm, 1-200 ppm, 1-190 ppm, 1-180 ppm, 1-170 ppm, 1-160 ppm, 1-150 ppm, 1-140 ppm, 1-130 ppm, 1-120 ppm, 1-100 ppm, 1-90 ppm, 1-80 ppm, 1-70 ppm, 1-60 ppm, 1-50 ppm, 1-60 ppm, 1-50 ppm, 1-40 ppm, 1-30 ppm, 1-20 ppm, 1-10 ppm, 1-9 ppm, 1-8 ppm, 1-7 ppm, 1-6 ppm, 1-5 ppm, 1-4 ppm, 1-3 ppm, or 1-2 ppm) of HCP.

[0151] In one embodiment, anti-α4β7 antibodies bound to protein A are eluted with an elution buffer with a pH greater 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 produce an elution buffer containing anti-α4β7 antibodies and reduced levels of HMW aggregates and / or an elution buffer containing anti-α4β7 antibodies and reduced levels of HCP. In one embodiment, the pH of the elution solution is 3.3 to 3.9. In one embodiment, the pH of the elution buffer is 3.3 to 3.8. In one embodiment, the pH of the elution buffer is 3.4 to 3.6. In one embodiment, the pH of the elution buffer is 3.4-4.0. In one embodiment, the elution buffer contains citric acid, such as sodium citrate, for example, 100 mM citric acid or 25 mM citric acid. In one embodiment, the protein A affinity column is washed and eluted in a buffer containing 25 mM sodium citrate, wherein the washing buffer has a pH of 5.6 to 6.2, 5.7 to 5.9, or 5.8, and the elution buffer has a pH of 3.3 to 3.9, 3.4 to 3.6, or 3.5.

[0152] In some embodiments, the material loaded onto the protein A resin is, for example, a clarified cell culture harvest from a recombinant cell line expressing an anti-α4β7 antibody. In some embodiments, the recombinant cell line (i.e., the host cell line) may be Chinese hamster ovary (CHO) cells. In some embodiments, the CHO cells may be GS-CHO cells lacking a gene encoding glutamine synthase. In some embodiments, the CHO cells may be DHFR-CHO cells lacking a gene encoding dihydrofolate reductase.

[0153] The pH and / or conductivity of the protein A eluent can be adjusted for subsequent purification steps. The protein A eluent can also be filtered via a depth filter to remove turbidity and / or various impurities from the target antibody prior to additional chromatographic purification steps.

[0154] Anti-α4β7 antibody was purified using HIC resin.

[0155] As detailed below and as stated in Example 2, antibodies, such as anti-α4β7 antibodies (e.g., vedolizumab or antibodies having a binding region corresponding to vedolizumab), can also be purified using downstream process techniques after protein A purification. The purification step in the downstream process is often referred to as a “refining” step and presents a unique challenge: the level of impurities may be relatively low, but given the nature of the antibody intended for human use, even lower levels are desirable.

[0156] In one aspect, the method described herein includes purifying the antibody from a liquid solution containing anti-α4β7 antibody and one or more impurities, such as a clarified harvest, using a hydrophobic interaction chromatography (HIC) resin.

[0157] In one embodiment, the present invention provides a way to reduce high molecular weight antibodies in anti-α4β7 antibody solutions.

[0158] A method for treating (HMW) aggregates, the method comprising contacting the antibody solution with a hydrophobic interaction chromatography (HIC) resin. Hydrophobic interaction chromatography (HIC) separates proteins based on differences in surface hydrophobicity by utilizing reversible interactions between proteins and the hydrophobic surface of a HIC resin (e.g., a polymer matrix modified with hydrophobic ligands). Given the hydrophobicity of anti-α4β7 antibodies, such as vedolizumab, highly hydrophobic HIC resins can be used during purification processes to remove impurities, including HMW aggregates, residual protein A, and / or host cell protein (HCP) contaminants, wherein the anti-α4β7 antibody flows through the HIC resin without binding. In some embodiments, the highly hydrophobic HIC resin suitable for use in the methods described herein comprises a polymethacrylate base material bonded to a C6 group, such as Toyopearl Hexyl-650C (Tosoh Biosciences).

[0159] In some implementations, HIC is used in “flow-through mode.” Therefore, as used herein, “flow-through fraction” refers to proteins collected in fractions in the mobile phase buffer that have passed through a resin-containing column as provided herein.

[0160] In some embodiments, a solution containing an anti-α4β7 antibody and at least one impurity is contacted with a hydrophobic interaction chromatography (HIC) resin under conditions that allow the anti-α4β7 antibody to flow 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 with a pH less than about 7.2. In one embodiment, the HIC resin is equilibrated with a buffer with a pH of about 5.5 to about 7.2. In one embodiment, the HIC resin is equilibrated with a buffer with 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 loading is about 55 to 75 mg / ml.

[0161] In one embodiment, the method for purifying anti-α4β7 antibody using a HIC column includes passing a solution containing anti-α4β7 antibody through the column, i.e., the purification includes collecting the anti-α4β7 antibody in the column flow-through while contaminants remain bound to the column, wherein the anti-α4β7 antibody and the column are in a solution containing phosphate, such as potassium phosphate, at a concentration of 150 to 300 mM, 175 to 250 mM, or about 200 mM and a pH of 5.2 to 6.5, 5.7 to 6.2, or about 5.9.

[0162] In some embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions comprising 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%) of HMW aggregates. In some embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions comprising anti-α4β7 antibodies and about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In certain embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions comprising anti-α4β7 antibody and about 0% to 2%, ≤2%, ≤1.9%, ≤1.8%, ≤1.7%, ≤1.6%, ≤1.5%, ≤1.4%, ≤1.3%, ≤1.2%, ≤1.1%, ≤1%, ≤0.9%, ≤0.8%, ≤0.7%, ≤0.6%, ≤0.5%, ≤0.4%, ≤0.3%, ≤0.2%, or ≤0.1% HMW aggregates. In some embodiments, such methods using highly hydrophobic HIC resins can be used to obtain compositions comprising anti-α4β7 antibody and less than 0.6% HMW aggregates. In one embodiment, a composition comprising anti-α4β7 antibody and less than 0.5% HMW aggregates is obtained. In one embodiment, a composition comprising anti-α4β7 antibody and less than 0.4% HMW aggregates is obtained. Furthermore, the composition may contain less than about 0.3 ppm of host cell protein (HCP), wherein the host cell is a Chinese hamster ovary (CHO) cell, such as GS-CHO cells. In one embodiment, the composition contains less than about 0.22 ppm of residual protein A.

[0163] Anti-α4β7 antibody was purified using a mixed-mode chromatographic resin.

[0164] In one aspect, this document provides a method for purifying an anti-α4β7 antibody (e.g., vedolizumab) from a liquid solution, such as a clarified cell culture harvest, containing an anti-α4β7 antibody and one or more impurities in a binding / elution mode using a mixed-mode chromatographic resin. In some embodiments, the mixed-mode chromatographic resin has properties suitable for high impurity removal and high capacity. In one embodiment, the mixed-mode chromatographic resin for purifying the anti-α4β7 antibody comprises strong anion exchange, hydrogen bonding, and hydrophobic bonding capabilities. In another embodiment, the mixed-mode chromatographic resin for purifying the anti-α4β7 antibody comprises beads with small diameters, such as beads of about 35-45 μm, exhibiting strong anion exchange, hydrogen bonding, and hydrophobic bonding capabilities. In some embodiments, the mixed-mode chromatographic resin used in the methods and compositions described herein is CAPTO. TM Adhere ImpRes (GE Healthcare Life Sciences, now Global Life Sciences Solutions, LLC). In some embodiments, the mixed-mode chromatographic resin used in the methods and compositions described herein is CAPTO. TM Adhere (GE Healthcare Life Sciences, now Global Life Sciences Solutions, LLC). Clarified cell culture harvests may be derived from host cells recombinantly expressing anti-α4β7 antibodies. In some embodiments, the host cells may be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells.

[0165] The mixed-mode chromatography method described herein involves 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), may be used before and / or after the mixed-mode chromatography method described herein. Therefore, in some embodiments, the loading material for the mixed-mode chromatography may comprise protein A eluent, AEX eluent, CEX eluent, or HIC eluent, or collected HIC flow material. In some embodiments, the mixed-mode chromatography method described herein may further include washing the mixed-mode resin with a washing solution and eluting the antibody from the resin.

[0166] In some embodiments, an antibody solution of 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) can be loaded onto a mixed-mode chromatography resin. For example, an antibody solution of at least 55 g / L can be loaded onto a mixed-mode chromatography resin. In some embodiments, antibody solutions of about 25 g / L to about 100 g / L, such as 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 a mixed-mode chromatography resin. For example, an antibody solution of about 55 g / L to about 80 g / L can be loaded onto a mixed-mode chromatography resin.

[0167] The resin may optionally be washed with a suitable washing buffer that will not elute the bound antibody from the resin. In one embodiment, the resin may optionally be washed with a sodium phosphate washing buffer. In some embodiments, the washing buffer may contain 10 mM sodium phosphate, 25 mM sodium phosphate, 50 mM sodium phosphate, or 75 mM sodium phosphate at a neutral or near-neutral pH (e.g., pH 6-8). Other suitable washing buffers compatible with mixed-mode chromatography are widely available.

[0168] This article describes buffers for increasing the yield of anti-α4β7 antibody preparations and / or reducing the level of its aggregates (e.g., HMW substance %) after elution from mixed-mode chromatography resins. To increase the yield of anti-α4β7 antibody and / or reduce the level of aggregates (e.g., HMW substance %), the pH and / or conductivity of the mixed-mode elution buffer can be adjusted. Suitable elution solutions compatible with mixed-mode chromatography are widely available. In some embodiments, the mixed-mode chromatography elution solution contains a buffer such as citrate, acetate, or phosphate.

[0169] In some embodiments, the elution buffer used with the mixed-mode chromatography resin in the methods described herein has a pH value equal to or higher than pH 3.5 (e.g., equal to or higher than pH 3.6, pH 3.7, pH 3.8, pH 3.9, pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, or pH 4.5). For example, the elution buffer used with the mixed-mode chromatography resin may have a pH value equal to or higher than pH 3.9. In some embodiments, the elution buffer used with the mixed-mode chromatography resin in the methods described herein has a pH of about pH 3.9 to about pH 4.5 (e.g., about pH 3.9 to about pH 4.5, about pH 3.9 to about pH 4.4, about pH 3.9 to about pH 4.3, about pH 3.9 to about pH 4.2, about pH 3.9 to about pH 4.1, or about pH 3.9 to about pH 4.0). In some embodiments, the elution buffer used for the mixed-mode chromatography provided herein may have a pH of about pH 3.9 to about pH 4.4.

[0170] In other or alternative embodiments, the elution buffer used with the mixed-mode chromatography resin in the methods described herein has a pH value equal to or lower than pH 4.5 (e.g., equal to or lower than pH 4.4, equal to or lower than pH 4.3, equal to or lower than pH 4.2, equal to or lower than pH 4.1, equal to or lower than pH 4.0, equal to or lower than pH 3.9, equal to or lower than pH 3.8, equal to or lower than pH 3.7, equal to or lower than pH 3.6, or equal to or lower than pH 3.5). For example, the elution buffer used with the mixed-mode chromatography resin may have a pH value equal to or lower than pH 4.2. In some embodiments, the elution buffer used with the 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, the pH of the elution buffer used with the mixed-mode chromatography resin may be about pH 4.2 to about pH 3.8.

[0171] In some embodiments, the elution buffer used with the mixed-mode chromatography resin in the methods described herein has a conductivity of about 40 mS / cm or lower (e.g., about 39 mS / cm, 38 mS / cm, 37 mS / cm, 36 mS / cm, 35 mS / cm, 34 mS / cm, 33 mS / cm, 32 mS / cm, 31 mS / cm, 30 mS / cm, 29 mS / cm, 28 mS / cm). (e.g., 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 lower). For example, the conductivity of the elution buffer used with the mixed-mode chromatography resin may be about 30 mS / cm or lower. In some embodiments, the conductivity of the elution buffer used with the mixed-mode chromatography resin in the methods described herein is about 10 mS / cm to about 40 mS / cm, for example, about 15 mS / cm to about 35 mS / cm or about 20 mS / cm to about 30 mS / cm. For example, the conductivity of the elution buffer used with mixed-mode chromatography resin can be from about 20 mS / cm to about 30 mS / cm.

[0172] In other or alternative embodiments, the conductivity of the elution buffer used with the mixed-mode chromatography resin in the methods described herein is equal to or less than 30 mS / cm (e.g., equal to or less than 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). For example, the conductivity of the elution buffer used with the mixed-mode chromatography resin may be equal to or less than 28 mS / cm. In some embodiments, the elution buffer used 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, 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 30 mS / cm). (e.g., approximately 17 mS / cm to approximately 30 mS / cm, approximately 18 mS / cm to approximately 30 mS / cm, approximately 19 mS / cm to approximately 30 mS / cm, approximately 20 mS / cm to approximately 30 mS / cm, approximately 21 mS / cm to approximately 30 mS / cm, approximately 22 mS / cm to approximately 30 mS / cm, approximately 23 mS / cm to approximately 30 mS / cm, approximately 24 mS / cm to approximately 30 mS / cm, approximately 25 mS / cm to approximately 30 mS / cm, approximately 26 mS / cm to approximately 30 mS / cm, or approximately 27 mS / cm to approximately 30 mS / cm). For example, in some embodiments, the conductivity of the elution buffer used with the mixed-mode chromatography resin may be approximately 18 mS / cm to approximately 28 mS / cm.

[0173] In some embodiments, the elution buffer used with the mixed-mode chromatography resin in the methods described herein may contain 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, the elution buffer used with the mixed-mode chromatography resin may have NaCl at a concentration of about 160 mM to about 240 mM. In some embodiments, the elution buffer used with the 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.

[0174] In some embodiments, the method of purifying anti-α4β7 antibodies, such as vedolizumab, from liquid solutions, such as clarified cell culture harvests, using mixed-mode chromatographic resins includes loading the anti-α4β7 antibody at a concentration of 40 to 90 g, 50 to 80 g, or about 65 g of protein per liter of resin onto a column containing the mixed-mode resin, washing the column, and eluting the column with an elution buffer, such as sodium citrate buffer, at a pH of 3.5 to 4.5, 3.9 to 4.4, or about 4.1. In some embodiments, the method further includes eluting the column with an ionic salt, such as NaCl, so that the conductivity of the elution buffer is 15 to 35 mS / cm, 20 to 30 mS / cm, or about 24 mS / cm. In some embodiments, the method includes loading the antibody at a concentration of 53-77 g of protein per liter of resin onto a column containing the mixed-mode resin. In some embodiments, the method includes eluting the antibody from the column using an elution buffer with a pH of about 3.9-4.4 and a conductivity of about 20-28 mS / cm.

[0175] In some implementations, purification of anti-α4β7 antibodies can be achieved using the mixed-mode chromatography combined with cation exchange (CEX) chromatography described herein.

[0176] In some implementations, the methods described herein can improve the yield of anti-α4β7 antibodies eluted from mixed-mode columns compared to the yield of a suitable control process using an elution buffer that does not have one or more parameters described herein, such as a process performed using an elution buffer with a pH of 3.7 or lower, 3.6 or lower, 3.5 or lower, 3.3 or lower, or 3.0 or lower. In some implementations, the yield is increased 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 higher percentage. In some embodiments, the buffers and methods described herein can achieve recoveries of 50% or higher (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher) of anti-α4β7 antibodies eluted from mixed-mode columns. In some embodiments, the buffers and methods described herein can achieve recoveries of 50%–95% (e.g., 55%–95%, 60%–95%, 65%–95%, 70%–95%, 75%–95%, 80%–95%, 85%–95%, 90%–95%, or higher) of anti-α4β7 antibodies eluted from mixed-mode columns.

[0177] In some embodiments, such compositions and methods using mixed-mode chromatographic resins can be used to obtain compositions comprising anti-α4β7 antibody and about 0% to 2.0% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%) of HMW aggregates. In some embodiments, such methods using mixed-mode chromatographic resins can be used to obtain compositions comprising anti-α4β7 antibody and about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of HMW aggregates. In a particular embodiment, such a method using a mixed-mode chromatographic resin can be used to obtain a composition comprising an anti-α4β7 antibody and about 0% to 2%, ≤2%, ≤1.9%, ≤1.8%, ≤1.7%, ≤1.6%, ≤1.5%, ≤1.4%, ≤1.3%, ≤1.2%, ≤1.1%, ≤1%, ≤0.9%, ≤0.8%, ≤0.7%, ≤0.6%, ≤0.5%, ≤0.4%, ≤0.3%, ≤0.2%, or ≤0.1% 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 higher percentage relative to the level of HMW aggregates in the load material. In some embodiments, the mixed-mode chromatography method provided herein can be used to reduce the level of HMW aggregates in compositions containing anti-α4β7 antibodies, relative to the level of HMW aggregates obtained from a suitable control process using an elution buffer that does not have one or more parameters described herein, for example, relative to a process performed using an elution buffer with a pH of 3.7 or lower, 3.6 or lower, 3.5 or lower, 3.3 or lower, or 3.0 or lower.In some implementations, 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 higher percentage relative to a suitable control.

[0178] Anti-α4β7 antibody was purified using cation exchange resin (CEX).

[0179] In one aspect, this document provides a method for purifying an anti-α4β7 antibody, such as vedolizumab, from a liquid solution, such as a clarified cell culture harvest, comprising a binding / elution mode using a cation exchange (CEX) resin, including an anti-α4β7 antibody, such as vedolizumab, and one or more impurities. In some embodiments, the CEX resin used for purifying the anti-α4β7 antibody is a strong cation exchange resin. In some embodiments, the CEX resin suitable for use in the methods and compositions described herein comprises -SO3-. - Functional groups. For example, in some embodiments, the CEX resin is Nuvia HR-S. The clarified cell culture harvest may be derived from host cells recombinantly expressing anti-α4β7 antibodies. In some embodiments, the host cells may be Chinese hamster ovary (CHO) cells, such as GS-CHO cells or DHFR-CHO cells.

[0180] The CEX method described herein involves 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), may be used before and / or after the CEX method described herein. Therefore, in some embodiments, the loading material for CEX chromatography may comprise a protein A eluent, an AEX eluent, a mixed-mode eluent, or a HIC eluent. In some embodiments, the CEX method described herein may further include washing the CEX resin with a washing solution and eluting the antibody from the resin. Solutions compatible with CEX chromatography and suitable for, for example, loading, washing, and eluting proteins, such as anti-α4β7 antibodies, are widely available. In some embodiments, the CEX chromatography solution comprises a buffer, such as citrate, acetate, or phosphate.

[0181] In some embodiments, an antibody solution of at least 20 g / L (e.g., at least 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, or 100 g / L) can be loaded onto CEX resin. For example, an antibody solution of at least 25 g / L can be loaded onto CEX resin. In some embodiments, an antibody solution of about 25-100 g / L (e.g., about 25-90 g / L, 25-80 g / L, 25-70 g / L, 25-60 g / L, 25-50 g / L, 25-40 g / L, or 25-30 g / L) can be loaded onto CEX resin. In some embodiments, an antibody solution of about 25-70 g / L (e.g., about 25-65 g / L, 30-60 g / L, 35-55 g / L, or 40-50 g / L) can be loaded onto CEX resin. For example, an antibody solution of about 30-60 g / L can be loaded onto CEX resin.

[0182] The resin may optionally be washed with a suitable washing buffer that will not elute the bound antibody from the resin. In some embodiments, the washing buffer has the same composition as the buffer used to load the antibody onto the resin. In one embodiment, the resin may optionally be washed with a sodium acetate buffer, such as 25 mM sodium acetate, 50 mM sodium acetate, 75 mM sodium acetate, or 100 mM sodium acetate. In some embodiments, the washing buffer has a pH range of pH 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 washing buffers compatible with CEX chromatography are widely available.

[0183] The pH and / or conductivity of the elution buffer can be adjusted to regulate the levels of HMW aggregates, the dominant isotype, the acidic isotype, and / or the basic isotype in the anti-α4β7 antibody formulation eluted from the CEX resin. In some embodiments, the pH of the elution buffer used with the CEX resin in the methods described herein is equal to or lower than pH 6.0 (e.g., equal to or lower than 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). In some embodiments, the elution buffer used with the 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, the pH of the elution buffer used with the CEX resin may be about pH 5.1 to about pH 5.8. In some embodiments, the pH of the CEX elution buffer is the same as that of the wash buffer.

[0184] In other or alternative embodiments, the elution buffer used with CEX resin in the methods described herein has a conductivity equal to or less than 20 mS / cm (e.g., equal to or less than 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). For example, the conductivity of the elution buffer used with CEX resin may be equal to or less than 16 mS / cm. In some embodiments, the elution buffer used with 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, the conductivity of the elution buffer used with CEX resin may be about 11 mS / cm to about 16 mS / cm. Alternatively or additionally, the conductivity of the elution buffer used with CEX resin may be equal to or less than 14 mS / cm. In some embodiments, the elution buffer used with CEX resin in the methods described herein has a conductivity of about 11 mS / cm to about 14 mS / cm, such as about 12 mS / cm to about 14 mS / cm or about 13 mS / cm to about 14 mS / cm. For example, the conductivity of the elution buffer used with CEX resin may be about 12 mS / cm to about 14 mS / cm. Alternatively or additionally, the conductivity of the elution buffer used with CEX resin may be equal to or greater than 11 mS / cm (e.g., equal to or greater than 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). For example, in some embodiments, the conductivity of the elution buffer used with CEX resin may be equal to or greater than 12 mS / cm.

[0185] In some embodiments, the elution buffer used with CEX resin in the methods described herein may have a concentration of about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM of NaCl. For example, the elution buffer used with CEX resin may have a concentration of about 90-120 mM of NaCl. In some embodiments, the elution buffer used with CEX resin in the methods described herein has a concentration of about 50-150 mM (e.g., about 50-140 mM, 60-130 mM, 70-120 mM, 80-110 mM, or 90-100 mM of NaCl). In certain embodiments, the elution buffer used with CEX resin in the methods described herein has a concentration of about 70-120 mM (e.g., about 70-110 mM, 70-100 mM, 70-90 mM, or 70-80 mM) of NaCl. For example, the elution buffer used with CEX resin may have a concentration of about 70-110 mM of NaCl.

[0186] In some embodiments, the method of purifying anti-α4β7 antibodies, such as vedolizumab, from liquid solutions, such as clarified cell culture harvests, using CEX resin, such as a strong cation exchange resin, includes loading the anti-α4β7 antibody at a concentration of 40 to 90 g, 50 to 65 g, or about 57 g of protein per liter of resin onto a column containing CEX resin, washing the column, and eluting the column with a buffer, such as sodium acetate buffer, at a pH of 5 to 6, 5.2 to 5.6, or about 5.4. In some embodiments, the method further includes an ionic salt, such as NaCl, so that the conductivity of the elution buffer is 5 to 25 mS / cm, 10 to 17 mS / cm, or about 13 mS / cm. In other embodiments, the method of purifying anti-α4β7 antibodies, such as vedolizumab, from liquid solutions, such as clarified cell culture harvests, using CEX resins, such as strong cation exchange resins, includes eluting the column with a buffer, such as sodium acetate buffer, with a pH of 5 to 6, 5.2 to 5.6, or about 5.4 and a conductivity of 5 to 25 mS / cm, 10 to 15 mS / cm, or about 13 mS / cm. In one embodiment, the method includes eluting the column with an elution buffer with a pH of about 5.4 and a conductivity of about 13 mS / cm. In some embodiments, the CEX resin is loaded with antibody at a concentration of about 57 g of protein per liter of resin.

[0187] In some implementations, the purification of anti-α4β7 antibodies can be achieved using the CEX resin combined with mixed-mode chromatography described herein.

[0188] In some embodiments, the CEX method described herein can be used to obtain compositions 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 CEX resin can be used to obtain compositions comprising anti-α4β7 antibody and about 2% or less (e.g., about 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less) of HMW aggregates. In a particular embodiment, such a method using CEX resin can be used to obtain a composition comprising anti-α4β7 antibody and about 0% to 2%, ≤2%, ≤1.9%, ≤1.8%, ≤1.7%, ≤1.6%, ≤1.5%, ≤1.4%, ≤1.3%, ≤1.2%, ≤1.1%, ≤1%, ≤0.9%, ≤0.8%, ≤0.7%, ≤0.6%, ≤0.5%, ≤0.4%, ≤0.3%, ≤0.2%, ≤0.1%, ≤0.09%, ≤0.08%, ≤0.07%, ≤0.06%, ≤0.05%, ≤0.04%, ≤0.03%, ≤0.02%, or ≤0.01% aggregates, such as 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 higher percentage relative to the level of HMW aggregates in the loaded material.In some embodiments, the CEX method described herein can be used to reduce the level of HMW aggregates in anti-α4β7 antibody formulations relative to the level obtained using an elution buffer that does not have one or more parameters as described herein, for example, relative to a method performed using an elution buffer with a pH of 6.3 or higher, 6.5 or higher, 6.7 or higher, or 6.9 or higher and / or a conductivity of 18 mS / cm or higher, 19 mS / cm or higher, 20 mS / cm or higher, 22 mS / cm or higher, or 24 mS / cm or higher. In some implementations, 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 higher percentage relative to a suitable control.

[0189] In some embodiments of the methods provided herein, the pH and / or conductivity of the elution buffer used to elute anti-α4β7 antibodies from CEX resin can be used to adjust the isotype distribution of anti-α4β7 antibodies present in the eluent. For example, the pH and / or conductivity of the elution buffer can be used to increase the percentage of the major (dominant) antibody isotype, decrease the percentage of acidic isotypes, and / or decrease the percentage of basic isotypes.

[0190] In some implementations, the pH of the selected elution buffer may be 6.0 or lower, such as 5.9 or lower, 5.8 or lower, 5.7 or lower, 5.6 or lower, 5.5 or lower, 5.4 or lower, 5.3 or lower, or 5.2 or lower, such as pH 4.5-6.0, pH 4.5-5.5, or pH 5.0-6.0. In some embodiments, the conductivity of the selected elution buffer may be at least 10 mS / cm, such as at least 11 mS / cm, at least 12 mS / cm, at least 13 mS / cm, at least 14 mS / cm, at least 15 mS / cm, at least 16 mS / cm or higher, such as 10-17 mS / cm, 12-17 mS / cm, 13-17 mS / cm, 14-17 mS / cm, 15-17 mS / cm, 16-17 mS / cm, 10-16 mS / cm, 12-16 mS / cm, 13-16 mS / cm, 14-16 mS / cm, or 15-16 mS / cm. In some embodiments, the aforementioned elution buffer conditions can be used to obtain compositions containing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% or more of a major isotype of anti-α4β7 antibody. In some embodiments, the aforementioned elution buffer conditions can be used to obtain compositions containing 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) of a basic isotype. In a particular embodiment, such a method using CEX resin can be used to obtain a composition comprising a major isotype of anti-α4β7 antibody and about ≤20%, ≤19%, ≤18%, ≤17%, ≤16%, ≤15%, ≤14%, ≤13%, ≤12%, ≤11%, ≤10%, ≤9%, ≤8%, ≤7%, ≤6%, ≤5%, ≤4%, ≤3%, ≤2%, or ≤1% of a basic isotype. In other embodiments, the level of basic isotype 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 higher percentage relative to the level of basic isotype species in the loaded material.

[0191] III. Analytical Methods

[0192] In some embodiments, the levels of aggregates, monomers, and fragments in a chromatographic sample produced using the techniques described herein are analyzed. In some embodiments, size exclusion chromatography (SEC) is used to measure aggregates, monomers, and fragments per molecule. For example, but not limitingly, is the TSK-gel G3000SWxL, 5μm. The 7.8×300mm column (Tosoh Bioscience) can be used in conjunction with certain implementation schemes, while the TSK-gel Super SW3000, 4μm, A 4.6 × 300 mm column (Tosoh Bioscience) can be used as an alternative implementation. In some implementations, the aforementioned column is used in conjunction with an Agilent or Shimazhu HPLC system. In some implementations, sample injection is performed under isocratic elution conditions using a mobile phase consisting of, for example, 100 mM sodium sulfate and 100 mM sodium phosphate at pH 6.8, and UV absorbance is detected at 214 nm. In some implementations, the mobile phase consists of 1X PBS at pH 7.4, and the elution profile is detected using UV absorbance at 280 nm. In some implementations, quantification is based on the relative area of ​​the detected peaks.

[0193] Any additional techniques, such as mass spectrometry, can be used to determine size variants.

[0194] The various parameters of the antibodies or their antigen-binding moieties reported in this article can be measured using standard analytical methods and techniques, such as those described below.

[0195] In the various embodiments described herein, cation exchange chromatography (CEX) can be used to determine the relative amounts of a group of antibodies or their antigen-binding moieties, such as the major isotype, basic isotype, and acidic isotype present in vedolizumab. The CEX method fractionates antibody species based on total surface charge. After dilution to a low ionic strength using a mobile phase, the test sample can be injected into a CEX column equilibrated in a suitable buffer, such as 10 mM sodium phosphate at pH 6.6, such as the Dionex Pro-Pac. TMOn a WCX-10 column (Thermo Fisher Scientific, Waltham, MA (USA)). Antibodies can be eluted using a sodium chloride gradient in the same buffer. Protein elution can be monitored at 280 nm, and peaks can be designated as acidic, basic, or major isotype. Acidic peaks eluted from the column have shorter retention times than major isotype peaks, while basic peaks eluted from the column have longer retention times than major isotype peaks. The percentages of major isotype, the sum of acidic percentages, and the sum of basic percentages are reported. The retention times of the major isotypes in the sample are compared with the retention times of the reference standard to determine concordance. In one embodiment, the CEX assay includes diluting the test sample to a low ionic strength, injecting it onto a CEX column equilibrated in 10 mM sodium phosphate at pH 6.6, eluting the column with a NaCl gradient in this buffer, monitoring peaks at 280 nm and designating the peaks as acidic, major, or basic species peaks, wherein the acidic species peaks are eluted first with the shortest retention time, the major species peaks are eluted second, and the basic species peaks are eluted with the longest retention time, and the peak areas are quantified and their amount is calculated as a percentage of the total peak area.

[0196] In the various embodiments described herein, size exclusion chromatography (SEC) can be used to determine the relative levels of monomers, high molecular weight (HMW) aggregates, and low molecular weight (LMW) degradation products present in a group of antibodies or their antigen-binding moieties, such as vedolizumab. The SEC method separates antibody monomers from HMW substances and LMW degradation products based on size. Commercially available SEC columns can be used, along with appropriate buffers to analyze test samples and reference standards. For example, in some embodiments, SEC analysis can be performed using a single G3000 SWxl column (Tosoh Bioscience, King of Prussia, PA (USA)) or two G3000 SWxl columns connected in tandem with an isocratic phosphate-sodium chloride buffer system at pH 6.8. Elution of protein species is monitored at 280 nm. The major species peak (monomer) and total peak area are evaluated to determine purity. In one implementation, the SEC analysis involves injecting the sample onto two G3000SWxl columns connected in tandem and running it in an isocratic phosphate-sodium chloride buffer system at pH 6.8, where elution of protein species is monitored at 280 nm and the major species peak (monomer) and total peak area are measured. Sample purity (%) (in monomer %), HMW aggregate % and / or LMW degradation product % are reported.

[0197] If necessary, standard techniques can be used to measure residual CHO host cell protein (HCP) impurities in the antibody preparation via enzyme-linked immunosorbent assay (ELISA). Many commercially available ELISA kits designed for this purpose are available, such as the CHO HCP ELISA Kit 3G from Cygnus Technologies (Southport, NC (USA)). The host cell protein in the test sample can be captured using an immobilized polyclonal anti-CHO HCP antibody. The captured protein can then be detected using a suitable detection agent, such as a horseradish peroxidase-labeled form of the same antibody. In this exemplary embodiment, the amount of captured peroxidase can be measured colorimetrically at 450 nm using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB), the amount of peroxidase being directly proportional to the concentration of CHO HCP. Therefore, the CHO HCP assay involves capturing HCP using a polyclonal anti-CHO HCP antibody, which is detected after binding to a horseradish peroxidase-labeled form of the polyclonal anti-CHO HCP antibody. This polyclonal anti-CHO HCP antibody converts the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a substance quantified colorimetrically at 450 nm. The HCP concentration can be determined by comparison with a CHO HCP standard curve, such as one included in the test kit, and is reported as a percentage of the total protein level in the antibody preparation.

[0198] IV. Downstream processing and formulation

[0199] Anti-α4β7 antibodies (e.g., vedolizumab or antibodies having a binding region corresponding to vedolizumab) can be further purified from contaminant soluble proteins and peptides, and the following procedures are examples of suitable purification procedures, which may be used alone or in combination with one or more methods provided herein: affinity chromatography, such as affinity chromatography of protein A using resins that bind to the Fc region of the antibody; and chromatography on ion-exchange columns or resins, such as cation-exchange chromatography (CEX), e.g., SP-Sepharose. TM Or CM-Sepharose TM Fractionation on hydroxyapatite; anion exchange chromatography (AEX); hydrophobic interaction chromatography (HIC); mixed-mode chromatography; ethanol precipitation; chromatographic focusing; ammonium sulfate precipitation; using, for example, Sephadex G-75 TMThe purification process involves gel filtration; ultrafiltration and / or percolation, or a combination thereof. Examples of purification methods are described in Liu et al., mAbs, 2:480-499 (2010). At the end of the purification process, the recombinant protein is of high purity and suitable for human therapeutic use, such as in pharmaceutical antibody formulations described below. After purification, the high-purity recombinant protein can be ultrafiltered / percolated (UF / DF) to prepare pharmaceutical formulations suitable for human administration.

[0200] Following percolation and ultrafiltration, the antibody preparation may be retained in liquid form or lyophilized into a dry antibody preparation. In one aspect, the dry lyophilized antibody preparation is provided in single-dose vials 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 the form of a stable liquid pharmaceutical composition stored at about 2°C–8°C in a container, such as a vial, syringe, or cartridge, until it is administered to a subject in need. In some embodiments, the reconstituted lyophilized preparation or stable liquid pharmaceutical composition of the anti-α4β7 antibody contains about 0% to 5.0%, 0% to 2%, ≤2%, ≤1%, ≤0.6%, or ≤0.5% aggregates.

[0201] Therefore, in some embodiments, this document provides a reconstituted lyophilized antibody formulation or a stable liquid pharmaceutical composition comprising a humanized anti-α4β7 antibody or its antigen-binding moiety. Examples of lyophilized formulations comprising anti-α4β7 antibodies, such as vedolizumab, are described in U.S. Patent No. 9,764,033, the contents of which are incorporated herein by reference. Examples of liquid formulations comprising anti-α4β7 antibodies, such as vedolizumab, are described in U.S. Patent No. 10,040,855, the contents of which are incorporated herein by reference. In some embodiments, the reconstituted lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody comprises about 11% to 16%, 12% to 15%, ...

[0202] ≤14%, ≤13%, ≤12%, or ≤11% of the basic isotype. In some embodiments, the lyophilized formulation or stable liquid pharmaceutical composition of the anti-α4β7 antibody contains 65% to 75%, 66% to 74%, 67% to 73%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, or at least 70% of the major isotype.

[0203] Purified antibodies, such as anti-α4β7 antibodies (e.g., vedolizumab or antibodies having a binding region corresponding to vedolizumab), can be concentrated to provide concentrated protein compositions, for example, compositions with antibody concentrations of at least 100 mg / mL, 125 mg / mL, or 150 mg / mL, or concentrations of about 100 mg / mL, 125 mg / mL, or 150 mg / mL. It should be understood that the concentrated antibody product can be concentrated to levels permissible under concentration conditions, for example, to concentrations where the peptide is no longer soluble in solution.

[0204] In some embodiments, the compositions obtained herein comprise purified anti-α4β7 antibodies, such as vedolizumab, and are subsequently formulated for human use. In one embodiment, the purified antibody is formulated into a dry lyophilized preparation, which can be reconstituted with a liquid, such as sterile water, for administration. The reconstituted preparation can be administered via one of the above-described routes, via parenteral injection. Intravenous injection can be performed by infusion, such as by further dilution with sterile isotonic saline, a buffer such as phosphate-buffered saline, or Ringer's solution (lactated or glucose). In some embodiments, the purified antibody is formulated into a liquid preparation, thereby administering, for example, a dose of about 54 mg, 108 mg, or about 165 mg or about 216 mg of anti-α4β7 antibody via subcutaneous injection.

[0205] Containers suitable for storing and freezing the purified compositions described herein include polycarbonate bottles (for IV formulations) or PETG bottles (for subcutaneous formulations). After the formulation is aliquoted into the bottles, it can be frozen (e.g., at -60°C or lower).

[0206] The following examples illustrate improved methods and compositions for purifying antibodies. Examples 1-5 describe various methods and compositions that can be used to obtain purified compositions of anti-α4β7 antibodies, particularly vedolizumab. This document includes the methods described in the following examples, including the various parameters described therein.

[0207] Example

[0208] The following examples describe a method for purifying vedozimab produced in cell cultures using CHO cells as the expression system.

[0209] Example 1: Effect of elution buffer on purification of vedolizumab using protein A resin

[0210] This embodiment demonstrates an antibody purification method using protein A resin, which can be used to produce therapeutic anti-α4β7 antibodies, such as vedolizumab. As described herein, adjusting the elution pH of the protein A resin reduces the level of aggregates in the purified vedolizumab composition.

[0211] Vedoizumab was produced by culturing recombinant Chinese hamster ovary (CHO) cells (GS-CHO) that were genetically engineered to express antibodies (for general cell culture methods, see Li et al. (2010) mAbs, 2:5, 466-477).

[0212] Following cell culture in CHO cells, vedolizumab was selectively captured after initial recovery using a protein A affinity column. Affinity chromatography was performed using recombinant protein A resin to selectively remove the antibody from the clarified harvest derived from the upstream initial recovery process. This step also removed process-related impurities, such as host cell proteins (HCPs).

[0213] First, equilibrate the protein A resin with PBS equilibration solution (pH 7.2). Then, load the clarified harvest. Perform three washes. The first wash is performed with the same PBS washing solution (pH 7.2) as the equilibration solution; the second wash is performed with PBS washing solution containing 1M NaCl (pH 7.2); and the third wash is performed with the same washing solution as the first wash (PBS) and the equilibration solution. Washing removes impurities from the antibody, which is still bound to the resin. Next, elute the antibody from the resin using an elution buffer with a pH range specified. Figure 1 As described, the elution buffer with a pH of 3 to 3.5 was tested. Figure 1 The results provided show that the percentage of aggregates decreases as pH increases. Figure 1 As described, eluting vedozimab from protein A at pH 3 yields an eluent with a high level of aggregates, i.e., about 1%-1.2% aggregates, while using an elution buffer with a higher pH, such as about 3.5, yields an eluent with about 0.6%-0.85% aggregates.

[0214] Another study was conducted to identify process parameters associated with protein A purification that significantly impact the product quality of vedolizumab. Clarified harvest from GS-CHO cells expressing recombinant vedolizumab was loaded onto MabSelect SuReLX resin (GE Healthcare, Pittsburgh, PA). The bound antibody was washed with PBS and sodium citrate buffer, followed by elution. Elution pH was evaluated within the range of pH 3.3 to pH 3.9. Elution was performed using sodium citrate buffer. The effect of elution buffer pH on aggregate levels (HMW substance%) and HCP levels in the purified vedolizumab composition is described in Table 1 and [Table data would be inserted here]. Figure 2 middle.

[0215] As shown in Table 1, the linear regression model revealed that elution pH had a significant impact on all assay results (p < 0.05). Although the data for LMW% and HCP varied slightly, loading rate affected HCP removal. The combination of loading rate and loading flow rate had a smaller effect on LMW%.

[0216] ·monomer%:

[0217] Elution pH had a significant effect on monomer percentage (p<0.05). No other input parameters showed a correlation with monomer percentage. Monomer percentage increased with increasing elution pH.

[0218] •HMW%:

[0219] Elution pH had a significant effect on HMW% (p<0.05). No other input parameters showed a correlation with HMW%. HMW% decreased as elution pH increased.

[0220] ·LMW%:

[0221] Elution pH and the combination of loading and loading flow rate affect LMW% (p<0.05), however, the effect of input parameters on LMW% is considered to be minimal.

[0222] HCP:

[0223] Elution pH and loading had a significant effect on HCP, and both were expressed as ppm and the logarithm of the reduction factor (p<0.05).

[0224] Table 1. Purification of vedozimab using protein A affinity chromatography – Evaluation of input parameters

[0225] Elution pH Monomer (%) HMW (%) LMW (%) HCP (ppm) HCPLRF Load material - - - 152590 - 3.34 97.55 1.61 0.84 113 3.13 3.34 97.38 1.76 0.86 88 3.24 3.34 97.35 1.81 0.84 101 3.18 3.34 97.26 1.93 0.81 179 2.93 3.34 97.36 1.83 0.80 85 3.25 3.34 97.38 1.81 0.82 94 3.21 3.34 97.27 1.88 0.85 139 3.04 3.34 97.22 1.95 0.83 108 3.15 3.60 97.65 1.57 0.78 105 3.16 3.60 97.31 1.88 0.81 122 3.10 3.95 98.67 0.62 0.72 168 2.96 3.95 98.78 0.42 0.79 115 3.12 3.95 98.74 0.47 0.79 157 2.99 3.95 98.92 0.38 0.71 217 2.85 3.95 98.60 0.60 0.80 185 2.92 3.95 98.60 0.62 0.78 116 3.12 3.95 98.45 0.71 0.84 123 3.09 3.95 98.55 0.66 0.79 207 2.87

[0226] Vedolizumab has unique properties that distinguish it from other IgG antibodies, such as high hydrophobicity. Figure 3 This provides a comparison of vedolizumab with three other IgG antibodies, and presents the amount (HMW%) of aggregates found in the eluent when each antibody (vedolizumab (MLN0002), IgG A, IgG B, or IgG C) is eluted from a cation exchange column (Nuvia S; Bio Rad) using elution buffers with increasing pH values ​​(pH increasing from left to right). The performance change of vedolizumab in clearing aggregates is greater than that of the other three tested IgGs under optimal conditions. Therefore, as in... Figure 3 As observed, pH can affect aggregation levels during the purification of vedolizumab.

[0227] Example 2: Purification of vedolizumab using hydrophobic HIC resin

[0228] Given the hydrophobic nature of vedolizumab, reducing HMW aggregates during downstream purification can be challenging. Furthermore, minimizing the amount of host cellular protein (HCP) is also important when vedolizumab is generated in mammals, such as CHO cells. High-throughput methods were used to screen HICs, mixed-mode resins, and anion exchange resins and membranes for performance. Subsequently, the ability of eight HIC resins to reduce aggregation and minimize HCP during vedolizumab purification was tested under various equilibration, loading, and elution conditions. Toyopearl's hexyl-650C (Tosoh Biosciences) hydrophobic HIC resin was the only resin capable of exhibiting acceptable aggregate clearance and minimizing HCP. More specifically, under suitable binding conditions (e.g., 0.5 M (NH4)2SO4, pH 6.7), hexyl-650C reduced the aggregation level from approximately 1.5% HMW aggregates to 0.35%. Other resins tested included butyl-650M, butyl-600M, ultrabutyl-55C, phenyl-650M, phenyl-600M, PPG-600M, and ether-650M, but none could achieve such low aggregate levels.

[0229] Compared to other resins tested, including ethers, PPG, phenyl, and butyl, hexyl-650C is the most hydrophobic resin. Hexyl-650C has an average pore size of approximately 1,000 Å and an average particle size of approximately 100 μm.

[0230] Further experiments were performed using hexyl-650C in both binding / elution and flow-through modes. For the binding / elution assays, hexyl-650C reduced aggregates to approximately 0.30% HMW, but with low binding capacity (approximately 20 mg / ml resin). In contrast, the flow-through mode using hexyl-650C and vedolizumab reduced aggregates while increasing loading capacity. Flow-through assays were performed with an initial unadjusted loading of 108 mg / ml and 10 mM sodium phosphate containing 0.2 M sodium chloride at pH 6.7. These conditions in the flow-through assays reduced HMW from 1.39% to 0.71%. Increasing the salt, including replacing sodium chloride with potassium phosphate, further improved aggregate reduction. A reduced loading of 250 mM potassium phosphate, 50 mM potassium chloride (for equilibration and loading adjustment), and 67.5 mg / ml resin reduced aggregates from approximately 0.72% HMW to approximately 0.3% (with a recovery of 95.5%).

[0231] A column-based design of experiment (DOE) study was performed to further evaluate the ability of hexyl-650C to reduce aggregate levels for vedolizumab purification in flow-through mode. As described in Table 2, low HMW% and low HCP levels (ppm) were obtained using low pH and increased phosphate loading / equilibrium conditions. The experiments in Table 2 were performed using a resin loading of 60 mg / ml. Low pH conditions reduced HMW from 1.0% to values ​​less than or equal to 0.34%. The average antibody recovery was approximately 91.5%. High pH combined with high phosphate appeared to increase the affinity of the major vedolizumab species to the resin, resulting in lower recovery. In contrast, higher phosphate levels and low pH resulted in increased HMW clearance. Low HMW, low HCP, and low residual protein A leaching were observed under low phosphate and low pH conditions (see, for example, row 12 below). Therefore, the highly hydrophobic HIC resin was able to successfully remove vedolizumab aggregates (HMW) at levels below 0.5%.

[0232] Table 2. DOE Design and Data

[0233]

[0234] Example 3: Effects of column loading, elution buffer pH, and conductivity on the purification of vedolizumab using mixed-mode chromatographic resin

[0235] To produce productive CHO cell lines expressing high titers of davidimonab (≥5.0 g / L), it is necessary to develop a purification method designed to accommodate large quantities of this highly hydrophobic antibody.

[0236] Capto Adhere ImpRes are mixed-mode (MXM) chromatographic resins that possess strong anion exchange, hydrogen bonding, and hydrophobic interactions at a small bead size, thereby allowing for improved impurity removal and increased capacity.

[0237] Capto Adhere ImpRes mixed-mode resins operated in flow-through mode were able to purify vedolizumab, but the yield and impurity removal were lower than desired. Pre-characterization experiments using Capto Adhere ImpRes in binding-elution mode showed that the significant reduction in step yield and / or impurity removal capacity was associated with three process input parameters: resin loading capacity, elution buffer pH, and elution buffer conductivity. This example describes a study designed to further examine the effects of variations in these parameters on the performance of Capto Adhere ImpRes for vedolizumab purification and their impact on various product quality properties.

[0238] Materials and methods

[0239] After purification of protein A, the clarified cell culture harvest was loaded onto a Capto Adhere ImpRes (GE Healthcare, Chicago, IL, USA) column. The mixed-mode resin was washed with sodium phosphate buffer at pH 7.8, and antibodies were eluted from the column under different conditions as described below.

[0240] Samples were immediately submitted to the SEC for analysis, stored at 2°C–8°C, and processed within one week. Other assays (CEX, CHO HCP ELISA) were performed using cryo-holding buffer (-80°C). The methods used for analysis are listed in Table 3 below and described in detail below. Loading materials sampled after a series of runs were analyzed to confirm that the quality properties of the loading materials had not undergone substantial change.

[0241] Table 3. Analytical Methods

[0242]

[0243] Experimental Design

[0244] A full factorial design was employed at three levels: resin loading, elution buffer pH, and elution buffer conductivity. Sodium citrate elution buffer was used in these experiments. The resulting experimental design comprised thirty runs under three centerpoint conditions. Additional centerpoint conditions were part of the experimental design specified in Table 5 as DOE Mode 222. All other process input parameters remained at the centerpoint conditions. Experiments were designed using elution buffer sodium chloride concentration, and elution buffer conductivity measurements were used as input parameters for statistical analysis. Tables 4 and 5 describe the parameter ranges studied in the design and provide an overview of the design, respectively.

[0245] Table 4. Parameter range for evaluation

[0246] Parameter Units Range evaluated Notes Protein load / resin volume g / L 53-77 - Elution buffer pH pH 3.8-4.4 - Elution buffer conductivity mS / cm 19.7–28.9 NaCl concentration range 160-240 mM

[0247] Table 5. Experimental Design

[0248]

[0249]

[0250] DOE mode (3): high level; (2): medium level; (1): low level; (0): center point of each input parameter range. Experiments were designed using NaCl concentration as input parameter and statistical analysis was performed using actual conductivity measurements.

[0251] calculate

[0252] HMW removal rate % = (1 - (eluent HMW / loaded HMW)) * 100

[0253] The logarithmic reduction factor (LRF) is determined as follows:

[0254] LRF = log 10 {Residual} 负载物 / residue 洗脱液}

[0255] [Residual] 洗脱液 [This refers to the concentration of CHO HCP or protein A in the eluent, and the residual concentration is...] 负载物 [The concentrations of CHO HCP or protein A in the same loading medium during the same operation are expressed in ppm relative to the corresponding MLN0002 concentration.]

[0256] Statistical analysis

[0257] The product quality, indicating the assay results and KPIs for all experiments in the design, was analyzed using JMP 11 statistical software (SAS Institute, Cary, NC). Each reaction was analyzed by fitting to a linear model, as shown in Equation 1:

[0258] Equation 1 General Linear Regression Model

[0259]

[0260] Where y u For the response at the u-th observation, x iu The variables are independent, and the various β terms are model coefficient estimates.

[0261] In statistical analysis and modeling, fitting a relatively small dataset to a model containing a relatively large number of potential inputs often leads to overfitting. Overfitting is characterized by a high R-value. 2 The algorithm generates models with several scientifically meaningless (and statistically insignificant) terms. To determine the optimal statistically significant model while avoiding overfitting, each model is developed using forward regression with input parameters and a target response. The regression is stopped by a p-value threshold, where the input parameters are incorporated into the model if the p-value of the model is ≤0.05. The model (i) generated by the analytical algorithm achieves the highest possible R-value. 2 The values ​​(ii) include as few input parameters as possible, and (iii) describe the physically possible behavior of the antibody after undergoing multi-peak chromatography processing (even if the findings appear inconsistent with the initial technical expectations).

[0262] Results and Discussion

[0263] The experimental results are described in Tables 6 and 7, which contain the parameter estimates, corresponding p-values, and R values ​​for each reaction model determined by statistical analysis. 2 value.

[0264] Table 6. Overview of Statistical Model, Predictive Expression Coefficients, and p-values ​​– Process Performance

[0265]

[0266] Table 7. Overview of Statistical Models, Predictive Expression Coefficients, and p-values—Product Quality (SEC and CEX)

[0267]

[0268] Effects of pH and conductivity of elution buffer on antibody yield

[0269] The effects of elution buffer pH and elution buffer conductivity on the recovery or yield of vedozimab are described in Figure 4 and Figure 5 In the middle. For example Figure 4 and Figure 5 As described, the observed step recovery rate data also fit well to the linear regression model, such as R0. 2 The value is 0.930.

[0270] Figure 4 and Figure 5The graph shows the relationship between Capto Adhere ImpRes step recovery and elution buffer pH and conductivity or loading. Recovery was significantly affected by elution buffer pH (p < 0.0001) and resin loading (p = 0.0021), and less affected by elution buffer conductivity (p = 0.0189). At any level of elution buffer conductivity and loading, a elution buffer pH of approximately 4.40 resulted in a step yield ≤ 83.91% (runs 1, 4, 11–14, 16, 19, and 27). Lower resin loading negatively impacted recovery. Breakthrough was observed in all runs with a resin loading of 77 g / L during the wash step following sample loading. The effect of loading depended on the elution buffer pH (p = 0.0170). At low elution buffer pH (i.e., pH 3.8), the resin loading had no practical effect on recovery. However, as the elution buffer pH increased, the resin loading decreased, leading to a lower recovery (the recovery was 73.36%–78.84% at a resin loading of 53 g / L when the elution buffer pH was approximately 4.4, compared to 81.94%–83.91% at a resin loading of 77 g / L). Based on experimental results and model predictions, the conductivity of the elution buffer had a minimal effect on recovery. The lowest recovery of 73.36% was observed when the elution buffer pH was 4.40, the elution buffer conductivity was 28.89 mS / cm, and the resin loading was 53 g / L (run 13, the model predicted a recovery of 76.22%).

[0271] Therefore, as Figure 4 and Figure 5 As described in the paper, the yield of vedolizumab increases when mixed-mode chromatographic resin is used with an elution buffer having optimal pH and conductivity.

[0272] Effects of elution buffer pH and conductivity on HMW aggregates

[0273] The effects of elution buffer pH and conductivity on aggregate size are described in Figure 6 and Figure 7 The figure shows the effect of input parameters on the HMW quantity.

[0274] According to the linear regression model (R) 2 =0.962), the amount of HMW in the eluent is affected by the pH of the elution buffer (p<0.0001), the conductivity of the elution buffer (p<0.0001), and the loading (p=0.0015). Increasing the elution pH and conductivity decreases the amount of HMW in the eluent, while increasing the loading increases its amount. The model also predicts a statistically significant (p=0.0462) interaction between the pH and conductivity of the elution buffer.

[0275] as follows Figure 6 and Figure 7 As described, HMW content of approximately 1% or higher was observed at an elution buffer pH of approximately 3.80. The highest HMW content of 1.23% was obtained at an elution pH of 3.80, an elution conductivity of 19.67 mS / cm (160 mM NaCl), and a resin loading of 65 g / L (Operation 2) (the model predicted a content of 1.19%). Under the same elution buffer conditions, the worst-case predicted HMW content at 77 g / L was 1.24%.

[0276] According to the linear regression model, the pH of the elution buffer (p < 0.0001), the conductivity of the elution buffer (p = 0.0003), and the loading (p = 0.0016) each had a statistically significant effect on HMW scavenging capacity. Under all conditions evaluated in this study, a certain level of scavenging capacity (12.50%–72.79%) was achieved. Among the inputs, the elution pH had the greatest impact. Increasing the pH of the elution buffer improved HMW scavenging, but its effect on recovery was opposite. According to the model, increasing the conductivity of the elution buffer and decreasing the loading increased HMW scavenging.

[0277] Therefore, as Figure 6 and Figure 7 As described above, the level of aggregates (HMW substance%) in the purified vedolizumab composition can be adjusted by selecting the pH and conductivity of the elution buffer 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 higher pH and / or higher conductivity.

[0278] Example 4: Effect of elution buffer on purification of vedolizumab using cation exchange (CEX) resin

[0279] Cation exchange chromatography (CEX) has also been developed as a means to further reduce the level of aggregates in vedolizumab formulations. The study described herein focuses on adapting CEX chromatography using Nuvia HR-S resin (Bio-Rad, Hercules, CA, USA) for the purification of vedolizumab, specifically aiming to reduce the level of aggregates in this hydrophobic antibody. Elution conditions for the CEX procedure operated in binding / elution mode were evaluated. A design-of-experiment (DoE) approach was used to evaluate the effects of several parameters, including elution buffer pH and elution buffer conductivity, on the process output.

[0280] Materials and methods

[0281] The loading materials and analytical methods used in this study are similar to those described in Example 3 above.

[0282] Experimental Design

[0283] Initial screening studies and preliminary risk assessments determined that the pH and conductivity of the elution buffer have known or potential effects on the performance output (PPO) of the Nuvia HR-S process when the resin is operated in binding / elution mode. This study aimed to characterize the effects of these process parameters. The ranges of study parameters and experimental design are listed in Tables 8 and 9, respectively. The conductivity of the elution buffer was varied by adjusting the concentration of sodium chloride (NaCl), and the evaluated ranges of NaCl are provided in Table 8.

[0284] Table 8. Study process parameters

[0285] Parameter Range evaluated Load 30-65 g mAb / L resin Elution buffer pH 5.1–5.7 Elution buffer conductivity 10.59-16.08 mS / cm Elution buffer [NaCl] 70-110 mM

[0286] Table 9. Experimental Design

[0287]

[0288]

[0289] Results and Discussion

[0290] The effects of elution buffer pH and elution buffer conductivity on Nuvia HR-S process performance output (PPO) are described in Figure 8-13 middle.

[0291] Effects of elution buffer pH and conductivity on HMW aggregates

[0292] The effects of elution buffer pH and conductivity on aggregate levels are described in Figure 8-10 The figure shows the effect of input parameters on the HMW quantity.

[0293] like Figure 8-10 As described, the changes in elution buffer HMW, monomer, and LMW were 0.01% to 0.88%, 98.33% to 99.27%, and 0.62% to 1.35%, respectively. The HMW model, in addition to including the interaction term between elution buffer pH and conductivity, also showed strong linear dependence on both parameters. The model surface (e.g.) Figure 8 As shown in the figure, HMW is lowest when the pH and conductivity of the elution buffer are extremely low, and highest when the pH and conductivity of the elution buffer are extremely high.

[0294] The HMW clearance rate for each run was determined to account for the variation in HMW content of the loaded material throughout the study. Similar to the eluent HMW, the HMW clearance rate varied considerably throughout the study (from -30.65% to 98.61%), and the HMW clearance rate model included linear terms and interaction terms for the pH and conductivity of the elution buffer. Figure 9 The model performance was shown to be as follows: while the highest HMW clearance values ​​were obtained under conditions of reduced elution buffer pH and conductivity, many test conditions exhibited HMW clearance rates >70%. However, the reported negative HMW clearance values ​​for runs 9, 20, and 24 (-1.18%, -20.55%, and -30.65%, respectively) indicate significant variations in aggregate removal observed across the evaluated conditions, and that some conditions may generate rather than remove aggregate material.

[0295] Runs 40 to 42 used Capto Adhere ImpRes elution buffer as the loading material, which contained aggregate levels higher than those typically used in loading materials during CEX processing under centerpoint conditions. HMW and HMW clearance models indicated that increasing the pH and conductivity of the elution buffer resulted in an increase in HMW content in the eluent. The conditions selected for runs 40 to 42 were intended to probe possible elution conditions using the “worst-case” aggregate levels of the CEX loading material. For elution buffer pH values ​​of 5.50 and 5.54 and an elution buffer conductivity of 13.40 mS / cm, the resulting eluents had HMW levels of 0.31% to 0.34%. However, when using an elution buffer at pH 5.60 (while maintaining constant conductivity), the eluent HMW increased to 0.59%. At these aggregate levels, further processing carries the risk of failing to meet the vedolizumab acceptance criteria regarding HMW.

[0296] It has been found that LMW decreases linearly with increasing elution buffer pH or elution buffer conductivity. The model also includes interaction terms for elution buffer pH / elution buffer conductivity, elution buffer pH / loading, and elution buffer conductivity / loading. Similar to the model for HMW, the monomer model exhibits a strong dependence on elution buffer pH and conductivity, which are presented as linear terms and interaction terms. The model surface (in...) Figure 10 The saddle-shaped function (shown in the figure) indicates that the minimum amount of monomer is obtained under conditions of extremely high combination of pH and conductivity of the elution buffer.

[0297] Therefore, as Figure 8-10 As described above, the pH and conductivity of the elution buffer can be used to adjust the level (HMW substance %) of aggregates in compositions containing vedolizumab purified using CEX resin. Furthermore, as... Figure 8-10As shown, when CEX resin is used with an elution buffer that has a low pH and / or low conductivity, the level of aggregates in the purified vedolizumab composition can be reduced.

[0298] Effects of pH and conductivity of elution buffer on basic isoform species

[0299] The effects of elution buffer pH and conductivity on the amount of basic isoform species are described in Figure 11-13 The figure shows the effect of input parameters on the content of acidic, primary, and basic isoforms.

[0300] like Figure 11-13 As described, the ranges for acidic, predominantly acidic, and alkaline content were 12.49% to 30.27%, 64.32% to 73.82%, and 5.42% to 18.04%, respectively. In addition to the interaction terms for all three parameters, the acidic content model also included linear terms for elution buffer pH, elution buffer conductivity, and loading. Elution buffer pH and elution buffer conductivity had the greatest impact on acidic content. Figure 11 The model surface shows that the acidity is highest when operating under a combination of extremely low pH and conductivity values ​​of the elution buffer.

[0301] The developed model for the content of major isotypes includes interaction terms between elution buffer pH, elution buffer conductivity, and loading, with the elution buffer pH / elution buffer conductivity interaction having the greatest impact on model expression. Figure 12 As shown, the highest major isotype content was obtained under the condition of the highest elution buffer conductivity and the lowest elution buffer pH; the lowest major isotype content was predicted under the condition of the combination of very low elution buffer pH and conductivity.

[0302] The linear terms of elution buffer pH, elution buffer conductivity, and loading had the greatest impact on the alkaline isoform content of the eluent; the interaction term showed a very small contribution. Figure 13 As can be seen from the model surface, the content of alkaline isoforms increases in response to increases in the pH and conductivity of the elution buffer.

[0303] Therefore, as Figure 11-13 As described above, the pH and conductivity of the elution buffer can be used to adjust the distribution of charged isoforms in compositions containing vedolizumab purified using CEX resin. Figure 11 As shown, when CEX resin is used with an elution buffer that increases pH and / or conductivity, the level of acidic isoforms in the purified vedolizumab composition can be reduced. Furthermore, as... Figure 13As shown, when CEX resin is used with an elution buffer that reduces pH and / or conductivity, the level of basic isoforms in the purified vedolizumab composition can be reduced.

[0304] Example 5: Determination of Product Quality Attributes

[0305] The following analytical assays and methods were used in the foregoing examples to determine the product quality properties of vedolizumab.

[0306] Cation exchange chromatography (CEX) fractionates vedolizumab antibody species (primarily isotypes, basic types, and acidic types) based on total surface charge. After dilution to low ionic strength using a mobile phase, the test sample is injected into Dionex Pro-Pac equilibrated at pH 6.6 with 10 mM sodium phosphate. TM Protein elution was performed on a WCX-10 column (Thermo Fisher Scientific, Waltham, MA (USA)) using a sodium chloride gradient in the same buffer. Protein elution was monitored at 280 nm, and each peak was designated as acidic, basic, or major isoform. The percentages of major isoforms, the sum of acidic percentages, and the sum of basic percentages were reported. The retention times of the major isoforms in the sample were compared with those in the reference standard to determine concordance.

[0307] Purity of vedolizumab was determined using size exclusion chromatography (SEC). Reference standards and test samples (75 μg) were analyzed using two tandem G3000SWxl columns (Tosoh Bioscience, King of Prussia, PA (USA)) and an isocratic phosphate-sodium chloride buffer system at pH 6.8. This method allowed for the separation of the antibody monomer from high molecular weight (HMW) and low molecular weight (LMW) degradation products. Elution of protein species was monitored at 280 nm. The major species peak (monomer) and total peak area were evaluated to determine purity. Sample purity (%) (in monomer %) and aggregate % were reported.

[0308] Equivalent solution

[0309] Those skilled in the art will recognize or be able to determine many equivalent embodiments of the invention described herein using only conventional experiments. Such equivalent embodiments are intended to be covered by the following claims. All references, patents, and published patent applications cited throughout this application are incorporated herein by reference.

[0310] Sequence Listing

[0311]

[0312]

Claims

1. A method for obtaining a composition comprising the anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising... The matrix containing protein A is contacted with the liquid solution containing the anti-α4β7 antibody and one or more impurities, thereby causing the anti-α4β7 antibody to bind to protein A; The matrix containing protein A is washed with a washing solution; and The anti-α4β7 antibody is eluted from the matrix by contacting the matrix containing protein A with an elution solution at a pH of 3.3 to 4, thereby obtaining a composition containing the anti-α4β7 antibody. The anti-α4β7 antibody is a humanized antibody, specifically an IgG1 antibody, comprising a heavy chain variable region containing a CDR3 domain as shown in SEQ ID NO:4, a CDR2 domain as shown in SEQ ID NO:3, and a CDR1 domain as shown in SEQ ID NO:2; and a light chain variable region containing a CDR3 domain as shown in SEQ ID NO:8, a CDR2 domain as shown in SEQ ID NO:7, and a CDR1 domain as shown in SEQ ID NO:

6. The antibody mentioned above is produced in Chinese hamster ovary (CHO) cells, and The composition containing the anti-α4β7 antibody contains less than 1% high molecular weight (HMW) aggregates.

2. The method of claim 1, wherein the protein A is immobilized on a solid phase.

3. The method of claim 2, wherein the solid phase comprises one or more of beads, gels, and resins.

4. The method according to any one of claims 1 to 3, wherein the pH value of the washing solution is 7.

5. The method of any one of claims 1 to 3, wherein the elution solution comprises citric acid.

6. The method of any one of claims 1 to 3, wherein the pH of the elution solution is 3.7 to 4.0 or 3.3 to 3.

8.

7. A method for producing a composition comprising the anti-α4β7 antibody from a liquid solution comprising an anti-α4β7 antibody and one or more impurities, the method comprising... The liquid solution containing the anti-α4β7 antibody and one or more impurities is contacted with a mixed-mode chromatography resin, thereby causing the anti-α4β7 antibody to bind to the resin; The mixed-mode chromatographic resin was washed with a washing solution; and The anti-α4β7 antibody is eluted from the mixed-mode chromatographic resin by contacting it with an elution solution at a pH of 3.9 to 4.4, thereby obtaining a composition containing the anti-α4β7 antibody. The anti-α4β7 antibody contains the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:5; The antibody mentioned above is produced in Chinese hamster ovary (CHO) cells, and The conductivity of the elution solution is 20 mS / cm to 30 mS / cm.

8. The method of claim 7, wherein the composition comprising the anti-α4β7 antibody contains less than 1% HMW aggregates.

9. The method of claim 7 or 8, wherein the pH of the elution solution is equal to or higher than pH 4.

1.

10. The method of claim 7 or 8, wherein the pH of the elution solution is from pH 4.1 to pH 4.

4.

11. The method of claim 7 or 8, wherein the elution solution comprises NaCl at a concentration of 160 mM to 240 mM.

12. The method of claim 7 or 8, wherein the mixed-mode chromatographic resin is Capto Adhere ImpRes.

13. The method of claim 7 or 8, wherein the method further comprises purifying the anti-α4β7 antibody using a cation exchange (CEX) resin.

14. The method of claim 13, wherein the CEX resin is operated in a binding / elution mode.

15. A method for producing a composition containing anti-α4β7 antibody from a liquid solution containing anti-α4β7 antibody and one or more impurities, the method comprising: The liquid solution containing the anti-α4β7 antibody and one or more impurities is contacted with a mixed-mode chromatography resin, thereby causing the anti-α4β7 antibody to bind to the resin; The mixed-mode chromatographic resin was washed with a washing solution; and The anti-α4β7 antibody is eluted from the mixed-mode chromatographic resin by contacting it with an elution solution having a pH of 3.8 to 4.2 and a conductivity of 18 mS / cm to 28 mS / cm, thereby obtaining a composition containing the anti-α4β7 antibody. The antibody mentioned above is produced in Chinese hamster ovary (CHO) cells, and The anti-α4β7 antibody contains the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:

5.

16. The method of claim 15, wherein, relative to a control composition containing anti-α4β7 antibody obtained in a similar manner using a control elution solution with a pH higher than pH 4.2 and / or a control conductivity higher than 28 mS / cm, the composition contains the anti-α4β7 antibody in increased yield.

17. The method of claim 15 or 16, wherein the pH of the elution solution is equal to or less than 4.

0.

18. The method of claim 15 or 16, wherein the elution solution comprises NaCl at a concentration of 160 mM to 240 mM.

19. The method of claim 15 or 16, wherein the mixed-mode chromatographic resin is contacted with at least 55 g of the anti-α4β7 antibody per liter of resin.

20. The method of claim 19, wherein the mixed-mode chromatographic resin is contacted with 55 g to 80 g of the anti-α4β7 antibody per liter of resin.

21. The method of claim 15 or 16, wherein the mixed-mode chromatographic resin has strong anion exchange, hydrogen bonding and hydrophobic interaction functions at a small bead size, optionally wherein the mixed-mode chromatographic resin is Capto Adhere ImpRes.

22. The method of claim 15 or 16, wherein the method further comprises purifying the anti-α4β7 antibody using a cation exchange (CEX) resin.

23. The method of claim 22, wherein the CEX resin is operated in a binding / elution mode.

24. The method according to any one of claims 1 to 3, 7 or 15, wherein the cells are GS-CHO cells.

25. The method of any one of claims 1, 7 or 15, wherein the resulting composition comprises a purified anti-α4β7 antibody, and wherein the method further comprises a subsequent step of formulating the anti-α4β7 antibody into a formulation suitable for human use.

26. The method of any one of claims 1, 7 or 15, wherein the method comprises formulating the purified anti-α4β7 antibody into a dried, lyophilized formulation.

27. The method of claim 26, wherein the method further comprises reconstituted the dried, lyophilized formulation with a liquid, thereby making the formulation suitable for administration.

28. The method of any one of claims 1, 7 or 15, wherein the method comprises formulating the purified anti-α4β7 antibody into a liquid preparation, thereby making the anti-α4β7 antibody suitable for administration by subcutaneous injection.

29. The method of claim 1, wherein the anti-α4β7 antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:1 and a light chain variable region sequence as shown in SEQ ID NO:

5.

30. The method of any one of claims 1, 7 or 15, wherein the anti-α4β7 antibody is vedolizumab.

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